Assessing Blood Clotting and Coagulation Factors in Mice.
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ABSTRACT:
SUBMITTER: Brake MA
PROVIDER: S-EPMC6771260 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
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Project description:Coagulation balance is maintained through fine-tuned interactions among clotting factors, whose physiological concentrations vary substantially. In particular, the concentrations of coagulation proteases (pM to nM) are much lower than their natural inactivator antithrombin (AT, ~ 3 μM), suggesting the existence of other coordinators. In the current study, we found that transferrin (normal plasma concentration ~40 μM) interacts with fibrinogen, thrombin, factor XIIa (FXIIa), and AT with different affinity to maintain coagulation balance. Normally, transferrin is sequestered by binding with fibrinogen (normal plasma concentration ~10 μM) at a molar ratio of 4:1. In atherosclerosis, abnormally up-regulated transferrin interacts with and potentiates thrombin/FXIIa and blocks AT's inactivation effect on coagulation proteases by binding to AT, thus inducing hypercoagulability. In the mouse model, transferrin overexpression aggravated atherosclerosis, whereas transferrin inhibition via shRNA knockdown or treatment with anti-transferrin antibody or designed peptides interfering with transferrin-thrombin/FXIIa interactions alleviated atherosclerosis. Collectively, these findings identify that transferrin is an important clotting regulator and an adjuster in the maintenance of coagulation balance and modifies the coagulation cascade.
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Project description:BackgroundTwenty-minute whole blood clotting test (20WBCT) and Modified Lee and White (MLW) method are the most routinely employed bedside tests for detecting coagulopathic snake envenomation. Our study compared the diagnostic utility of MLW and 20WBCT for snakebite victims at a tertiary care hospital in Central Kerala, South India.MethodsThis single-center study recruited 267 patients admitted with snake bites. 20WBCT and MLW were performed simultaneously at admission along with the measurement of Prothrombin Time (PT). The diagnostic utility of 20WBCT and MLW was determined by comparing the sensitivity (Sn), specificity (Sp), positive and negative predictive values, likelihood ratios, and accuracy at admission with an INR value > 1.4.ResultsOut of 267 patients, 20 (7.5%) patients had VICC. Amongst those who had venom-induced consumption coagulopathy (VICC), MLW was prolonged for 17 patients, (Sn 85% 95% confidence interval [CI]: 61.1-96.0) whereas 20WBCT was abnormal for 11 patients (Sn 55%, 95% CI: 32.04-76.17). MLW and 20WBCT were falsely positive for the same patient (Sp 99.6%, 95% CI: 97.4-99.9%).ConclusionMLW is more sensitive than 20WBCT to detect coagulopathy at the bedside amongst snakebite victims. However, further studies are necessary for standardizing bedside coagulation tests in snakebite cases.
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Project description:Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.
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Project description:A hallmark of hemostasis is that cells and proteins involved in the formation of a blood clot remain in a quiescent state and are only activated following an appropriate stimulus. The homologous proteins factors V and VIII cannot participate to any significant degree in their macromolecular enzyme complexes and are thus considered procofactors. Activity is generated following limited proteolysis, indicating that the conversion of the procofactors to factor Va and factor VIIIa must result in structural changes that impart cofactor function. The proteolytic events that lead to the activation of these proteins have been extensively characterized over the past three decades. However, a fundamental understanding of the mechanism(s) by which these proteins are kept as inactive procofactors and how specific bond cleavage facilitates the conversion to the active cofactor state is only starting to become known. These molecular processes undoubtedly play critical regulatory roles, evolved to maintain normal hemostasis since factor Va and factor VIIIa have a tremendous influence on thrombin generation. This review will detail our current understanding of the molecular process of procofactor activation and highlight structural features that play a major role in factor V and factor VIII activation.
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Project description:Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.
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Project description:Recombinant blood clotting factor VIII is one of the most complex proteins for industrial manufacturing due to the low efficiency of its gene transcription, massive intracellular loss of its proprotein during post-translational processing, and the instability of the secreted protein. Improvement in hemophilia A therapy requires a steady increase in the production of factor VIII drugs despite tightening standards of product quality and viral safety. More efficient systems for heterologous expression of factor VIII can be created on the basis of the discovered properties of its gene transcription, post-translational processing, and behavior in the bloodstream. The present review describes the deletion variants of factor VIII protein with increased secretion efficiency and the prospects for the pharmaceutical development of longer acting variants and derivatives of factor VIII.
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Project description:Most of the steps in the blood clotting cascade require clotting proteins to bind to membrane surfaces with exposed phosphatidylserine. In spite of the importance of these protein-membrane interactions, we still lack a detailed understanding of how clotting proteins interact with membranes and how membranes contribute so profoundly to catalysis. Our laboratories are using multidisciplinary approaches to explore, at atomic-resolution, how blood clotting protein complexes assemble and function on membrane surfaces.
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2023-10-03 | GSE206543 | GEO