Clinical Focus


  • Cancer > Hematology
  • Hematology

Academic Appointments


Administrative Appointments


  • Senior Associate Dean for Veterans Affairs, School of Medicine (2006 - 2020)
  • Chief of Staff, VA Palo Alto Health Care System (2006 - 2020)

Boards, Advisory Committees, Professional Organizations


  • Editor-in-Chief, Hematology, UpToDate (2008 - Present)
  • Member, Association of American Physicians (2004 - Present)
  • Member, American Society for Clinical Investigation (1987 - Present)

Professional Education


  • Board Certification: American Board of Internal Medicine, Hematology (1980)
  • Residency: New York Presbyterian Cornell Campus Internal Medicine Residency (1978) NY
  • Internship: New York Presbyterian Cornell Campus Internal Medicine Residency (1976) NY
  • Fellowship: New York Presbyterian Cornell Campus Hematology Oncology Fellowship (1981) NY
  • Medical Education: Columbia University Vagelos College of Physicians and Surgeons Registrar (1975) NY
  • Board Certification: American Board of Internal Medicine, Internal Medicine (1978)
  • Board Certification: American Board of Internal Medicine, Medical Oncology (1981)

Current Research and Scholarly Interests


Our long-term interest is to have a better understanding of the natural antithrombotic pathways and the pathophysiology of vascular thrombosis. We have focused on thrombin, the key enzyme in the blood clotting cascade. Using a library of thrombin mutants generated by site-directed mutagenesis, we have mapped the critical functional sites on thrombin for its interactions with its diverse substrates. We are studying a thrombin-activatable carboxypeptidase that plays a key role in the physiologic regulation of thrombin’'s pro-thrombotic and pro-inflammatory properties. Our goal is to develop new antithrombotic agents and devise new diagnostic tests for vascular thrombotic disorders.

2024-25 Courses


All Publications


  • Methods to Investigate Thrombin Cleavage of Osteopontin (OPN). Methods in molecular biology (Clifton, N.J.) Zhao, L., Leung, L. L., Morser, J. 2024; 2747: 95-117

    Abstract

    Osteopontin (OPN) is a matricellular protein containing binding sites for a variety of ligands including an RGD sequence for binding to αvβ3 integrins. OPN is a conserved substrate for thrombin, the effector protease of the coagulation cascade. Thrombin cleaves OPN at a single site revealing new functionalities such as a previously cryptic α4β1 and α9β1 integrin-binding site. That integrin-binding site is abolished upon treatment with a basic carboxypeptidase. The thrombin cleavage of OPN has been demonstrated to play a role in regulating tumor growth.This report describes methods for production of full-length OPN as well as the enzymatically cleaved OPN fragments resulting from thrombin and carboxypeptidase treatments. Quantification procedures for the various OPN proteins are described as well as functional assays on mouse melanoma and myeloid cell lines.

    View details for DOI 10.1007/978-1-0716-3589-6_9

    View details for PubMedID 38038935

    View details for PubMedCentralID 9917417

  • Chemerin Levels in Individuals with Type 2 Diabetes and a Normal Weight versus Individuals with Type 2 Diabetes and Obesity: An Observational, Cross-Sectional Study. Biomedicines Mukherji, A. B., Idowu, V., Zhao, L., Leung, L. L., Shen, S., Palaniappan, L., Morser, J. 2024; 12 (5)

    Abstract

    Chemerin acts as both a chemotactic agent and an adipokine that undergoes proteolytic cleavage, converting inactive precursors into their active forms before being subsequently inactivated. Elevated chemerin levels are linked to obesity and type 2 diabetes mellitus (T2D). This study aimed to elucidate the effects of T2D and obesity on chemerin levels by comparing plasma samples from individuals with a normal weight and T2D (BMI < 25; NWD group n = 22) with those from individuals who are overweight or obese and have T2D (BMI ≥ 25; OWD group n = 39). The total chemerin levels were similar in the NWD and OWD groups, suggesting that T2D may equalize the chemerin levels irrespective of obesity status. The cleavage of chemerin has been previously linked to myocardial infarction and stroke in NWD, with potential implications for inflammation and mortality. OWD plasma exhibited lower levels of cleaved chemerin than the NWD group, suggesting less inflammation in the OWD group. Here, we showed that the interaction between obesity and T2D leads to an equalization in the total chemerin levels. The cleaved chemerin levels and the associated inflammatory state, however, differ significantly, underscoring the complex relationship between chemerin, T2D, and obesity.

    View details for DOI 10.3390/biomedicines12050983

    View details for PubMedID 38790945

  • Chemerin in Participants with or without Insulin Resistance and Diabetes. Biomedicines Zhao, L., Zhou, J., Abbasi, F., Fathzadeh, M., Knowles, J. W., Leung, L. L., Morser, J. 2024; 12 (4)

    Abstract

    Chemerin is a chemokine/adipokine, regulating inflammation, adipogenesis and energy metabolism whose activity depends on successive proteolytic cleavages at its C-terminus. Chemerin levels and processing are correlated with insulin resistance. We hypothesized that chemerin processing would be higher in individuals with type 2 diabetes (T2D) and in those who are insulin resistant (IR). This hypothesis was tested by characterizing different chemerin forms by specific ELISA in the plasma of 18 participants with T2D and 116 without T2D who also had their insulin resistance measured by steady-state plasma glucose (SSPG) concentration during an insulin suppression test. This approach enabled us to analyze the association of chemerin levels with a direct measure of insulin resistance (SSPG concentration). Participants were divided into groups based on their degree of insulin resistance using SSPG concentration tertiles: insulin sensitive (IS, SSPG ≤ 91 mg/dL), intermediate IR (IM, SSPG 92-199 mg/dL), and IR (SSPG ≥ 200 mg/dL). Levels of different chemerin forms were highest in patients with T2D, second highest in individuals without T2D who were IR, and lowest in persons without T2D who were IM or IS. In the whole group, chemerin levels positively correlated with both degree of insulin resistance (SSPG concentration) and adiposity (BMI). Participants with T2D and those without T2D who were IR had the most proteolytic processing of chemerin, resulting in higher levels of both cleaved and degraded chemerin. This suggests that increased inflammation in individuals who have T2D or are IR causes more chemerin processing.

    View details for DOI 10.3390/biomedicines12040924

    View details for PubMedID 38672278

    View details for PubMedCentralID PMC11048116

  • Thrombin Cleavage of Osteopontin and the Host Anti-Tumor Immune Response. Cancers Leung, L. L., Myles, T., Morser, J. 2023; 15 (13)

    Abstract

    Osteopontin (OPN) is a multi-functional protein that is involved in various cellular processes such as cell adhesion, migration, and signaling. There is a single conserved thrombin cleavage site in OPN that, when cleaved, yields two fragments with different properties from full-length OPN. In cancer, OPN has tumor-promoting activity and plays a role in tumor growth and metastasis. High levels of OPN expression in cancer cells and tumor tissue are found in various types of cancer, including breast, lung, prostate, ovarian, colorectal, and pancreatic cancer, and are associated with poor prognosis and decreased survival rates. OPN promotes tumor progression and invasion by stimulating cell proliferation and angiogenesis and also facilitates the metastasis of cancer cells to other parts of the body by promoting cell adhesion and migration. Furthermore, OPN contributes to immune evasion by inhibiting the activity of immune cells. Thrombin cleavage of OPN initiates OPN's tumor-promoting activity, and thrombin cleavage fragments of OPN down-regulate the host immune anti-tumor response.

    View details for DOI 10.3390/cancers15133480

    View details for PubMedID 37444590

  • Chemerin Forms: Their Generation and Activity. Biomedicines Zhao, L., Leung, L. L., Morser, J. 2022; 10 (8)

    Abstract

    Chemerin is the product of the RARRES2 gene which is secreted as a precursor of 143 amino acids. That precursor is inactive, but proteases from the coagulation and fibrinolytic cascades, as well as from inflammatory reactions, process the C-terminus of chemerin to first activate it and then subsequently inactivate it. Chemerin can signal via two G protein-coupled receptors, chem1 and chem2, as well as be bound to a third non-signaling receptor, CCRL2. Chemerin is produced by the liver and secreted into the circulation as a precursor, but it is also expressed in some tissues where it can be activated locally. This review discusses the specific tissue expression of the components of the chemerin system, and the role of different proteases in regulating the activation and inactivation of chemerin. Methods of identifying and determining the levels of different chemerin forms in both mass and activity assays are reviewed. The levels of chemerin in circulation are correlated with certain disease conditions, such as patients with obesity or diabetes, leading to the possibility of using chemerin as a biomarker.

    View details for DOI 10.3390/biomedicines10082018

    View details for PubMedID 36009565

  • Simultaneous Cases of Carfilzomib-Induced Thrombotic Microangiopathy in 2 Patients With Multiple Myeloma. Federal practitioner : for the health care professionals of the VA, DoD, and PHS Myall, N. J., Wang, S. X., Hall, E. T., Witteles, W. H., Leung, L., Dunn, T. J., Hong, W. J. 2022; 39 (Suppl 3): S56-S62

    Abstract

    In patients with multiple myeloma, thrombotic microangiopathy is a rare adverse event associated with proteasome inhibitors, such as bortezomib, carfilzomib, and ixazomib.Two patients with multiple myeloma who presented with carfilzomib-induced thrombotic microangiopathy received eculizumab with subsequent stabilization of renal function.Given the overall rarity of this adverse event, the simultaneous presentation of these 2 cases was unexpected. These cases underscores the need for heightened awareness in clinical practice of thrombotic microangiopathy. The potential role of eculizumab as a therapeutic treatment in the setting of thrombotic microangiopathy requires further investigation.

    View details for DOI 10.12788/fp.0284

    View details for PubMedID 36426106

    View details for PubMedCentralID PMC9662305

  • Thrombin cleavage of osteopontin initiates osteopontin's tumor-promoting activity. Journal of thrombosis and haemostasis : JTH Peraramelli, S., Zhou, Q., Zhou, Q., Wanko, B., Zhao, L., Nishimura, T., Leung, T. H., Mizuno, S., Ito, M., Myles, T., Stulnig, T. M., Morser, J., Leung, L. L. 1800

    Abstract

    BACKGROUND: Osteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for alpha4 beta1 and alpha9 beta1 integrins.METHODS: Thrombin cleavage-resistant OPNR153A knock-in (OPN-KI) mice were generated and compared to OPN deficient mice (OPN-KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes.RESULTS: OPN-KI mice engineered with a thrombin cleavage-resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN-KI mouse was identical to that observed in OPN-KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN-KI mice had increased tumor-associated macrophages with an altered activation phenotype. Immunodeficient OPN-KI mice (NOG-OPN-KI) or macrophage-depleted OPN-KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin-cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor-associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN-KI and OPN-KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells.CONCLUSIONS: Thrombin cleavage of OPN, derived from the host and the tumor, initiates OPN's tumor-promoting activity in vivo.

    View details for DOI 10.1111/jth.15663

    View details for PubMedID 35108449

  • Both plasma basic carboxypeptidases, carboxypeptidase B2 and carboxypeptidase N, regulate vascular leakage activity in mice. Journal of thrombosis and haemostasis : JTH Zhou, Q., Zhao, L., Shao, Z., Declerck, P., Leung, L. L., Morser, J. 2021

    Abstract

    BACKGROUND: Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2 (B2R) causing vascular leakage. BK is inactivated by proteolysis by the plasma carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2.OBJECTIVES: Determine the role of CPB2 and CPN in the regulation of vascular leakage.METHODS: Mice deficient in CPB2, CPN or both (Cpb2-/- , Cpn-/- and Cpb2-/- /Cpn-/- ) were compared to wild type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments mice were pretreated with antibodies that prevent activation of proCPB2.RESULTS: Skin irritation increased vascular leakage most in Cpb2-/- /Cpn-/- , less in Cpb2-/- and Cpn-/- and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin (TM) complex, increased vascular leakage to the level seen in Cpb2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis.CONCLUSIONS: BK is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma carboxypeptidases have more vascular leak than ones lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.

    View details for DOI 10.1111/jth.15551

    View details for PubMedID 34626062

  • Chemerin regulates formation and function of brown adipose tissue: Ablation results in increased insulin resistance with high fat challenge and aging. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Zhang, Y., Shen, W., Qiu, S., Yang, P., Dempsey, G., Zhao, L., Zhou, Q., Hao, X., Dong, D., Stahl, A., Kraemer, F. B., Leung, L. L., Morser, J. 2021; 35 (7): e21687

    Abstract

    Apart from its role in inflammation and immunity, chemerin is also involved in white adipocyte biology. To study the role of chemerin in adipocyte metabolism, we examined the function of chemerin in brown adipose tissue. Brown and white adipocyte precursors were differentiated into adipocytes in the presence of Chemerin siRNA. Chemerin-deficient (Chem-/- ) mice were compared to wild-type mice when fed a high-fat diet. Chemerin is expressed during brown adipocyte differentiation and knock down of chemerin mRNA results in decreased brown adipocyte differentiation with reduced fatty acid uptake in brown adipocytes. Chem-/- mice are leaner than wild-type mice but gain more weight when challenged with high-fat diet feeding, resulting in a larger increase in fat deposition. Chem-/- mice develop insulin resistance when on a high-fat diet or due to age. Brown adipose depots in Chem-/- mice weigh more than in wild-type mice, but with decreased mitochondrial content and function. Compared to wild-type mice, male Chem-/- mice have decreased oxygen consumption, CO2 production, energy expenditure, and a lower respiratory exchange ratio. Additionally, body temperature of Chem-/- mice is lower than that of wild-type mice. These results revealed that chemerin is expressed during brown adipocyte differentiation and has a pivotal role in energy metabolism through brown adipose tissue thermogenesis.

    View details for DOI 10.1096/fj.202100156R

    View details for PubMedID 34089273

  • Antibody-mediated targeting of cleavage-specific OPN-T cell interactions PLOS ONE Wanko, B., Tardelli, M., Juerets, A., Neuhofer, A., Prager, G., Morser, J., Leung, L. L., Staffler, G., Zeyda, M., Stulnig, T. M. 2019; 14 (4)
  • Antibody-mediated targeting of cleavage-specific OPN-T cell interactions. PloS one Wanko, B., Tardelli, M., Jurets, A., Neuhofer, A., Prager, G., Morser, J., Leung, L. L., Staffler, G., Zeyda, M., Stulnig, T. M. 2019; 14 (4): e0214938

    Abstract

    T cells are crucial players in obesity-mediated adipose tissue inflammation. We hypothesized that osteopontin (OPN), an inflammatory protein with enhanced activity when proteolytically cleaved, affects the number of viable T cells in adipose tissue and assessed inhibition of the interaction between T cells and thrombin and matrix metalloproteinases-cleaved OPN using antibodies and postimmune sera. Gene expression of T cell markers in adipose tissue from wild-type (wt) and Spp1-/- (OPN deficient) mice was analyzed after 16 weeks of high fat diet (HFD) or low fat diet (LFD) feeding. CD3, CD8 and OPN gene expression in omental adipose tissue from individuals with obesity was measured. OPN-T cell interactions were assessed with a fluorescence-based adhesion assay and blocked with antibodies targeting OPN. Comparison of T cell gene expression in adipose tissue from wt and Spp1-/- mice showed that OPN affected the number of T cells while in humans, levels of OPN correlated with T cell markers in omental adipose tissue. The interaction between T cells and cleaved OPN was blocked by postimmune sera following OPN peptide vaccinations and with monoclonal antibodies. In conclusion, levels of OPN affected the number of T cells in obesity and antibodies against cleaved OPN antagonize OPN-T cell interactions.

    View details for PubMedID 30951532

  • Decoding the Genomics of Abdominal Aortic Aneurysm. Cell Li, J., Pan, C., Zhang, S., Spin, J. M., Deng, A., Leung, L. L., Dalman, R. L., Tsao, P. S., Snyder, M. 2018; 174 (6): 1361

    Abstract

    A key aspect of genomic medicine is to make individualized clinical decisions from personal genomes. We developed a machine-learning framework to integrate personal genomes and electronic health record (EHR) data and used this framework to study abdominal aortic aneurysm (AAA), a prevalent irreversible cardiovascular disease with unclear etiology. Performing whole-genome sequencing on AAA patients and controls, we demonstrated its predictive precision solely from personal genomes. By modeling personal genomes with EHRs, this framework quantitatively assessed the effectiveness of adjusting personal lifestyles given personal genome baselines, demonstrating its utility as a personal health management tool. We showed that this new framework agnostically identified genetic components involved in AAA, which were subsequently validated in human aortic tissues and in murine models. Our study presents a new framework for disease genome analysis, which can be used for both health management and understanding the biological architecture of complex diseases. VIDEO ABSTRACT.

    View details for PubMedID 30193110

  • Decoding the Genomics of Abdominal Aortic Aneurysm CELL Li, J., Pan, C., Zhang, S., Spin, J. M., Deng, A., Leung, L. K., Dalman, R. L., Tsao, P. S., Snyder, M. 2018; 174 (6): 1361-+
  • Dynamic and tissue-specific proteolytic processing of chemerin in obese mice PLOS ONE Zhao, L., Yamaguchi, Y., Shen, W., Morser, J., Leung, L. K. 2018; 13 (8)
  • Chemerin 156F, generated by chymase cleavage of prochemerin, is elevated in joint fluids of arthritis patients. Arthritis research & therapy Zhao, L., Yamaguchi, Y., Ge, X., Robinson, W. H., Morser, J., Leung, L. L. 2018; 20 (1): 132

    Abstract

    BACKGROUND: Chemerin is a chemoattractant involved in immunity that also functions as an adipokine. Chemerin is secreted as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C-terminus, involving proteases in coagulation, fibrinolysis, and inflammation. Previously, we found chem158K was the dominant chemerin form in synovial fluids from patients with arthritis. In this study, we aimed to characterize a distinct cleaved chemerin form, chem156F, in osteoarthritis (OA) and rheumatoid arthritis (RA).METHODS: Purified chem156F was produced in transfected CHO cells. To quantify chem156F in OA and RA samples, we developed a specific ELISA for chem156F using antibody raised against a peptide representing the C-terminus of chem156F.RESULTS: Ca2+ mobilization assays showed that the EC50 values for chem163S, chem156F, and chem157S were 252±141nM, 133±41.5nM, and 5.83±2.48nM, respectively. chem156F was more active than its precursor, chem163S, but very much less potent than chem157S, the most active chemerin form. Chymase was shown to be capable of cleaving chem163S at a relevant rate. Using the chem156F ELISA we found a substantial amount of chem156F present in synovial fluids from patients with OA and RA, 24.06±5.51ng/ml and 20.35±5.19ng/ml (mean±SEM, n=25) respectively, representing 20% of total chemerin in OA and 76.7% of chemerin in RA synovial fluids.CONCLUSIONS: Our data show that chymase cleavage of chem163S to partially active chem156F can be found in synovial fluids where it can play a role in modulation of the inflammation in joints.

    View details for PubMedID 29973268

  • Chemerin 156F, generated by chymase cleavage of prochemerin, is elevated in joint fluids of arthritis patients ARTHRITIS RESEARCH & THERAPY Zhao, L., Yamaguchi, Y., Ge, X., Robinson, W. H., Morser, J., Leung, L. K. 2018; 20
  • Men and Women Differ in the Biochemical Composition of Platelet-Rich Plasma AMERICAN JOURNAL OF SPORTS MEDICINE Xiong, G., Lingampalli, N., Koltsov, J. B., Leung, L. L., Bhutani, N., Robinson, W. H., Chu, C. R. 2018; 46 (2): 409–19

    Abstract

    Autologous platelet-rich plasma (PRP) is widely used for a variety of clinical applications. However, clinical outcome studies have not consistently shown positive effects. The composition of PRP differs based on many factors. An improved understanding of factors influencing the composition of PRP is important for the optimization of PRP use.Age and sex influence the PRP composition in healthy patients.Controlled laboratory study.Blood from 39 healthy patients was collected at a standardized time and processed into leukocyte-poor PRP within 1 hour of collection using the same laboratory centrifuge protocol and frozen for later analysis. Eleven female and 10 male patients were "young" (aged 18-30 years), while 8 male and 10 female patients were "older" (aged 45-60 years). Thawed PRP samples were assessed for cytokine and growth factor levels using a multiplex assay and enzyme-linked immunosorbent assay. The platelet count and high-sensitivity C-reactive protein levels were measured. Two-way analysis of variance determined age- and sex-based differences.Platelet and high-sensitivity C-reactive protein concentrations were similar in PRP between the groups ( P = .234). Male patients had higher cytokine and growth factor levels in PRP compared with female patients for inflammatory cytokines such as interleukin-1 beta (IL-1β) (9.83 vs 7.71 pg/mL, respectively; P = .008) and tumor necrosis factor-alpha (TNF-α) (131.6 vs 110.5 pg/mL, respectively; P = .048); the anti-inflammatory IL-1 receptor antagonist protein (IRAP) (298.0 vs 218.0 pg/mL, respectively; P < .001); and growth factors such as fibroblast growth factor-basic (FGF-basic) (237.9 vs 194.0 pg/mL, respectively; P = .01), platelet-derived growth factor (PDGF-BB) (3296.2 vs 2579.3 pg/mL, respectively; P = .087), and transforming growth factor-beta 1 (TGF-β1) (118.8 vs 92.8 ng/mL, respectively; P = .002). Age- but not sex-related differences were observed for insulin-like growth factor-1 (IGF-1) ( P < .001). Age and sex interaction terms were not significant. While mean differences were significant, there was also substantial intragroup variability.This study in healthy patients shows differences in the composition of PRP between men and women, with sex being a greater factor than age. There was also proteomic variability within the groups. These data support a personalized approach to PRP treatment and highlight the need for a greater understanding of the relationships between proteomic factors in PRP and clinical outcomes.Variability in the proteomic profile of PRP may affect tissue and clinical responses to treatment. These data suggest that clinical studies should account for the composition of PRP used.

    View details for PubMedID 29211968

  • Prochemerin cleavage by factor XIa links coagulation and inflammation BLOOD Ge, X., Yamaguchi, Y., Zhao, L., Bury, L., Gresele, P., Berube, C., Leung, L. L., Morser, J. 2018; 131 (3): 353–64

    Abstract

    Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg162 was not required for cleavage at Lys158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

    View details for PubMedID 29158361

    View details for PubMedCentralID PMC5774209

  • Dynamic and tissue-specific proteolytic processing of chemerin in obese mice. PloS one Zhao, L., Yamaguchi, Y., Shen, W., Morser, J., Leung, L. L. 2018; 13 (8): e0202780

    Abstract

    Chemerin is a chemoattractant involved in immunity as well as an adipokine, whose activity is regulated by successive proteolytic cleavages at its C-terminus. Chemerin's C-terminal sequence and its proteolytic cleavage sites are highly conserved between human and mouse, as well as in other species. We produced, purified and characterized different mouse chemerin forms. Ca2+ mobilization assay showed that the EC50 values for mchem161T and mchem157R were 135.8 ± 158 nM and 71.2 ± 55.4 nM, respectively, whereas mchem156S and mchem155F had a 20-fold higher potency with an EC50 of 4.6 ± 1.8 nM and 3.6 ± 3.0 nM, respectively, likely representing the two physiologically active forms of chemerin. No agonist activity was found for mchem154A. Similar results were obtained in a chemotaxis assay. To identify and quantify the in vivo mouse chemerin forms in biological samples, we developed specific ELISAs for mchem162K, mchem157R, mchem156S, mchem155F and mchem154A, using antibodies raised against peptides from the C-terminus of the different mouse chemerin forms. The prochemerin form, mchem162K, was the major chemerin form in plasma with its increase matching the increase of total plasma chemerin in obese mice. During the onset of obesity in high-fat diet fed mice, mchem156S was elevated in plasma. In contrast, mchem155F was the dominant form in epididymal fat extracts. Our study provides the first direct evidence that mouse chemerin undergoes extensive, dynamic and tissue-specific proteolytic processing in vivo, similar to human chemerin, underlining the importance of measuring individual chemerin forms in studies of chemerin biology in mouse models.

    View details for PubMedID 30161155

  • Deficiency of Either Carboxypeptidase B2 or Carboxypeptidase N Exacerbated Disease Activity in a Mouse Model of Hemolytic Uremic Syndrome Leung, L. L., Shao, Z., Nishimura, T., Zhou, Q., Gigliello, L., Castro, P., Higgins, J., Morser, J. AMER SOC HEMATOLOGY. 2016
  • Chemerin Activation in Human Obesity OBESITY Chang, S., Eisenberg, D., Zhao, L., Adams, C., Leib, R., Morser, J., Leung, L. 2016; 24 (7): 1522-1529

    Abstract

    Chemerin is an inflammatory adipokine, whose activity is regulated by successive proteolytic cleavages at its C-terminus. It is secreted as an inactive precursor (chem163S); cleavage at Lys158 converts it to chem158K with modest activity. Chem157S is the most potent form and chem155A is inactive. The aim of this study was to determine if chemerin was activated in samples from patients with obesity.Using specific ELISAs for different chemerin forms and a pan-chemerin ELISA, chemerin forms in human obesity were characterized.Plasma chemerin from patients with obesity (BMI 44.3 ± 1.3 kg/m(2) , n = 29) was significantly higher than in lean controls (BMI 20.9 ± 0.7 kg/m(2) , n = 10) (160 ± 11 vs. 76.2 ± 5.5 ng/mL, respectively, P < 0.0001). This increase in chemerin was due to increased previously unattributed chemerin, with further C-terminal truncation demonstrated by mass spectrometry, accounting for ∼35% of total plasma chemerin. Chemerin forms in adipose tissue showed a different profile, with minimal chem163S and significant levels of chem157S. Chem155A was present in omental but not in subcutaneous adipose tissue. Unattributed chemerin forms were undetectable in adipose tissue.Chemerin is activated in adipose tissue of subjects with obesity, and further C-terminal processing occurs during the disposition of chemerin from adipose tissue, resulting in substantial levels of novel degraded forms in plasma that correlate with obesity.

    View details for DOI 10.1002/oby.21534

    View details for Web of Science ID 000379303200017

    View details for PubMedID 27222113

  • Plasmin as a complement C5 convertase. EBioMedicine Leung, L. L., Morser, J. 2016; 5: 20-21

    View details for DOI 10.1016/j.ebiom.2016.03.015

    View details for PubMedID 27077104

    View details for PubMedCentralID PMC4816833

  • Thrombin Cleavage of Osteopontin Disrupts a Pro-chemotactic Sequence for Dendritic Cells, Which Is Compensated by the Release of Its Pro-chemotactic C-terminal Fragment JOURNAL OF BIOLOGICAL CHEMISTRY Shao, Z., Morser, J., Leung, L. L. 2014; 289 (39): 27146-27158

    Abstract

    Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.

    View details for DOI 10.1074/jbc.M114.572172

    View details for Web of Science ID 000342853900040

    View details for PubMedCentralID PMC4175350

  • Thrombin cleavage of osteopontin disrupts a pro-chemotactic sequence for dendritic cells, which is compensated by the release of its pro-chemotactic C-terminal fragment. journal of biological chemistry Shao, Z., Morser, J., Leung, L. L. 2014; 289 (39): 27146-27158

    Abstract

    Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved (168)RSKSKKFRR(176) sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.

    View details for DOI 10.1074/jbc.M114.572172

    View details for PubMedID 25112870

    View details for PubMedCentralID PMC4175350

  • Open aortic valve replacement in a patient with Glanzmann's thrombasthenia: a multidisciplinary strategy to minimize perioperative bleeding. Transfusion Sheikh, A. Y., Hill, C. C., Goodnough, L. T., Leung, L. L., Fischbein, M. P. 2014; 54 (2): 300-305

    Abstract

    BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive disorder in which the platelet (PLT) glycoprotein IIb/IIIa complex is either deficient or dysfunctional. In its most severe form, GT may result in spontaneous bleeding, although most cases are first detected in the setting of an invasive procedure. CASE REPORT: A 59-year-old male with Type I GT and a history of transfusion reactions to PLT infusions developed severe aortic stenosis secondary to bicuspid valve disease. He successfully underwent open aortic valve replacement with cardiopulmonary bypass without perioperative bleeding complications. RESULTS: A multidisciplinary team (anesthesia, hematology, cardiac surgery, and transfusion medicine) was established to optimize perioperative hematologic management. Bleeding risk was assessed given the patient's prior history and a dosing timeline for administration of blood products and recombinant clotting factors was established. Successful management was achieved during the operation by prophylactic administration of HLA-matched PLTs and Factor VIIa. Prophylactic PLT administration was continued through the immediate postoperative period and no bleeding complications occurred. Thromboelastograms (TEGs) were used in conjunction with traditional hematologic laboratory analysis to optimize clinical management. CONCLUSION: Patients with GT requiring cardiac surgical procedures are at high risk for perioperative bleeding complications. This case report illustrates the importance of multidisciplinary planning, TEG analysis, and the judicious use of recombinant factors to minimize operative bleeding risk.

    View details for DOI 10.1111/trf.12275

    View details for PubMedID 23710629

  • Brief report: carboxypeptidase B serves as a protective mediator in osteoarthritis. Arthritis & rheumatology (Hoboken, N.J.) Lepus, C. M., Song, J. J., Wang, Q., Wagner, C. A., Lindstrom, T. M., Chu, C. R., Sokolove, J., Leung, L. L., Robinson, W. H. 2014; 66 (1): 101-106

    Abstract

    We previously demonstrated that carboxypeptidase B (CPB) protects against joint erosion in rheumatoid arthritis by inactivating complement component C5a. We also found that levels of CPB are abnormally high in the synovial fluid of individuals with another joint disease, osteoarthritis (OA). We undertook this study to investigate whether CPB plays a role in the pathogenesis of OA.We compared the development of OA in CPB-deficient (Cpb2(-/-) ) mice and wild-type mice by subjecting them to medial meniscectomy and histologically assessing cartilage damage, osteophyte formation, and synovitis in the stifle joints 4 months later. We measured levels of proCPB, proinflammatory cytokines, and complement components in synovial fluid samples from patients with symptomatic and radiographic knee OA. Finally, we used enzyme-linked immunosorbent assay, flow cytometry, and hemolytic assays to assess the effect of CPB on formation of membrane attack complex (MAC)-a complement effector critical to OA pathogenesis.Cpb2(-/-) mice developed dramatically greater cartilage damage than did wild-type mice (P < 0.01) and had a greater number of osteophytes (P < 0.05) and a greater degree of synovitis (P < 0.05). In synovial fluid samples from OA patients, high levels of proCPB were associated with high levels of proinflammatory cytokines and complement components, and levels of proCPB correlated positively with those of MAC. In in vitro complement activation assays, activated CPB suppressed the formation of MAC as well as MAC-induced hemolysis.Our data suggest that CPB protects against inflammatory destruction of the joints in OA, at least in part by inhibiting complement activation.

    View details for DOI 10.1002/art.38213

    View details for PubMedID 24449579

  • Carboxypeptidase B Serves as a Protective Mediator in Osteoarthritis ARTHRITIS & RHEUMATOLOGY Lepus, C. M., Song, J. J., Wang, Q., Wagner, C. A., Lindstrom, T. M., Chu, C. R., Sokolove, J., Leung, L. L., Robinson, W. H. 2014; 66 (1): 101-106

    Abstract

    We previously demonstrated that carboxypeptidase B (CPB) protects against joint erosion in rheumatoid arthritis by inactivating complement component C5a. We also found that levels of CPB are abnormally high in the synovial fluid of individuals with another joint disease, osteoarthritis (OA). We undertook this study to investigate whether CPB plays a role in the pathogenesis of OA.We compared the development of OA in CPB-deficient (Cpb2(-/-) ) mice and wild-type mice by subjecting them to medial meniscectomy and histologically assessing cartilage damage, osteophyte formation, and synovitis in the stifle joints 4 months later. We measured levels of proCPB, proinflammatory cytokines, and complement components in synovial fluid samples from patients with symptomatic and radiographic knee OA. Finally, we used enzyme-linked immunosorbent assay, flow cytometry, and hemolytic assays to assess the effect of CPB on formation of membrane attack complex (MAC)-a complement effector critical to OA pathogenesis.Cpb2(-/-) mice developed dramatically greater cartilage damage than did wild-type mice (P < 0.01) and had a greater number of osteophytes (P < 0.05) and a greater degree of synovitis (P < 0.05). In synovial fluid samples from OA patients, high levels of proCPB were associated with high levels of proinflammatory cytokines and complement components, and levels of proCPB correlated positively with those of MAC. In in vitro complement activation assays, activated CPB suppressed the formation of MAC as well as MAC-induced hemolysis.Our data suggest that CPB protects against inflammatory destruction of the joints in OA, at least in part by inhibiting complement activation.

    View details for DOI 10.1002/art.38213

    View details for Web of Science ID 000337356300014

  • Carboxypeptidase B2 Down-Regulates Osteoclast Activation In Inflammation. Song, J., Lee, S., Park, Y., Lee, S., Leung, L., Robinson, W. H. WILEY-BLACKWELL. 2013: S781
  • Targeting complement component 5a promotes vascular integrity and limits airway remodeling. Proceedings of the National Academy of Sciences of the United States of America Khan, M. A., Maasch, C., Vater, A., Klussmann, S., Morser, J., Leung, L. L., Atkinson, C., Tomlinson, S., Heeger, P. S., Nicolls, M. R. 2013; 110 (15): 6061-6066

    Abstract

    Increased microvascular dilatation and permeability is observed during allograft rejection. Because vascular integrity is an important indicator of transplant health, we have sought to limit injury to blood vessels by blocking complement activation. Although complement component 3 (C3) inhibition is known to be vasculoprotective in transplantation studies, we recently demonstrated the paradoxical finding that, early in rejection, C3(-/-) transplant recipients actually exhibit worse microvascular injury than controls. In the genetic absence of C3, thrombin-mediated complement component 5 (C5) convertase activity leads to the generation of C5a (anaphylatoxin), a promoter of vasodilatation and permeability. In the current study, we demonstrated that microvessel thrombin deposition is significantly increased in C3(-/-) recipients during acute rejection. Thrombin colocalization with microvessels is closely associated with remarkably elevated plasma levels of C5a, vasodilatation, and increased vascular permeability. Administration of NOX-D19, a specific C5a inhibitor, to C3(-/-) recipients of airway transplants significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia, even in the absence of conventional T-cell-directed immunosuppression. As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit.

    View details for DOI 10.1073/pnas.1217991110

    View details for PubMedID 23530212

    View details for PubMedCentralID PMC3625314

  • Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage JOURNAL OF BIOLOGICAL CHEMISTRY Yamaguchi, Y., Shao, Z., Sharif, S., Du, X., Myles, T., Merchant, M., Harsh, G., Glantz, M., Recht, L., Morser, J., Leung, L. L. 2013; 288 (5): 3097-3111

    Abstract

    Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

    View details for DOI 10.1074/jbc.M112.362954

    View details for Web of Science ID 000314397900019

    View details for PubMedID 23204518

    View details for PubMedCentralID PMC3561533

  • Microfluidic impedance cytometer for platelet analysis LAB ON A CHIP Evander, M., Ricco, A. J., Morser, J., Kovacs, G. T., Leung, L. L., Giovangrandi, L. 2013; 13 (4): 722-729

    Abstract

    We present the design and performance characteristics of a platelet analysis platform based on a microfluidic impedance cytometer. Dielectrophoretic focusing is used to centre cells in a fluid stream, which then forms the core of a two-phase flow (dielectric focusing). This flow then passes between electrodes for analysis by differential impedance spectroscopy at multiple frequencies from 280 kHz to 4 MHz. This approach increases the signal-to-noise ratio relative to a single-phase, unfocused stream, while minimising the shear forces to which the cells are subjected. The percentage of activated platelets before and after passage through the chip was measured using flow cytometry, and no significant change was measured. Measuring the in-phase amplitude at a single frequency is sufficient to distinguish platelets from erythrocytes. Using multi-frequency impedance measurements and discriminant analysis, resting platelets can be discriminated from activated platelets. This multifrequency impedance cytometer therefore allows ready determination of the degree of platelet activation in blood samples.

    View details for DOI 10.1039/c2lc40896a

    View details for Web of Science ID 000313971300028

    View details for PubMedID 23282651

  • Differential Gene Expression in Thrombomodulin (TM; CD141)(+) and TM(-) Dendritic Cell Subsets. PloS one Toda, M., Shao, Z., Yamaguchi, K. D., Takagi, T., D'Alessandro-Gabazza, C. N., Taguchi, O., Salamon, H., Leung, L. L., Gabazza, E. C., Morser, J. 2013; 8 (8)

    Abstract

    Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.

    View details for DOI 10.1371/journal.pone.0072392

    View details for PubMedID 24009678

    View details for PubMedCentralID PMC3751914

  • Chemerin Bioactivity Is Regulated by Factor XIa: A Novel Interface Linking Between Coagulation, Hemostasis and Immunity 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Yamaguchi, Y., Morser, J., Leung, L. L. AMER SOC HEMATOLOGY. 2011: 981–82
  • Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but Not in Plasma JOURNAL OF BIOLOGICAL CHEMISTRY Zhao, L., Yamaguchi, Y., Sharif, S., Du, X., Song, J. J., Lee, D. M., Recht, L. D., Robinson, W. H., Morser, J., Leung, L. L. 2011; 286 (45): 39520-39527

    Abstract

    Chemerin is a chemoattractant involved in immunity that may also function as an adipokine. Chemerin circulates as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C terminus, involving proteases involved in coagulation, fibrinolysis, and inflammation. However, the key proteolytic steps in prochemerin activation in vivo remain to be established. Previously, we have shown that C-terminal cleavage of chem163S by plasmin to chem158K, followed by a carboxypeptidase cleavage, leads to the most active isoform, chem157S. To identify and quantify the in vivo chemerin isoforms in biological specimens, we developed specific ELISAs for chem163S, chem158K, and chem157S, using antibodies raised against peptides from the C terminus of the different chemerin isoforms. We found that the mean plasma concentrations of chem163S, chem158K, and chem157S were 40 ± 7.9, 8.1 ± 2.9, and 0.7 ± 0.8 ng/ml, respectively. The total level of cleaved and noncleaved chemerins in cerebrospinal fluids was ∼10% of plasma levels whereas it was elevated ∼2-fold in synovial fluids from patients with arthritis. On the other hand, the fraction of cleaved chemerins was much higher in synovial fluid and cerebrospinal fluid samples than in plasma (∼75%, 50%, and 18% respectively). Chem158K was the dominant chemerin isoform, and it was not generated by ex vivo processing, indicating that cleavage of prochemerin at position Lys-158, whether by plasmin or another serine protease, represents a major step in prochemerin activation in vivo. Our study provides the first direct evidence that chemerin undergoes extensive proteolytic processing in vivo, underlining the importance of measuring individual isoforms.

    View details for DOI 10.1074/jbc.M111.258954

    View details for PubMedID 21930706

  • Proteolytic Cleavage of Chemerin Protein Is Necessary for Activation to the Active Form, Chem157S, Which Functions as a Signaling Molecule in Glioblastoma JOURNAL OF BIOLOGICAL CHEMISTRY Yamaguchi, Y., Du, X., Zhao, L., Morser, J., Leung, L. L. 2011; 286 (45): 39510-39519

    Abstract

    Chemerin is a chemoattractant involved in innate and adaptive immunity as well as an adipokine implicated in adipocyte differentiation. Chemerin circulates as an inactive precursor in blood whose bioactivity is closely regulated through proteolytic processing at its C terminus. We developed methodology for production of different recombinant chemerin isoforms (chem163S, chem157S, and chem155A) which allowed us to obtain large quantities of these proteins with purity of >95%. Chem158K was generated from chem163S by plasmin cleavage. Characterization by mass spectrometry and Edman degradation demonstrated that both the N and C termini were correct for each isoform. Ca(2+) mobilization assays showed that the EC(50) values for chem163S and chem158K were 54.2 ± 19.9 nm and 65.2 ± 13.2 nm, respectively, whereas chem157S had a ∼50-fold higher potency with an EC(50) of 1.2 ± 0.7 nm. Chem155A had no agonist activity and weak antagonist activity, causing a 50% reduction of chem157S activity at a molar ratio of 100:1. Similar results were obtained in a chemotaxis assay. Because chem158K is the dominant form in cerebrospinal fluid from patients with glioblastoma (GBM), we examined the significance of chemerin in GBM biology. In silico analysis showed chemerin mRNA was significantly increased in tissue from grade III and IV gliomas. Furthermore, U-87 MG cells, a human GBM line, express the chemerin receptors, chemokine-like receptor 1 and chemokine receptor-like 2, and chem157S triggered Ca(2+) flux. This study emphasized the necessity of appropriate C-terminal proteolytic processing to generate the likely physiologic form of active chemerin, chem157S, and suggested a possible role in malignant GBM.

    View details for DOI 10.1074/jbc.M111.258921

    View details for Web of Science ID 000296759800068

    View details for PubMedID 21949124

    View details for PubMedCentralID PMC3234774

  • Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis JOURNAL OF CLINICAL INVESTIGATION Song, J. J., Hwang, I., Cho, K. H., Garcia, M. A., Kim, A. J., Wang, T. H., Lindstrom, T. M., Lee, A. T., Nishimura, T., Zhao, L., Morser, J., Nesheim, M., Goodman, S. B., Lee, D. M., Bridges, S. L., Gregersen, P. K., Leung, L. L., Robinson, W. H. 2011; 121 (9): 3517-3527

    Abstract

    The immune and coagulation systems are both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma carboxypeptidase B (CPB), which is activated by the thrombin/thrombomodulin complex, plays a procoagulant role during fibrin clot formation. However, an antiinflammatory role for CPB is suggested by the recent observation that CPB can cleave proinflammatory mediators, such as C5a, bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerbated inflammatory arthritis in a mouse model of RA, and cleavage of C5a by CPB suppressed the ability of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at abrogating the proinflammatory properties of C5a. Additionally, expression of both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link between inflammation and coagulation in autoimmune arthritis.

    View details for DOI 10.1172/JCI46387

    View details for PubMedID 21804193

  • Enhanced Abdominal Aortic Aneurysm Formation in Thrombin-Activatable Procarboxypeptidase B-Deficient Mice ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Schultz, G., Tedesco, M. M., Sho, E., Nishimura, T., Sharif, S., Du, X., Myles, T., Morser, J., Dalman, R. L., Leung, L. L. 2010; 30 (7): 1363-1370

    Abstract

    To determine whether procarboxypeptidase B (pCPB)(-/-) mice are susceptible to accelerated abdominal aortic aneurysm (AAA) development secondary to unregulated OPN-mediated mural inflammation in the absence of CPB inhibition.Thrombin/thrombomodulin cleaves thrombin-activatable pCPB or thrombin-activatable fibrinolysis inhibitor, activating CPB, which inhibits the generation of plasmin and inactivates proinflammatory mediators (complement C5a and thrombin-cleaved osteopontin [OPN]). Apolipoprotein E(-/-)OPN(-/-) mice are protected from experimental AAA formation. Murine AAAs were created via intra-aortic porcine pancreatic elastase (PPE) infusion. Increased mortality secondary to AAA rupture was observed in pCPB(-/-) mice at the standard PPE dose. At reduced doses of PPE, pCPB(-/-) mice developed larger AAAs than wild-type controls (1.01+/-0.27 versus 0.68+/-0.05 mm; P=0.02 [mean+/-SD]). C5(-/-) and OPN(-/-) mice were not protected against AAA development. Treatment with tranexamic acid inhibited plasmin generation and abrogated enhanced AAA progression in pCPB(-/-) mice.This study establishes the role of CPB in experimental AAA disease, indicating that CPB has a broad anti-inflammatory role in vivo. Enhanced AAA formation in the PPE model is the result of increased plasmin generation, not unregulated C5a- or OPN-mediated mural inflammation.

    View details for DOI 10.1161/ATVBAHA.109.202259

    View details for Web of Science ID 000278856600013

    View details for PubMedID 20431069

  • What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse? JOURNAL OF THROMBOSIS AND HAEMOSTASIS Morser, J., Gabazza, E. C., Myles, T., Leung, L. L. 2010; 8 (5): 868-876

    Abstract

    Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

    View details for DOI 10.1111/j.1538-7836.2010.03787.x

    View details for Web of Science ID 000277328600002

    View details for PubMedID 20128866

  • Thrombospondin Type I Domain Containing 7A (THSD7A) Mediates Endothelial Cell Migration and Tube Formation JOURNAL OF CELLULAR PHYSIOLOGY Wang, C., Su, P., Du, X., Kuo, M., Lin, C., Yang, C., Chan, H., Chang, S., Kuo, C., Seo, K., Leung, L. L., Chuan, Y. 2010; 222 (3): 685-694

    Abstract

    Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data-mining against other non-ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl-terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha(V)beta(3) integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti-angiogenic therapy.

    View details for DOI 10.1002/jcp.21990

    View details for Web of Science ID 000274273700025

    View details for PubMedID 20020485

  • Heparin-induced thrombosis without thrombocytopenia JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Tibayan, F. A., Leung, L. L., Burdon, T. A., Fann, J. I. 2010; 139 (2): E6-E7

    View details for DOI 10.1016/j.jtcvs.2008.07.006

    View details for Web of Science ID 000274014300050

    View details for PubMedID 19660256

  • Prochemerin Is a New Substrate for Thrombin 51st Annual Meeting and Exposition of the American-Society-of-Hematology Du, X., Myles, T., Morser, J., Leung, L. L. AMER SOC HEMATOLOGY. 2009: 1391–91
  • Thrombin-Activatable Carboxypeptidase B Cleavage of Osteopontin Regulates Neutrophil Survival and Synoviocyte Binding in Rheumatoid Arthritis ARTHRITIS AND RHEUMATISM Sharif, S. A., Du, X., Myles, T., Song, J. J., Price, E., Lee, D. M., Goodman, S. B., Nagashima, M., Morser, J., Robinson, W. H., Leung, L. L. 2009; 60 (10): 2902-2912

    Abstract

    Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal alpha4beta1 and alpha9beta1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA.Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels.Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to alpha4beta1, whereas OPN-L did not.Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion.

    View details for DOI 10.1002/art.24814

    View details for PubMedID 19790060

  • Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets JOURNAL OF BIOLOGICAL CHEMISTRY Du, X., Zabel, B. A., Myles, T., Allen, S. J., Handel, T. M., Lee, P. P., Butcher, E. C., Leung, L. L. 2009; 284 (2): 751-758

    Abstract

    Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH(2)-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH(2)-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

    View details for DOI 10.1074/jbc.M805000200

    View details for Web of Science ID 000262122900008

    View details for PubMedID 19010784

    View details for PubMedCentralID PMC2613638

  • Differentially Cleaved Forms of Osteopontin by Thrombin and Carboxypeptidase B (TAF1a) Regulates Neutrophil Viability and Synoviocyte Binding in Rheumatoid Arthritis 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Sharif, S., Du, X., Myles, T., Song, J. J., Price, E. A., Lee, D., Goodman, S., Morser, J., Robinson, W., Leung, L. L. AMER SOC HEMATOLOGY. 2008: 1219–19
  • Platelet Interaction with Bone Marrow Derived CD34(+) Stem Cells: Potential Role of Chemoattractant Chemerin 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Du, X., Leung, L. L. AMER SOC HEMATOLOGY. 2008: 1339–39
  • Enhanced Abdominal Aortic Aneurysm (AAA) Formation in Procarboxypeptidase B (pCPB)-Deficient Mice 81st Annual Scientific Session of the American-Heart-Association Tedesco, M. M., Sho, E., Nishimura, T., Sharif, S., Du, X., Dalman, R. L., Leung, L. L. LIPPINCOTT WILLIAMS & WILKINS. 2008: S310–S310
  • A central anti-inflammatory role of carboxypeptidase B in autoimmune arthritis 72nd Annual Scientific Meeting of the American-College-of-Rheumatology/43rd Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals Song, J. J., Nishimura, T., Garcia, M., Myles, T., Du, X., Sharif, S., Leung, L., Robinson, W. WILEY-BLACKWELL. 2008: S264–S265
  • Thrombin hydrolysis of human osteopontin is dependent on thrombin anion-binding exosites JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Leung, L. L. 2008; 283 (26): 17789-17796

    Abstract

    The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic alpha(4)beta(1)/alpha(9)beta(1) integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k(cat)/K(m) value of 1.14 x 10(5) m(-1) s(-1). Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC(50) = 1.2 +/- 0.2 microm), unfractionated heparin (IC(50) = 56.6 +/- 8.4 microg/ml) and low molecular weight (5 kDa) heparin (IC(50) = 31.0 +/- 7.9 microg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162-197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162-197) with a specificity constant of k(cat)/K(m) = 1.64 x 10(4) m(-1) s(-1). Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (-4.1 +/- 1.0) and OPN-(162-197) (-2.4 +/- 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.

    View details for DOI 10.1074/jbc.M708629200

    View details for Web of Science ID 000256949200006

    View details for PubMedID 18413297

    View details for PubMedCentralID PMC2440630

  • Thrombin-cleaved osteopontin is regulated by thrombin-activatable carboxypeptidase B in rheumatoid arthritis. 49th Annual Meeting of the American-Society-of-Hematology Myles, T., Sharif, S. A., Du, X., Song, J. J., Elizabeth, P., Robinson, W. H., Leung, L. L. AMER SOC HEMATOLOGY. 2007: 519A–519A
  • Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (TAFIa) and platelets 49th Annual Meeting of the American-Society-of-Hematology Du, X., Zabel, B., Allen, S. J., Handel, T. M., Lee, P. P., Butcher, E. C., Leung, L. L. AMER SOC HEMATOLOGY. 2007: 126A–127A
  • Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin JOURNAL OF THROMBOSIS AND HAEMOSTASIS Fortenberry, Y. M., Whinna, H. C., Cooper, S. T., Myles, T., Leung, L. L., Church, F. C. 2007; 5 (7): 1486-1492

    Abstract

    Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin.Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT.Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM.Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.

    View details for Web of Science ID 000247980000024

    View details for PubMedID 17635698

  • Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo BLOOD Nishimura, T., Myles, T., Piliposky, A. M., Kao, P. N., Berry, G. J., Leung, L. L. 2007; 109 (5): 1992-1997

    Abstract

    Plasma procarboxypeptidase B (proCPB) is activated by the endothelial thrombin-thrombomodulin [corrected] complex. Activated proCPB [corrected] (CPB) functions as a fibrinolysis inhibitor, but it may play a broader role by inactivating inflammatory mediators. To test this hypothesis, C5a-induced alveolitis was studied in wild-type (WT) and proCPB-deficient mice (proCPB-/-). C5a-induced alveolitis, as measured by cell counts and total protein contents in bronchoalveolar lavage fluids, was markedly enhanced in the proCPB-/- mice. E229K thrombin, a thrombin mutant with minimal clotting activity but retaining its ability to activate protein C and proCPB, attenuated C5a-induced alveolitis in WT but not in proCPB-/- mice, indicating that its beneficial effect is mediated primarily by its activation of proCPB. Lung tissue histology confirmed these cellular inflammatory responses. Delayed administration of E229K thrombin after the C5a instillation was ineffective in reducing alveolitis in WT mice, suggesting that the beneficial effect of E229K thrombin is due to the direct inhibition of C5a by CPB. Our studies show that thrombin-activatable proCPB, in addition to its role in fibrinolysis, has intrinsic anti-inflammatory functions. Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury.

    View details for DOI 10.1182/blood-2006-03-012567

    View details for PubMedID 17105819

  • Thrombin-activatabte carboxypeptidase B prevents anti-collagen antibody induced arthritis 7th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Song, J., Nishimura, T., Garcia, M., Myles, T., Ho, P., Du, X., Sharif, S., Leung, L., Robinson, W. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2007: S98–S98
  • Enhanced abdominal aortic aneurysm formation in procarboxypeptidase B-deficient mice 79th Annual Scientific Session of the American-Heart-Association Tedesco, M. M., Sho, E., Nishimura, T., Sho, M., Dalman, R. L., Leung, L. L. LIPPINCOTT WILLIAMS & WILKINS. 2006: 39–40
  • Antiangiogenic antithrombin induces global changes in the gene expression profile of endothelial cells CANCER RESEARCH Zhang, W., Chuang, Y., Jin, T., Swanson, R., Xiong, Y., Leung, L., Olson, S. T. 2006; 66 (10): 5047-5055

    Abstract

    Antithrombin, a serpin family protease inhibitor crucial to hemostasis, acquires antiangiogenic properties on undergoing conformational alterations induced by limited proteolysis or elevated temperature. To better understand the biochemical mechanisms underlying antithrombin antiangiogenic activity, we did genome-wide expression profiling, coupled with quantitative reverse transcription-PCR, Northern blot, and Western blot analyses, to characterize the gene expression patterns that are induced by antiangiogenic antithrombin in cultured primary human umbilical vein endothelial cells. Overall, 35 genes with significantly increased expression and 93 genes with significantly reduced expression (> or =2-fold changes) due to antiangiogenic antithrombin treatment were identified. More than half of the down-regulated genes have well-established proangiogenic functions in endothelial cells, including cell-surface and matrix proteoglycans (e.g., perlecan, biglycan, and syndecans 1 and 3) and mitogenesis-related signaling proteins (e.g., mitogen-activated protein kinase 3, signal transducers and activators of transcription 2, 3, and 6, and early growth response factor 1). In contrast, most up-regulated genes (e.g., caspase-3, p21, tissue inhibitor of metalloproteinases 1, 2, and 3, and adenomatosis polyposis coli) are known for their antiangiogenic functions which include the promotion of cell apoptosis and cell cycle arrest and the inhibition of tumor growth and metastasis. These results show that the antiangiogenic activity of antithrombin is mediated at least in part by a global genetic reprogramming of endothelial cells and strongly implicate an endothelial cell ligand-receptor signaling mechanism in this reprogramming.

    View details for DOI 10.1158/0008-5472.CAN-05-4449

    View details for Web of Science ID 000237679900011

    View details for PubMedID 16707426

  • Perioperative evaluation of bleeding diathesis. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Leung, L. L. 2006: 457-461

    Abstract

    The differential diagnosis of a long APTT with a normal prothrombin time can be due to either a clotting factor deficiency or the presence of an inhibitor, which can be distinguished by using a plasma-mixing study. The various clotting factor deficiency states are reviewed. Clinical bleeding following cardiac bypass surgery due to acquired factor V and thrombin antibodies is also reviewed.

    View details for PubMedID 17124099

  • Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain JOURNAL OF BIOLOGICAL CHEMISTRY Nogami, K., Zhou, Q., Myles, T., Leung, L. L., Wakabayashi, H., Fay, P. J. 2005; 280 (18): 18476-18487

    Abstract

    Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.

    View details for DOI 10.1074/jbc.M412778200

    View details for Web of Science ID 000228807200111

    View details for PubMedID 15746105

  • Thrombin-activatable fibrinolysis inhibitor (TAFI) regulates activated complement C5a in vivo. 46th Annual Meeting of the American-Society-of-Hematology Nishimura, T., Myles, T., Nagashima, M., MORSER, J., Pearl, R. G., Leung, L. L. AMER SOC HEMATOLOGY. 2004: 812A–812A
  • Mechanism of interaction between the heavy chain of factor VIII and thrombin 46th Annual Meeting of the American-Society-of-Hematology Nogami, K., Zhou, Q., Wakabayashi, H., Myles, T., Leung, L. L., Fay, P. J. AMER SOC HEMATOLOGY. 2004: 475A–475A
  • Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery JOURNAL OF BIOLOGICAL CHEMISTRY Carter, W. J., Myles, T., Gibbs, C. S., Leung, L. L., Huntington, J. A. 2004; 279 (25): 26387-26394

    Abstract

    Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.

    View details for DOI 10.1074/jbc.M402364200

    View details for Web of Science ID 000222003000059

    View details for PubMedID 15075325

  • Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells BLOOD Zhang, W. Q., Chuang, Y. J., Swanson, R., Li, J., Seo, K. G., Leung, L., Lau, L. F., Olson, S. T. 2004; 103 (4): 1185-1191

    Abstract

    Antithrombin, a key serpin family regulator of blood coagulation proteases, is transformed into a potent antiangiogenic factor by limited proteolysis or mild heating. Here, we show by cDNA microarray, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting analyses that the expression of the proangiogenic heparan sulfate proteoglycan (HSPG), perlecan, but not other HSPGs, is dramatically down-regulated in human umbilical vein endothelial cells (HUVECs) treated with antiangiogenic cleaved and latent forms of antithrombin but not with the native form. Down-regulation of perlecan expression by cleaved and latent antithrombins was observed in both basic fibroblast growth factor (bFGF)-stimulated and unstimulated cells, whereas the antiangiogenic antithrombins inhibited the proliferation of only bFGF-stimulated HUVECs by arresting cells at the G(1) cell cycle phase. The importance of perlecan expression levels in mediating the antiproliferative effect of the antiangiogenic antithrombins was suggested by the finding that transforming growth factor-beta 1, a potent stimulator of perlecan expression in endothelial cells, blocked the down-regulation of perlecan expression and antiproliferative activity of cleaved antithrombin on endothelial cells. The previously established key role of perlecan in mediating bFGF stimulation of endothelial cell proliferation and angiogenesis suggests that a primary mechanism by which antiangiogenic antithrombins exert their effects is through the down-regulation of perlecan expression.

    View details for DOI 10.1182/blood-2003-08-2920

    View details for Web of Science ID 000188828200011

    View details for PubMedID 14563633

  • Molecular cloning of nonsecreted endothelial cell-derived lipase isoforms GENOMICS Ishida, T., Zheng, Z., Dichek, H. L., Wang, H. J., Moreno, I., Yang, E., Kundu, R. K., Talbi, S., Hirata, K. I., Leung, L. L., Quertermous, T. 2004; 83 (1): 24-33

    Abstract

    To expand our knowledge of factors involved in lipid metabolism in the blood vessel wall, we have cloned unique molecular isoforms of endothelial cell-derived lipase (EDL) (HGMW-approved symbol/LIPG). One isoform encoded a truncated protein (EDL2a) lacking the first 80 amino acid residues of the previously characterized EDL1a isoform, including the signal peptide. A similar second clone (EDL2b) was identified that lacked not only the first 80 amino acids, but also a 74-amino-acid region that encodes a portion of the lid domain. RT-PCR analysis confirmed expression of EDL2a/2b isoforms in several human tissues and cultured cells, including endothelial cells. Western blot and immunofluorescence studies using stable transfectants revealed that EDL2a and EDL2b were localized in the cytosol, while, EDL1a was secreted into the culture medium. Cell extracts of EDL2a/2b transfectants did not have triglyceride or phospholipase activity. Thus endothelial cells express three EDL isoforms, two of which remain intracellular and do not function as lipases.

    View details for DOI 10.1016/S0888-7543(03)00181-2

    View details for PubMedID 14667806

  • Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Nishimura, T., Yun, T. H., Nagashima, M., MORSER, J., Patterson, A. J., Pearl, R. G., Leung, L. L. 2003; 278 (51): 51059-51067

    Abstract

    The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.

    View details for DOI 10.1074/jbc.M306977200

    View details for Web of Science ID 000187206300029

    View details for PubMedID 14525995

  • Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively (Retracted Article. See vol 282, pg 29067, 2007) JOURNAL OF BIOLOGICAL CHEMISTRY Yun, T. H., Baglia, F. A., Myles, T., Navaneetham, D., Lopez, J. A., Walsh, P. N., Leung, L. L. 2003; 278 (48): 48112-48119

    Abstract

    Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.

    View details for DOI 10.1074/jbc.M306925200

    View details for Web of Science ID 000186731400092

  • Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ib alpha with thrombin anion-binding exosites I and II, respectively. The Journal of biological chemistry Yun, T. H., Baglia, F. A., Myles, T., Navaneetham, D., López, J. A., Walsh, P. N., Leung, L. L. 2003; 278 (48): 48112-9

    Abstract

    Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ibalpha (GPIb alpha). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb alpha molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI alpha molecule, via ABE-I on its anterior surface. GPIb alpha plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.

    View details for DOI 10.1074/jbc.M306925200

    View details for PubMedID 12968031

  • Quantitative transcript profiling and the identification of a novel vascular endothelial cell marker, placental endothelial protein-1, by serial analysis of gene expression. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Chuang, Y. J., Seo, K., Gray, J., Druzin, M., Kuo, C., Leung, L. AMER SOC HEMATOLOGY. 2003: 10A–11A
  • Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation - Competition for cofactor sites on thrombin determines its fate JOURNAL OF BIOLOGICAL CHEMISTRY Philippou, H., Rance, J., Myles, T., Hall, S. W., Ariens, R. A., Grant, P. J., Leung, L., Lane, D. A. 2003; 278 (34): 32020-32026

    Abstract

    Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.

    View details for DOI 10.1074/jbc.M305364200

    View details for Web of Science ID 000184782100072

    View details for PubMedID 12794066

  • Structural requirements for the activation of human factor VIII by thrombin BLOOD Myles, T., Yun, T. H., Leung, L. L. 2002; 100 (8): 2820-2826

    Abstract

    The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion-binding exosite-I (Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and anion-binding exosite-II (Arg98Ala), the Na(+)-binding site (Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the 50-insertion loop (Trp50Ala), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I appears to be important for cleavage at both sites, whereas the anion-binding exosite-II residue Arg98Ala is important for cleavage at Arg372 alone. The Glu229Ala mutant, which contributes to the Na(+)-binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at Arg372 and Arg1689. This suggests that the integrity of the active site and the Na(+)-bound form of thrombin are important for its procoagulant activity against FVIII. Detailed mutagenic analysis of thrombin can assist in understanding the pathogenesis of bleeding disorders and may lead to the rational design of selective thrombin inhibitors.

    View details for Web of Science ID 000178519100023

    View details for PubMedID 12351390

  • Thrombin inhibition by a reactive center L444R mutant of heparin cofactor II Fortenberry, Y. M., Mitchell, J. W., Myles, T., Leung, L. L., Church, F. C. FEDERATION AMER SOC EXP BIOL. 2002: A1193–A1193
  • Identification of critical residues on thrombin mediating its interaction with fibrin THROMBOSIS AND HAEMOSTASIS HALL, S. W., Gibbs, C. S., Leung, L. L. 2001; 86 (6): 1466-1474

    Abstract

    Thrombin binding to fibrin may be important in localizing thrombin to the site of vascular injury. However, fibrin-bound thrombin retains its catalytic activity toward fibrinogen, and may be prothrombotic under certain conditions. A collection of 52 purified thrombin mutants was used to identify those residues mediating the thrombin-fibrin interaction. Comparison of fibrinogen clotting activity with fibrin binding activity identified twenty residues involved in fibrinogen recognition with four of these residues important in fibrin binding (Lys65, His66, Tyr71, Arg73). No mutant was identified with normal clotting activity and deficient fibrin binding, suggesting that these two properties are not readily dissociable. A DNA thrombin aptamer that binds to these residues was able to inhibit the thrombin-fibrin interaction, and displace thrombin that was already bound. Mapping of these fibrin-binding residues on thrombin revealed that they are localized within exosite I, and comprise a subset of the residues important in fibrinogen recognition.

    View details for Web of Science ID 000172857400020

    View details for PubMedID 11776315

  • Immunological consequences of topical bovine thrombin AMERICAN JOURNAL OF PATHOLOGY Zehnder, J. L., Leung, L. L. 2001; 159 (6): 2371-2371

    View details for Web of Science ID 000172457400040

    View details for PubMedID 11733385

    View details for PubMedCentralID PMC1850577

  • Dextran sulfate-mediated thrombin activation of factor XI requires anion binding exosite II. Yun, T. H., Myles, T., Leung, L. L. AMER SOC HEMATOLOGY. 2001: 529A–529A
  • Molecular mapping of the thrombin-heparin cofactor II complex. Fortenberry, Y. M., Gentry, H. R., Myles, T., Leung, L. L., Church, F. C. AMER SOC HEMATOLOGY. 2001: 825A–825A
  • An extensive interaction interface between thrombin and factor V is required for factor V activation JOURNAL OF BIOLOGICAL CHEMISTRY Myles, T., Yun, T. H., HALL, S. W., Leung, L. L. 2001; 276 (27): 25143-25149

    Abstract

    The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.

    View details for Web of Science ID 000169800700096

    View details for PubMedID 11312264

  • A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ramakrishnan, V., Deguzman, F., Bao, M., HALL, S. W., Leung, L. L., Phillips, D. R. 2001; 98 (4): 1823-1828

    Abstract

    Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.

    View details for Web of Science ID 000166949200092

    View details for PubMedID 11172035

    View details for PubMedCentralID PMC29341

  • An extensive interaction interface between thrombin and factor V is revealed by alanine scanning mutagenesis of thrombin. Myles, T., Yun, T. H., HALL, S. W., Leung, L. L. AMER SOC HEMATOLOGY. 2000: 632A-?
  • GP Ib is a functional thrombin receptor on platelets. Ramakrishnan, V., Deguzman, F., Bao, M., HALL, S. W., Leung, L. L., Phillips, D. R. AMER SOC HEMATOLOGY. 2000: 446A–447A
  • Anti-apoptosis is a prominent response of endothelial cells to inflammatory cytokine stimulation. Zheng, Z., Ross, D. T., Brown, P. O., Leung, L. L. AMER SOC HEMATOLOGY. 1999: 226A–226A
  • Identification of critical residues in thrombin mediating platelet activation. HALL, S. W., Venkataraman, G., Thompson, D., Leung, L. L. AMER SOC HEMATOLOGY. 1999: 648A–648A
  • Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains JOURNAL OF BIOLOGICAL CHEMISTRY Hall, S. W., Nagashima, M., Zhao, L., MORSER, J., Leung, L. L. 1999; 274 (36): 25510-25516

    Abstract

    A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.

    View details for Web of Science ID 000082554200046

    View details for PubMedID 10464282

  • Dissociation of thrombin's protein C and TAFI activities by site-directed mutagenesis. HALL, S. W., Nagashima, M., Zhao, L., MORSER, J., Leung, L. L. AMER SOC HEMATOLOGY. 1998: 184A–184A
  • Strategies for development of novel antithrombotics: modulating thrombin's procoagulant and anticoagulant properties CELLULAR AND MOLECULAR LIFE SCIENCES HALL, S. W., Gibbs, C. S., Leung, L. L. 1997; 53 (9): 731-736

    Abstract

    Thrombin is a serine proteinase that can interact with a large number of diverse macromolecular substrates, which results in either a procoagulant or anticoagulant effect. These divergent properties are physiologically regulated by the endogenous protein thrombomodulin. This review summarizes recent work on a variety of methods used to exploit the allosteric nature of the enzyme. The procoagulant and anticoagulant functions of thrombin can be modulated by sodium binding, site-directed mutagenesis, and a small synthetic molecule. Modulation of thrombin's intrinsic properties represents a novel approach to the development of unique antithrombotic agents.

    View details for Web of Science ID A1997YB75600002

    View details for PubMedID 9368669

  • Modulation of thrombin's procoagulant and anticoagulant properties THROMBOSIS AND HAEMOSTASIS Leung, L. L., Gibbs, C. S. 1997; 78 (1): 577-580

    Abstract

    The procoagulant and anticoagulant functions of thrombin are controlled physiologically by allosteric changes induced by Na+ and vascular cell-surface TM. Key residues that mediate Na+ interaction with thrombin have been identified. Based on a site-directed mutagenesis approach, E229K thrombin is found to be the most optimal and potent PC activator with a marked shift in substrate specificity for PC over fibrinogen. E229K thrombin demonstrates significant anticoagulant and antithrombotic efficacy in animal models in vivo. Alternatively, a synthetic organic molecule (LY254603) has been discovered which interacts with thrombin and effectively modulates its functions in vitro. This new class of antithrombotic agents exploits the powerful natural PC anticoagulant pathway and may have a superior therapeutic profile than direct thrombin inhibitors.

    View details for Web of Science ID A1997XE83800102

    View details for PubMedID 9198219

  • Identification of a domain (155-183) on CD36 implicated in the phagocytosis of apoptotic neutrophils JOURNAL OF BIOLOGICAL CHEMISTRY Navazo, M. D., Daviet, L., Savill, J., Ren, Y., Leung, L. L., McGregor, J. L. 1996; 271 (26): 15381-15385

    Abstract

    Clearance of apoptotic neutrophils by macrophages is a crucial event following the resolution of acute inflammation. CD36, together with alphavbeta3, has been identified as one of the adhesion molecules on the surface of macrophages implicated in the clearance of polymorphonuclear leukocytes. The domain on CD36 implicated in the phagocytosis of aged neutrophils remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA had a significantly higher capacity to phagocytose human apoptotic neutrophils compared with murine CD36 cDNA. Moreover, monoclonal antibodies 10/5 or OKM5 (epitopes identified on amino acids 155-183) but not monoclonal antibody 13/10 (epitope identified on amino acids 30-76) inhibited phagocytosis of apoptotic neutrophils by COS cells transfected by human CD36. Swapping the human CD36 155-183 domain from human to murine CD36 (human-murine CD36 chimera) imparted to murine CD36-transfected COS cells an increased capacity to phagocytose apoptotic neutrophils. Conversely, when the murine domain 155-183 was inserted in human CD36, a decreased phagocytic capacity was observed. In addition, a synthetic peptide(155-169) but not its scrambled form significantly inhibited phagocytosis. These results identify for the first time a functional domain encompassing amino acids 155-183 on human CD36 implicated in the recognition and phagocytosis of apoptotic neutrophils.

    View details for DOI 10.1074/jbc.271.26.15381

    View details for Web of Science ID A1996UV29900015

    View details for PubMedID 8663130

  • SELECTION OF A SUPPRESSOR MUTATION THAT RESTORES AFFINITY OF AN OLIGONUCLEOTIDE INHIBITOR FOR THROMBIN USING IN-VITRO GENETICS JOURNAL OF BIOLOGICAL CHEMISTRY Tsiang, M., Gibbs, C. S., Griffin, L. C., Dunn, K. E., Leung, L. L. 1995; 270 (33): 19370-19376

    Abstract

    The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70-E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T-->A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin.

    View details for Web of Science ID A1995RP70300030

    View details for PubMedID 7642616

  • FUNCTIONAL MAPPING OF THE SURFACE RESIDUES OF HUMAN THROMBIN JOURNAL OF BIOLOGICAL CHEMISTRY Tsiang, M., Jain, A. K., Dunn, K. E., Rojas, M. E., Leung, L. L., Gibbs, C. S. 1995; 270 (28): 16854-16863

    Abstract

    Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.

    View details for Web of Science ID A1995RJ34700062

    View details for PubMedID 7622501

  • Conversion of thrombin into an anticoagulant by protein engineering Nature Leung LLK, Gibbs CS, Coutre SE, Tsiang M, Li WX, Jain AK, Dunn KE, Law VS, Mao CT, Matsumura SY, Mejsa SJ, Paborsky LR 1995
  • PILOT-STUDY OF THE EFFICACY OF A THROMBIN INHIBITOR FOR USE DURING CARDIOPULMONARY BYPASS 30th Annual Meeting of the Society-of-Thoracic-Surgeons DeAnda, A., Coutre, S. E., Moon, M. R., Vial, C. M., Griffin, L. C., Law, V. S., Komeda, M., Leung, L. L., Miller, D. C. ELSEVIER SCIENCE INC. 1994: 344–50

    Abstract

    Heparin is normally used for anticoagulation during cardiopulmonary bypass (CPB), but its use is contraindicated in patients with a history of heparin-induced thrombocytopenia, heparin-provoked thrombosis, or both. Heparin therapy can also be ineffective due to heparin resistance. A short-acting, oligonucleotide-based thrombin inhibitor (thrombin aptamer) may potentially serve as a substitute for heparin in these and other clinical situations. We tested a novel thrombin aptamer in a canine CPB pilot study to determine its anticoagulant efficacy, the resultant changes in coagulation variables, and the aptamer's clearance mechanisms and pharmacokinetics. Seven dogs were studied initially: Four received varied doses of the aptamer (to establish the pharmacokinetic profile) and 3 received heparin. Subsequently, 4 other dogs underwent CPB, receiving a constant infusion of the aptamer before CPB (to characterize the baseline coagulation status), with partial CPB and hemodilution, during 60 minutes of total CPB, and, finally, after a 2-hour recovery period. At a 0.5 mg.kg-1.min-1 dose, the activated clotting time rose with aptamer infusion from 106 +/- 12 seconds to 187 +/- 8 seconds (+/- 1 standard deviation) (p = 0.014), increased further with hemodilution (to 259 +/- 41 seconds; p = 0.017), and was even more prolonged during total CPB (> 1,500 seconds; p < 0.001). This later increase in the activated clotting time paralleled a rise in the plasma concentration of the thrombin aptamer during total CPB, as determined by high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994PD58900014

    View details for PubMedID 8067830