Anandi Krishnan

All Publications

• Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens. bioRxiv : the preprint server for biology Seetharam, S. M., Liu, Y., Wu, J., Fechter, L., Murugesan, K., Maecker, H., Gotlib, J., Zehnder, J., Paulmurugan, R., Krishnan, A. 2023

  Abstract

  Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34 + cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin ( ENKUR ), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20 . Silencing of ENKUR by shRNA in CD34 + derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.VISUAL ABSTRACT: Key Points: Enkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs.MPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20.Likely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.

  View details for DOI 10.1101/2023.01.07.523111

  View details for PubMedID 36712071

• IFITM3 regulates fibrinogen endocytosis and platelet reactivity in non-viral sepsis. The Journal of clinical investigation Campbell, R. A., Manne, B. K., Banerjee, M., Middleton, E. A., Ajanel, A., Schwertz, H., Denorme, F., Stubben, C., Montenont, E., Saperstein, S., Page, L., Tolley, N. D., Lim, D. L., Brown, S. M., Grissom, C. K., Sborov, D. W., Krishnan, A., Rondina, M. T. 2022

  Abstract

  Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We examined if non-viral sepsis induces differential platelet gene expression and reactivity. Non-viral sepsis upregulated IFITM3, an interferon responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we examined if IFITM3 promoted endocytosis of alpha granule proteins. Interferon stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacts with clathrin and alphaIIb and altered their plasma membrane localization into lipid rafts. In vivo interferon administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from interferon-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on interferon-alpha and IFITMs. Platelets from patients with non-viral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.

  View details for DOI 10.1172/JCI153014

  View details for PubMedID 36194487

• Evaluation of Immune Evasion in SARS-CoV-2 Delta and Omicron variants. Computational and structural biotechnology journal Chaudhari, A. M., Joshi, M., Kumar, D., Patel, A., Bharat Lokhande, K., Krishnan, A., Hanack, K., Filipek, S., Liepmann, D., Renugopalakrishnan, V., Paulmurugan, R., Joshi, C. 2022

  Abstract

  Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibitsimmune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.

  View details for DOI 10.1016/j.csbj.2022.08.010

  View details for PubMedID 35965661

• Inhaled Gold Nano-star Carriers for Targeted Delivery of Triple Suicide Gene Therapy and Therapeutic MicroRNAs to Lung Metastases: Development and Validation in a Small Animal Model. Advanced therapeutics Liu, Y., Sukumar, U. K., Jugniot, N., Seetharam, S. M., Rengaramachandran, A., Sadeghipour, N., Mukherjee, P., Krishnan, A., Massoud, T. F., Paulmurugan, R. 2022; 5 (8)

  Abstract

  Pulmonary metastases pose significant treatment challenges for many cancers, including triple-negative breast cancer (TNBC). We developed and tested a novel suicide gene and therapeutic microRNAs (miRs) combination therapy against lung metastases in vivo in mouse models after intranasal delivery using nontoxic gold nanoparticles (AuNPs) formulated to carry these molecular therapeutics. We used AuNPs coated with chitosan-β-cyclodextrin (CS-CD) and functionalized with a urokinase plasminogen activator (uPA) peptide to carry triple cancer suicide genes (thymidine kinase-p53-nitroreductase: TK-p53-NTR) plus therapeutic miRNAs (antimiR-21, antimiR-10b and miR-100). We synthesized three AuNPs: 20nm nanodots (AuND), and 20nm or 50nm nanostars (AuNS), then surface coated these with CS-CD using a microfluidic-optimized method. We sequentially coated the resulting positively charged AuNP-CS-CD core with synthetic miRNAs followed by TK-p53-NTR via electrostatic interactions, and added uPA peptide through CD-adamantane host-guest chemistry. A comparison of transfection efficiencies for different AuNPs showed that the 50nm AuNS allowed ∼4.16-fold higher gene transfection than other NPs. The intranasal delivery of uPA-AuNS-TK-p53-NTR-microRNAs NPs (pAuNS@TK-p53-NTR-miRs) in mice predominantly accumulated in lungs and facilitated ganciclovir and CB1954 prodrug-mediated gene therapy against TNBC lung metastases. This new nanosystem may serve as an adaptable-across-cancer-type, facile, and clinically scalable platform to allow future inhalational suicide gene-miR combination therapy for patients harboring pulmonary metastases.

  View details for DOI 10.1002/adtp.202200018

  View details for PubMedID 36212523

  View details for PubMedCentralID PMC9543365

• Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPN. Blood Jutzi, J. S., Marneth, A. E., Ciboddo, M., Guerra-Moreno, A., Jiménez-Santos, M. J., Kosmidou, A., Dressman, J. W., Liang, H., Hamel, R. S., Lozano, P. R., Rumi, E., Doench, J., Gotlib, J. R., Krishnan, A., Elf, S., Al-Shahrour, F., Mullally, A. 2022

  Abstract

  Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and normalization of key MPN disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow, as compared to bone marrow-derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPN.

  View details for DOI 10.1182/blood.2022015629

  View details for PubMedID 35763665

• Platelet Heterogeneity in Myeloproliferative Neoplasms ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Thomas, S., Krishnan, A. 2021; 41 (11): 2661-2670

  Abstract

  [Figure: see text].

  View details for DOI 10.1161/ATVBAHA.121.316373

  View details for Web of Science ID 000710623300004

  View details for PubMedID 34615371

  View details for PubMedCentralID PMC8551046

• Platelet transcriptome identifies progressive markers and potential therapeutic targets in chronic myeloproliferative neoplasms CELL REPORTS MEDICINE Shen, Z., Du, W., Perkins, C., Fechter, L., Natu, V., Maecker, H., Rowley, J., Gotlib, J., Zehnder, J., Krishnan, A. 2021; 2 (10)

  View details for DOI 10.1016/j.xcrm.2021.100425

  View details for Web of Science ID 000709912800003

• Camouflaged Hybrid Cancer Cell-Platelet Fusion Membrane Nanovesicles Deliver Therapeutic MicroRNAs to Presensitize Triple-Negative Breast Cancer to Doxorubicin. Advanced functional materials Liu, Y., Sukumar, U. K., Kanada, M., Krishnan, A., Massoud, T. F., Paulmurugan, R. 2021; 31 (41)

  Abstract

  Camouflaged cell-membrane-based nanoparticles have been gaining increasing attention owing to their improved biocompatibility and immunomodulatory properties. Using nanoparticles prepared from the membranes of specific cell types, or fusions derived from different cells membranes, can improve their functional performance in several aspects. Here, we used cell membranes extracted from breast cancer cells and platelets to fabricate a hybrid-membrane vesicle fusion (cancer cell-platelet-fusion-membrane vesicle, CPMV) in which we loaded therapeutic microRNAs (miRNAs) for the treatment of triple-negative breast cancer (TNBC). We used a clinically scalable microfluidic platform for the fusion of cell membranes. The reconstitution process during synthesis allows for efficient loading of miRNAs into CPMVs. We systematically optimized the conditions for preparation of miRNA-loaded CPMVs and demonstrated their property of homing to source cells using in vitro experiments, and by therapeutic evaluation in vivo. In vitro, the CPMVs exhibited significant recognition of their source cells and avoided engulfment by macrophages. After systemic delivery in mice, the CPMVs showed a prolonged circulation time and site-specific accumulation at implanted TNBC-xenografts. The delivered antimiRNAs sensitized TNBCs to doxorubicin, resulting in an improved therapeutic response and survival rate. This strategy has considerable potential for clinical translation to improve personalized therapy for breast cancer and other malignancies.

  View details for DOI 10.1002/adfm.202103600

  View details for PubMedID 34899115

  View details for PubMedCentralID PMC8664068

• Toward platelet transcriptomics in cancer diagnosis, prognosis and therapy British Journal of Cancer Krishnan, A., Thomas, S. 2021

  View details for DOI 10.1038/s41416-021-01627-z

• Increased Mortality and Bleeding in a Large Cohort of Patients on Heparin Anticoagulation Therapy with Discordant Anti-Factor Xa Activity and Activated Partial Thromboplastin Time (PTT); Implications for Clinical Management Gombar, S., Boothroyd, D., Krishnan, A., Sharifi, H., Hsu, J., Zehnder, J. AMER SOC HEMATOLOGY. 2019

  View details for DOI 10.1182/blood-2019-132112

  View details for Web of Science ID 000518218500781

• Platelet Transcriptomic Signatures in Myeloproliferative Neoplasms Blood Krishnan, A., Zhang, Y., Perkins, C., Gotlib, J., Zehnder, J. 2017; 130: 5288

  View details for DOI 10.1182/blood.V130.Suppl_1.5288.5288

• The effect of surface contact activation and temperature on plasma coagulation with an RNA aptamer directed against factor IXa JOURNAL OF THROMBOSIS AND THROMBOLYSIS Krishnan, A., Vogler, E. A., Sullenger, B. A., Becker, R. C. 2013; 35 (1): 48-56

  Abstract

  The anticoagulant properties of a novel RNA aptamer that binds FIXa depend collectively on the intensity of surface contact activation of human blood plasma, aptamer concentration, and its binding affinity for FIXa. Accordingly, anticoagulation efficiency of plasma containing any particular aptamer concentration is low when coagulation is strongly activated by hydrophilic surfaces compared to the anticoagulation efficiency in plasma that is weakly activated by hydrophobic surfaces. Anticoagulation efficiency is lower at hypothermic temperatures possibly because aptamer-FIXa binding decreases with decreasing temperatures. Experimental results demonstrating these trends are qualitatively interpreted in the context of a previously established model of anticoagulation efficiency of thrombin-binding DNA aptamers that exhibit anticoagulation properties similar to the FIXa aptamer. In principle, FIXa aptamer anticoagulants should be more efficient and therefore more clinically useful than thrombin-binding aptamers because aptamer binding to FIXa competes only with FX that is at much lower blood concentration than fibrinogen (FI) that competes with thrombin-binding aptamers. Our findings may have translatable relevance in the application of aptamer anticoagulants for clinical conditions in which blood is in direct contact with non-biological surfaces such as those encountered in cardiopulmonary bypass circuits.

  View details for DOI 10.1007/s11239-012-0778-7

  View details for Web of Science ID 000312784500007

  View details for PubMedID 23054460

• Antithrombotic therapy for ischemic stroke: guidelines translated for the clinician JOURNAL OF THROMBOSIS AND THROMBOLYSIS Krishnan, A., Lopes, R. D., Alexander, J. H., Becker, R. C., Goldstein, L. B. 2010; 29 (3): 368-377

  Abstract

  Acute ischemic stroke is the result of abrupt interruption of focal cerebral blood flow. The majority of ischemic strokes are caused by embolic or thrombotic arterial occlusions. Acute stroke management is complex, in part because of the varying etiologies of stroke and the very brief window of time for reperfusion therapy. Efforts to optimize stroke care have also encountered barriers including low public awareness of stroke symptoms. As initiatives move forward to improve stroke care worldwide, health care providers and institutions are being called onto deliver the most current evidence-based care. Updated versions of three major guidelines were published in 2008 by the American College of Chest Physicians, the American Heart Association, and the European Stroke Organization. This article presents a concise overview of current recommendations for the use of fibrinolytic therapy for acute ischemic stroke and antithrombotic therapy for secondary prevention. Future directions are also reviewed, with particular emphasis on improving therapeutic options early after stroke onset.

  View details for DOI 10.1007/s11239-010-0439-7

  View details for Web of Science ID 000275555100018

  View details for PubMedID 20127273

• Mixology of protein solutions and the Vroman effect LANGMUIR Krishnan, A., Siedlecki, C. A., Vogler, E. A. 2004; 20 (12): 5071-5078

  Abstract

  Mixing rules stipulating both concentration and distribution of proteins adsorbed to the liquid-vapor (LV) interphase from multicomponent aqueous solutions are derived from a relatively straightforward protein-adsorption model. Accordingly, proteins compete for space within an interphase separating bulk-vapor and bulk-solution phases on a weight, not molar, concentration basis. This results in an equilibrium weight-fraction distribution within the interphase that is identical to bulk solution. However, the absolute interphase concentration of any particular protein adsorbing from an m-component solution is 1/mth that adsorbed from a pure, single-component solution of that protein due to competition with m - 1 constituents. Applied to adsorption from complex biological fluids such as blood plasma and serum, mixing rules suggest that there is no energetic reason to expect selective adsorption of any particular protein from the mixture. Thus, dilute members of the plasma proteome are overwhelmed at the hydrophobic LV surface by the 30 classical plasma proteins occupying the first 5 decades of physiological concentration. Mixing rules rationalize the experimental observations that (i) concentration-dependent liquid-vapor interfacial tension, gammalv, of blood plasma and serum (comprised of about 490 different proteins) cannot be confidently resolved, even though serum is substantially depleted of coagulable proteins (e.g., fibrinogen), and (ii) gammalv of plasma is startlingly similar to that of purified protein constituents. Adsorption-kinetics studies of human albumin (66.3 kDa) and IgM (1000 kDa) binary mixtures revealed that relatively sluggish IgM molecules displace faster-moving albumin molecules adsorbing to the LV surface. This Vroman-effect-like process leads to an equilibrium gammalv reflecting the linear combination of weight/volume concentrations at the surface predicted by theory. Thus, the Vroman effect is interpreted as a natural outcome of protein reorganization to achieve an equilibrium interphase composition dictated by a firm set of mixing rules.

  View details for DOI 10.1021/la036218r

  View details for Web of Science ID 000221846000042

  View details for PubMedID 15984270

• Platelet transcriptome yields progressive markers in chronic myeloproliferative neoplasms and identifies putative targets of therapy bioRxiv Shen, Z., Du, W., Perkins, C., Fechter, L., Natu, V., Maecker, H., Rowley, J., Gotlib, J., Zehnder, J. L., Krishnan, A. 2021

  View details for DOI 10.1101/2021.03.12.435190

• Platelet Transcriptomic Signatures in Myeloproliferative Neoplasms Krishnan, A., Zhang, Y., Perkins, C., Gotlib, J., Zehnder, J. L. AMER SOC HEMATOLOGY. 2017

  View details for Web of Science ID 000432419705465

• Effects of limb posture on reactive hyperemia EUROPEAN JOURNAL OF APPLIED PHYSIOLOGY Krishnan, A., Lucassen, E. B., Hogeman, C., Blaha, C., Leuenberger, U. A. 2011; 111 (7): 1415-1420

  Abstract

  To examine the role of limb posture on vascular conductance during rapid changes in vascular transmural pressure, we determined brachial (n = 10) and femoral (n = 10) artery post-occlusive reactive hyperemic blood flow (RHBF, ultrasound/Doppler) and vascular conductance in healthy humans with each limb at three different positions-horizontal, up and down. Limb posture was varied by raising or lowering the arm or leg from the horizontal position by 45°. In both limbs, peak RHBF and vascular conductance were highest in the down or horizontal position and lowest in the up position (arm up 338 ± 38, supine 430 ± 52, down 415 ± 52 ml/min, P < 0.05; leg up 1,208 ± 88, supine 1,579 ± 130, down 1,767 ± 149 ml/min, P < 0.05). In contrast, the maximal dynamic fall in blood flow following peak RHBF (in ml/s/s) in both limbs was highest in the limb-down position and lowest with the limb elevated (P < 0.05). These data suggest that the magnitude and temporal pattern of limb reactive hyperemia is in part related to changes in vascular transmural pressure and independent of systemic blood pressure and sympathetic control.

  View details for DOI 10.1007/s00421-010-1769-z

  View details for Web of Science ID 000291602200018

  View details for PubMedID 21161263

  View details for PubMedCentralID PMC3285391

• Volumetric interpretation of protein adsorption: Interfacial packing of protein adsorbed to hydrophobic surfaces from surface-saturating solution concentrations BIOMATERIALS Kao, P., Parhi, P., Krishnan, A., Noh, H., Haider, W., Tadigadapa, S., Allara, D. L., Vogler, E. A. 2011; 32 (4): 969-978

  Abstract

  The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square or hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square or hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM.

  View details for DOI 10.1016/j.biomaterials.2010.09.075

  View details for Web of Science ID 000285675200003

  View details for PubMedID 21035180

  View details for PubMedCentralID PMC3040988

• A real-time device for converting Doppler ultrasound audio signals into fluid flow velocity AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Herr, M. D., Hogeman, C. S., Koch, D. W., Krishnan, A., Momen, A., Leuenberger, U. A. 2010; 298 (5): H1626-H1632

  Abstract

  A Doppler signal converter has been developed to facilitate cardiovascular and exercise physiology research. This device directly converts audio signals from a clinical Doppler ultrasound imaging system into a real-time analog signal that accurately represents blood flow velocity and is easily recorded by any standard data acquisition system. This real-time flow velocity signal, when simultaneously recorded with other physiological signals of interest, permits the observation of transient flow response to experimental interventions in a manner not possible when using standard Doppler imaging devices. This converted flow velocity signal also permits a more robust and less subjective analysis of data in a fraction of the time required by previous analytic methods. This signal converter provides this capability inexpensively and requires no modification of either the imaging or data acquisition system.

  View details for DOI 10.1152/ajpheart.00713.2009

  View details for Web of Science ID 000277301400037

  View details for PubMedID 20173048

  View details for PubMedCentralID PMC2867441

• Interfacial energetics of protein adsorption from aqueous buffer to surface with varying hydrophilicity LANGMUIR Cha, P., Krishnan, A., Fiore, V. F., Vogler, E. A. 2008; 24 (6): 2553-2563

  Abstract

  Adsorption isotherms constructed from time-and-concentration-dependent advancing contact angles thetaa show that the profound biochemical diversity among ten different blood proteins with molecular weight spanning 10-1000 kDa has little discernible effect on the amount adsorbed from aqueous phosphate-buffered saline (PBS) solution after 1 h contact with a particular test surface selected from the full range of observable water wettability (as quantified by PBS adhesion tension tauoa=gammaolv cos thetaoa; where gammaolv is the liquid-vapor interfacial tension and thetaoa is the advancing PBS contact angle). The maximum advancing spreading pressure, Pimaxa, determined from adsorption isotherms decreases systematically with tauoa for methyl-terminated self-assembled monolayers (CH3 SAM, tauo=-15 mN/m), polystyrene spun-coated onto electronic-grade SiOx wafers (PS, tauo=7.2 mN/m), aminopropyltriethoxysilane-treated SiOx surfaces (APTES, tauo = 42 mN/m), and fully water wettable SiOx (tauo=72 mN/m). Likewise, the apparent Gibbs' surface excess [Gammasl-Gammasv], which measures the difference in the amount of protein adsorbed Gamma (mol/cm2) at solid-vapor (SV) and solid-liquid (SL) interfaces, decreases with tauo from maximal values measured on the CH3 SAM surface through zero (no protein adsorption in excess of bulk solution concentration) near tauo=30 mN/m (thetaa=65 degrees). These latter results corroborate the conclusion drawn from independent studies that water is too strongly bound to surfaces with tauo>or=30 mN/m to be displaced by adsorbing protein and that, as a consequence, protein does not accumulate within the interfacial region of such surfaces at concentrations exceeding that of bulk solution ([Gammasl-Gammasv]=0 at tauo=30 mN/m). Results are collectively interpreted to mean that water controls protein adsorption to surfaces and that the mechanism of protein adsorption can be understood from this perspective for a diverse set of proteins with very different amino acid compositions.

  View details for DOI 10.1021/la703310k

  View details for Web of Science ID 000253941000042

  View details for PubMedID 18229964

• Interfacial energetics of protein adsorption from aqueous buffer to surfaces with varying hydrophilicity Langmuir P Cha, A Krishnan, VF Fiore, EA Vogler 2008; 24 (6): 2553
• The role of the cyclooxygenase products in evoking sympathetic activation in exercise AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Cui, J., McQuillan, P., Momen, A., Blaha, C., Moradkhan, R., Mascarenhas, V., Hogeman, C., Krishnan, A., Sinoway, L. I. 2007; 293 (3): H1861-H1868

  Abstract

  Animal studies suggest that prostaglandins in skeletal muscles stimulate afferents and contribute to the exercise pressor reflex. However, human data regarding a role for prostaglandins in this reflex are varied, in part because of systemic effects of pharmacological agents used to block prostaglandin synthesis. We hypothesized that local blockade of prostaglandin synthesis in exercising muscles could attenuate muscle sympathetic nerve activity (MSNA) responses to fatiguing exercise. Blood pressure (Finapres), heart rate, and MSNA (microneurography) were assessed in 12 young healthy subjects during static handgrip and postexercise muscle ischemia (PEMI) before and after local infusion of 6 mg of ketorolac tromethamine in saline via Bier block (regional intravenous anesthesia). In the second experiment (n = 10), the same amount of saline was infused via the Bier block. Ketorolac Bier block decreased the prostaglandins synthesis to approximately 33% of the baseline. After ketorolac Bier block, the increases in MSNA from the baseline during the fatiguing handgrip was significantly lower than that before the Bier block (before ketorolac: Delta502 +/- 111; post ketorolac: Delta348 +/- 62%, P = 0.016). Moreover, the increase in total MSNA during PEMI after ketorolac was significantly lower than that before the Bier block (P = 0.014). Saline Bier block had no similar effect. The observations indicate that blockade of prostaglandin synthesis attenuates MSNA responses seen during fatiguing handgrip and suggest that prostaglandins contribute to the exercise pressor reflex.

  View details for DOI 10.1152/ajpheart.00258.2007.

  View details for Web of Science ID 000249237800068

  View details for PubMedID 17604332

  View details for PubMedCentralID PMC2559802

• Interfacial energetics of blood plasma and serum adsorption to a hydrophobic self-assembled monolayer surface BIOMATERIALS Krishnan, A., Cha, P., Liu, Y. H., Allara, D., Vogler, E. A. 2006; 27 (17): 3187-3194

  Abstract

  Interfacial energetics of blood plasma and serum adsorption to a hydrophobic, methyl-terminated self-assembled monolayer (SAM) surface (solid-liquid SL interface) are shown to be essentially the same as to the buffer-air interface (liquid-vapor LV interface). Specifically, spreading pressure (Pia) isotherms scaled on a w/v concentration basis constructed from advancing contact angles (thetaa) of serially diluted plasma/serum derived from four different mammalian species (bovine, equine, human, and ovine) on the SAM surface are not resolvable at the 99% confidence level and furthermore are found to be strikingly similar to isotherms of purified human-blood proteins. Maximum advancing spreading pressures Piamax for protein mixtures fall within a relatively narrow 17

  View details for DOI 10.1016/j.biomaterials.2005.12.032

  View details for Web of Science ID 000236564000004

  View details for PubMedID 16494939

• Interfacial rheology of blood proteins adsorbed to the aqueous-buffer/air interface BIOMATERIALS Ariola, F. S., Krishnan, A., Vogler, E. A. 2006; 27 (18): 3404-3412

  Abstract

  Concentration-dependent, interfacial-shear rheology and interfacial tension of albumin, IgG, fibrinogen, and IgM adsorbed to the aqueous-buffer/air surface is interpreted in terms of a single viscoelastic layer for albumin but multi-layers for the larger proteins. Two-dimensional (2D) storage and loss moduli G(') and G(''), respectively, rise and fall as a function of bulk-solution concentration, signaling formation of a network of interacting protein molecules at the surface with viscoelastic properties. Over the same concentration range, interfacial spreading pressure Pi(LV) identical with gamma(lv)(o)-gamma(lv) rises to a sustained maximum Pi(LV)(max). Mixing as little as 25 w/v% albumin into IgG at fixed total protein concentration substantially reduces peak G('), strongly suggesting that albumin acts as rheological modifier by intercalating with adsorbed IgG molecules. By contrast to purified-protein solutions, serially diluted human blood serum shows no resolvable concentration-dependent G(')and G('').

  View details for DOI 10.1016/j.biomaterials.2006.02.005

  View details for Web of Science ID 000236783000009

  View details for PubMedID 16504286

• Interfacial energetics of globular?blood protein adsorption to a hydrophobic interface from aqueous-buffer solution J. Royal Soc. Interface Krishnan A, YH Liu, P Cha, D Allara, EA Vogler 2006; 3 (7): 283
• Scaled interfacial activity of proteins at a hydrophobic solid/aqueous-buffer interface. Journal of biomedical materials research. Part A Krishnan, A., Liu, Y., Cha, P., Allara, D., Vogler, E. A. 2005; 75 (2): 445-457

  Abstract

  Contact-angle goniometry confirms that interfacial energetics of protein adsorption to the hydrophobic solid/aqueous-buffer (solid-liquid, SL) surface is not fundamentally different than adsorption to the aqueous-buffer/air (liquid-vapor, LV) interface measured by pendant-drop tensiometry. Adsorption isotherms of 9 globular blood proteins with molecular weight (MW) spanning from 10 to 1000 kDa on methyl-terminated self-assembled monolayer surfaces demonstrate that (i) proteins are weak surfactants, reducing contact angles by no more than about 15 degrees at maximum solution concentrations ( approximately 10 mg/mL); (ii) the corresponding dynamic range of spreading pressure Pi(a) < 20 mN/m; and (iii) the maximum spreading pressure Pi(max) (a) for these diverse proteins falls within a relatively narrow 5 mN/m band. As with adsorption to the LV interface, we find that concentration scaling substantially alters perception of protein interfacial activity measured by Pi(a). Proteins appear more similar than dissimilar on a weight/volume basis whereas molarity scaling reveals a systematic ordering by MW, suggesting that adsorption is substantially driven by solution concentration rather than diversity in protein amphilicity. Scaling as a ratio-to-physiological-concentration demonstrates that certain proteins exhibit Pi(max)(a) at-and-well-below physiological concentration whereas others require substantially higher solution concentration to attain Pi(max)(a). Important among this latter category of proteins is blood factor XII, assumed by the classical biochemical mechanism of plasma coagulation to be highly surface active, even in the presence of overwhelming concentrations of other blood constituents such as albumin and immunoglobulin that are shown by this work to be among the class of highly surface-active proteins at physiologic concentration. The overarching interpretation of this work is that water plays a dominant, controlling role in the adsorption of globular-blood proteins to hydrophobic surfaces and that energetics of hydration control the amount of protein adsorbed to poorly water-wettable biomaterials.

  View details for PubMedID 16104049

• An evaluation of methods for contact angle measurement COLLOIDS AND SURFACES B-BIOINTERFACES Krishnan, A., Liu, Y. H., Cha, P., Woodward, R., Allara, D., Vogler, E. A. 2005; 43 (2): 95-98

  Abstract

  A systematic comparison of Wilhelmy-balance tensiometry (WBT), tilting-plate goniometry (TPG) and captive-drop goniometry (CDG), using a variety of silane-treated glass surfaces of variable wetting characteristics is reported. WBT was assumed to be the benchmark of comparison (gold standard). Advancing angles measured by TPG and CDG were in statistical agreement with corresponding WBT measurements. In contrast, receding angles from both goniometric techniques were systematically higher than WBT.

  View details for DOI 10.1016/j.colsurfb.2005.04.003

  View details for Web of Science ID 000230572600004

  View details for PubMedID 15922578

• Liquid-vapor interfacial tension of blood plasma, serum and purified protein constituents thereof BIOMATERIALS Krishnan, A., Wilson, A., Sturgeon, J., Siedlecki, C. A., Vogler, E. A. 2005; 26 (17): 3445-3453

  Abstract

  A systematic study of water-air (liquid-vapor, LV) interfacial tension gamma(lv) of blood plasma and serum derived from four different mammalian species (human, bovine, ovine and equine) reveals nearly identical concentration-dependence (dgamma(lv)/dlnC(B); where C(B) is plasma/serum dilution expressed in v/v concentration units). Comparison of results to a previously-published survey of purified human-blood proteins further reveals that dgamma(lv)/dlnC(B) of plasma and serum is surprisingly similar to that of purified protein constituents. It is thus concluded that any combination of blood-protein constituents will be substantially similar because dgamma(lv)/dlnC(B) of individual proteins are very similar. Experimental results are further interpreted in terms of a recently-developed theory emphasizing the controlling role of water in protein adsorption. Accordingly, the LV interphase saturates with protein adsorbed from bulk solution at a fixed weight-volume concentration ( approximately 436 mg/mL) independent of protein identity or mixture. As a direct consequence, dgamma(lv)/dlnC(B) of purified proteins closely resembles that of mixed solutions and does not depend on the relative proportions of individual proteins comprising a mixture. Thus variations in the plasma proteome between species are not reflected in dgamma(lv)/dlnC(B) nor is serum different from plasma in this regard, despite being depleted of coagulation proteins (e.g. fibrinogen). A comparison of pendant-drop and Wilhelmy-balance tensiometry as tools for assessing protein gamma(lv) shows that measurement conditions employed in the typical Wilhelmy plate approach fails to achieve the steady-state adsorption state that is accessible to pendant-drop tensiometry.

  View details for DOI 10.1016/j.biomaterials.2004.09.016

  View details for Web of Science ID 000226968200008

  View details for PubMedID 15621233

• An Evaluation of Goniometric Methods J. Coll. Interface A Krishnan, YH Liu, P Cha, DL Allara, EA Vogler 2005; 43: 95
• Scaled interfacial activity of proteins at the liquid-vapor interface. Journal of biomedical materials research. Part A Krishnan, A., Sturgeon, J., Siedlecki, C. A., Vogler, E. A. 2004; 68 (3): 544-557

  Abstract

  A principal conclusion drawn from observations of time- and concentration-dependent liquid-vapor (LV) interfacial tension gamma(lv) of a diverse selection of proteins ranging from albumin to ubiquitin spanning nearly three decades in molecular weight (MW) is that concentration scaling substantially alters perception of protein interfacial activity as measured by reduction in gamma(lv). Proteins appear more similar than dissimilar on a weight/volume basis, whereas molarity scaling reveals a "Traube-rule" ordering by MW, suggesting that adsorption is substantially driven by solution concentration rather than diversity in protein amphilicity. Scaling as a ratio-to-physiological-concentration demonstrates that certain proteins exhibit the full possible range of interfacial activity at and well-below physiological concentration, whereas others are only weakly surface active within this range, requiring substantially higher solution concentration to achieve reduction in gamma(lv). Important among this latter category of proteins are the blood factors XII and XIIa, assumed by the classical biochemical mechanism of plasma coagulation to adsorb to procoagulant surfaces, even in the presence of overwhelming concentrations of other blood constituents such as albumin and immunoglobulin that are shown by this work to be among the class of highly surface-active proteins at physiologic concentration. A comparison of pendant drop and Wilhelmy balance tensiometry as tools for assessing protein interfacial activity shows that measurement conditions employed in the typical Wilhelmy plate approach fails to achieve the steady-state adsorption condition that is accessible to pendant drop tensiometry.

  View details for PubMedID 14762935

• Traube-rule interpretation of protein adsorption at the liquid-vapor interface LANGMUIR Krishnan, A., Siedlecki, C. A., Vogler, E. A. 2003; 19 (24): 10342-10352

  View details for DOI 10.1021/la035308t

  View details for Web of Science ID 000186694500053

• Traube-rule interpretation of protein adsorption at the liquid-vapor interface Langmuir Krishnan A, Siedlecki CA, Vogler EA 2003; 19 (24): 10342