Kenneth G. Mann

Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation

Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)-activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor-treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A-positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

The role of the red cell membrane in thrombin generation

Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization.

Anticoagulant effects of statins and their clinical implications

There is evidence indicating that statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may produce several cholesterol-independent antithrombotic effects. In this review, we provide an update on the current understanding of the interactions between statins and blood coagulation and their potential relevance to the prevention of venous thromboembolism (VTE). Anticoagulant properties of statins reported in experimental and clinical studies involve decreased tissue factor expression resulting in reduced thrombin generation and attenuation of pro-coagulant reactions catalysed by thrombin, such as fibrinogen cleavage, factor V and factor XIII activation, as well as enhanced endothelial thrombomodulin expression, resulting in increased protein C activation and factor Va inactivation. Observational studies and one randomized trial have shown reduced VTE risk in subjects receiving statins, although their findings still generate much controversy and suggest that the most potent statin rosuvastatin exerts the largest effect.

Thrombin Generation in Hemorrhage Control and Vascular Occlusion

Hippocrates and Aristotle recognized that blood clots outside the body, but it was not until 1720 that John Louis Petit recognized that control of hemorrhage after amputation was associated with the blood clotting process.1 One hundred thirty-eight years later, Rudolph Virchow formulated his postulates regarding clots in venous thrombosis.2 The association of blood clots with acute arterial occlusion was advanced by James B. Herrick3 in 1912, but the significance of blood clotting in acute arterial events was only resolved by studies in the 1980s.4–6 As a consequence, our knowledge base has largely been driven by studies of the hemostatic process that have been extrapolated to describe the pathological occlusive clotting events in venous and arterial thrombosis.nnVascular integrity and blood fluidity are maintained by complex interplay between procoagulant and anticoagulant properties provided by the blood, the vasculature, and subvascular elements. Our knowledge of the inventory and connectivity between the blood-supplied components of the hemostatic and anticoagulant matrix was initially provided by genetic accidents that produced hemostatic and thrombotic defects, most of which have been ratified by experiments conducted with transgenic mice. Additional blood and vascular elements were discovered by use of in vitro assays that identified new entities and required additions to the coagulation/anticoagulation circuits. Pathways for the clotting process were described that were dependent on the plasma coming into contact with a foreign surface (the intrinsic pathway)7–9 or that were associated with the introduction of tissue components (now identified as membrane displayed tissue factor [TF]) into the plasma (the extrinsic pathway; Figure 1A).10,11 However, the absence of bleeding pathology with individuals or transgenic mice that display molecular abnormalities in the factor (F) XII12 and high-molecular-weight kininogen13 components of the contact enzyme (dotted box in …

Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism.

Background: The key source of prothrombin activation in vivo is prothrombinase assembled on the activated platelet surface. Results: Platelet-associated prothrombinase utilizes the prethrombin-2 pathway of prothrombin activation and a concerted enzyme mechanism. Conclusion: Platelet-associated prothrombinase activates prothrombin with a concerted mechanism in which no anticoagulant intermediates are released. Significance: Platelet-associated prothrombinase promotes coagulation by avoiding the release of catalytically active meizothrombin. The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.

Guided Antithrombotic Therapy: Current Status and Future Research Direction Report on a National Heart, Lung and Blood Institute Working Group

The National Heart, Lung, and Blood Institute (NHLBI) convened a working group to develop a research agenda to enhance the understanding and effectiveness of antithrombotic therapy. The working group brought together cardiologists, hematologists, interventionalists, clinical trialists, genetic epidemiologists, basic scientists, and other stakeholders to review (1) coagulation, platelet activation and aggregation, and antithrombotic therapy; (2) issues surrounding antithrombotic therapy failure – how to define it, how to predict and diagnose it, available tests and how to optimize them; (3) the factors that affect the efficacy, safety, and predictability of antithrombotic therapies; (4) how to optimize antithrombotic therapy, improve on present interventions, and individually tailor therapy to increase efficacy and safety and to avoid failure; and (5) the clinical applicability and cost-effectiveness of individually tailored antithrombotic therapy based on functional and genetic testing. The working group characterized and discussed challenges for guided antithrombotic therapy in 4 domains: therapeutic strategies, antithrombotic metrics, pharmacology and pharmacogenetics, and stakeholders roles. Overall, the working group identified and prioritized the most pressing clinical needs to focus future research and translational efforts. This report presents highlights of these reviews and a summary of suggested research directions.nnThere has been tremendous progress in the field of thrombosis in the past 2 decades.1–5 The ramifications on cardiovascular care have been profound. A greater appreciation of the central role of platelets in atherothrombosis and an increased understanding of the receptors involved in platelet activation and aggregation have led to pivotal randomized controlled trials (RCTs) of novel agents.6 Many of these agents have been associated with substantial reductions in adverse cardiovascular outcomes. Simultaneously, an appreciation of the complexity of the coagulation cascade and the artificiality of separating it from cellular and platelet interactions has promoted a deeper understanding of thrombosis and, consequently, identification of pharmacological targets to …

Membrane binding events in the initiation and propagation phases of tissue factor initiated zymogen activation under flow

Background: Much of our understanding of blood coagulation comes from “test tube” experiments that neglect the dynamics of the open vasculature. Results: Membrane binding is critical in the activation of factor X under flow. Conclusion: Different shear rates throughout the vasculature regulate factor Xa and thrombin presentation. Significance: Determining flows effects on coagulation will help us to understand differences between venous and arterial clots. This study investigates the dynamics of zymogen activation when both extrinsic tenase and prothrombinase are assembled on an appropriate membrane. Although the activation of prothrombin by surface-localized prothrombinase is clearly mediated by flow-induced dilutional effects, we find that when factor X is activated in isolation by surface-localized extrinsic tenase, it exhibits characteristics of diffusion-mediated activation in which diffusion of substrate to the catalytically active region is rate-limiting. When prothrombin and factor X are activated coincident with each other, competition for available membrane binding sites masks the diffusion-limiting effects of factor X activation. To verify the role of membrane binding in the activation of factor X by extrinsic tenase under flow conditions, we demonstrate that bovine lactadherin competes for both factor X and Xa binding sites, limiting factor X activation and forcing the release of bound factor Xa from the membrane at a venous shear rate (100 s−1). Finally, we present steady-state models of prothrombin and factor X activation under flow showing that zymogen and enzyme membrane binding events further regulate the coagulation process in an open system representative of the vasculature geometry.

Rivaroxaban Delivery and Reversal at a Venous Flow Rate

Objective—Rivaroxaban is an oral anticoagulant that directly targets both free factor Xa and factor Xa in complex with its protein cofactor, factor Va, in the prothrombinase complex. It is approved in the United States for the prophylaxis of deep vein thrombosis and stroke in patients with atrial fibrillation; however, it also carries a black box warning regarding the risk of thrombosis after discontinuation of treatment. The purpose of this study was to determine the degree to which rivaroxaban, over a range of physiologically relevant free plasma concentrations, inhibits preassembled prothrombinase at a typical venous shear rate (100 s−1) and to determine the dynamics of rivaroxaban washout. Methods and Results—Prothrombinase was assembled on phospholipid-coated glass capillaries. Its activity was characterized with respect to the activation of prothrombin (mean plasma concentration, 1.4 &mgr;mol/L) in the absence and presence of rivaroxaban (2, 5, and 10 nmol/L). The degree of inactivation of preassembled prothrombinase is sensitive to the solution-phase rivaroxaban concentration; however, prothrombinase unmasking upon removal of rivaroxaban is concentration independent. Conclusion—The model system presented suggests that when rivaroxaban plasma concentrations decrease after cessation of therapy, there will be an unmasking of thrombus-associated prothrombinase that may be related to the reported rebound phenomena.

From principle to practice: bridging the gap in patient profiling.

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as “global assays” of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individuals thrombotic and bleeding risk during “normal” processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.

Disulfide reduction abolishes tissue factor cofactor function

BACKGROUNDnTissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186-Cys209, has been hypothesized to be essential for an allosteric decryption phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.nnnMETHODSnThe status of disulfides in recombinant TF1-263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.nnnRESULTSnIn native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.nnnCONCLUSIONnThe reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.nnnGENERAL SIGNIFICANCEnResults of this study advance our knowledge on TF structure/function relationships.