Thrombin Generation in Hemorrhage Control and Vascular Occlusion
2011; Lippincott Williams & Wilkins; Volume: 124; Issue: 2 Linguagem: Inglês
10.1161/circulationaha.110.952648
ISSN1524-4539
Autores Tópico(s)Hemophilia Treatment and Research
ResumoHomeCirculationVol. 124, No. 2Thrombin Generation in Hemorrhage Control and Vascular Occlusion Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBThrombin Generation in Hemorrhage Control and Vascular Occlusion Kenneth G. Mann Kenneth G. MannKenneth G. Mann From the Department of Biochemistry, University of Vermont, Colchester, VT. Originally published12 Jul 2011https://doi.org/10.1161/CIRCULATIONAHA.110.952648Circulation. 2011;124:225–235IntroductionHippocrates 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.Vascular 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 Figure 1) led to the conclusion that this catalyst is not in the primary pathway that maintains hemostasis. Thus, although the contact activation catalyst most likely does not have a role in hemostasis, recent data obtained with FXII knockout animals12 and in human inflammatory syndromes14 suggest this pathway may be relevant in some thrombotic disorders.Download figureDownload PowerPointFigure 1. A, Diagram illustrating the surface-bound complex enzymes of the intrinsic and extrinsic coagulation pathways. The surface-bound complex of the intrinsic pathway is outlined by a dashed line. Amplification by thrombin feedback activations of factor (F) V, FVIII, FVII, and FXI is also illustrated. B, Coagulation system regulation by the stoichiometric inhibitors (antithrombin and tissue factor pathway inhibitor), which downregulate the system by binding the serine proteases, and the dynamic activated protein C (APC) system, which causes proteolytic inactivation the cofactors (FVa and FVIIIa). HMW indicates high molecular weight; PC, protein C; AT, antithrombin; and TFPI, tissue factor pathway inhibitor. Reproduced from Mann10 with permission of the publisher. Copyright © 2003, American College of Chest Physicians.The coagulation process is strictly regulated by stoichiometric and dynamic inhibitory processes; the former are primarily a consequence of antithrombin and tissue factor pathway inhibitor (TFPI), the latter a consequence of the dynamic protein C (PC) system, the function of which is directly linked to the generation of thrombin (Figure 1B). Antithrombin inhibits all the serine proteases of the coagulation system, with its function accelerated by heparin and by heparan sulfate proteoglycans15 expressed on the vascular endothelium. TFPI is a Kunitz-type inhibitor16 that can inhibit both FXa and FVIIa-TF independently; however, its primary target is the FXa-FVIIa-TF product complex.17 TFPI is a very high-affinity inhibitor, but is present in blood at very low concentrations and on the vascular endothelium, from which it is secreted by heparin.18The dynamic PC system integrates endothelial cell thrombomodulin19,20 and its cofactor, the endothelial PC receptor (EPCR), with thrombin generation.21,22 The thrombomodulin-EPCR-thrombin vascular membrane–bound catalyst activates plasma PC to the enzyme-activated protein C.21 The anticoagulant activated PC cleaves and inactivates FVa and FVIIIa.23,24 Antithrombin acts in synergy with TFPI and the PC system to provide a more potent anticoagulant effect.25,26 The combination of the stoichiometric and dynamic inhibitors produces "go/no-go" thresholds for the clotting process, which ensures that the triggering insult is of sufficient magnitude to warrant initiation of a potentially thrombotic response.Hemostasis and Vascular OcclusionFrom the biochemical perspective, common molecular and cellular events are associated with thrombin generation whether involved in protective or pathological clotting processes. However, it is also clear that the biomechanics of flow and the vascular architecture and composition are not equivalent in the venous and arterial circuits. Furthermore, in hemorrhage control, the source of tissue factor is extravascular, whereas it is intravascular in thrombosis. Nonetheless, for all coagulation processes, the essential enzyme, produced by the choreography of the blood and vascular components, is thrombin, which is produced from prothrombin as a consequence of 3 surface-bound catalysts (Figure 1A). In the present review, the initial focus is on the process of hemorrhage control, with more speculative extensions to clotting in venous and arterial circuits.Good Clots: Hemorrhage ControlHemorrhage control involves extravascular thrombin generation that leads to a stable fibrin-platelet dam that stops blood flow. The environment of the clot is primarily extravascular tissue that elicits thrombin formation after a vascular perforation by platelet adhesion, secretion, and aggregation via interactions with connective tissue collagen and blood-derived von Willebrand factor.27,28 Thrombin production is initiated with the presentation of plasma FVIIa, a biologically inactive serine protease,29 to extraluminal subendothelial TF as a consequence of the vascular perforation (Figure 2A). The FVIIa-TF complex that generates the initial burst of FXa is primarily regulated by TFPI. The FVIIa-TF complex activates 2 substrates, FX and FIX, to form their respective products, FXa and FIXa31–33 (Figure 1A). Small amounts of FXa in combination with platelet and tissue membrane surfaces activate a small amount of prothrombin to produce thrombin.34 Thrombin amplifies its own generation in the adverse environment of overwhelming inhibitor concentrations by activating additional platelets35,36 and the procofactors (FV and FVIII)37,38 to produce the cofactors (FVa and FVIIIa) essential to the formation of these membrane-bound catalysts (Figure 2B). The membrane binding sites for the intrinsic factor Xase (FVIIIa-FIXa),39 which becomes the major FX activator and prothrombinase (FVa-FXa), include those expressed on the activated platelets, damaged cells at the site of injury, and other peripheral blood cells.40,41 The complex catalysts are 105-fold to 107-fold more active than their constituent serine proteases, and their membrane binding localizes their reactivity to the site of vascular injury.40,42 The formation of these surface-bound catalysts leads to local explosive generation of thrombin in the blood leaked into extravascular space. These catalysts anchored at the wound site are protected from inactivation by antithrombin. The catalysts continue to build up in the wound site as more platelets and plasma substrates are delivered by blood leaking through the site. The α-thrombin end product of the coagulation reaction system cleaves fibrinopeptides A and B from fibrinogen, which leads to expanding fibrin clot formation that is stabilized by the concomitant thrombin activation of FXIII to FXIIIa, which produces cross-links between the aggregated fibrin monomers. The dynamics of fibrin formation aggregation and cross-linking are tightly integrated and lead to a stable clot of sufficient mechanical strength to prevent blood loss.43–45 The reaction continues to proceed as long as more reactants are delivered to the wound site. The intravascular/extravascular 2-compartment reaction system will produce thrombin, fibrin, and aggregated platelets until the extravascular compartment becomes starved of reactants or after closure of the wound site (Figure 2C) by the thrombus. Coagulant enzymes that escape from the wound site by complex dissociation are inactivated by antithrombin. Thrombin downregulates its intravascular production through activation of the PC system. Thrombin binds to endothelial cell thrombomodulin, and this thrombin-thrombomodulin complex, aided by EPCR, activates plasma PC to become activated PC. Activated PC cleaves FVa and FVIIIa, which inactivates the cofactors24,46 (Figure 2D). Systemic thrombotic occlusion does not occur because of the potent negative regulatory elements provided by the vascular wall and plasma. Thrombin also initiates secretion of plasminogen activators that initiate clot dissolution concurrent with vascular repair.47Download figureDownload PowerPointFigure 2. Schema of a 2-compartment model of the regulation of tissue factor (TF)–initiated blood coagulation. A cross section of a blood vessel showing the luminal space, endothelial cell layer, and extravascular region is presented at the site of a perforation. The blood coagulation process in response is depicted in 4 stages. TF·VIIa indicates TF-factor VIIa complex; Xa·Va, prothrombinase complex; VIIIa·IXa, intrinsic factor Xase; HS·ATIII·(IIa or Xa), ATIII–endothelial cell heparan sulfate proteoglycan complex bound to thrombin or factor Xa; and TM·IIa·PC, protein C bound to thrombomodulin-thrombin. A, Perforation results in delivery of blood, and with it circulating factor VIIa and platelets, to an extravascular space rich in membrane-bound TF. Platelets adhere to collagen and von Willebrand factor associated with the extravascular tissue, and TF binds factor VIIa, which initiates the process of factor IX and factor X activation. Factor Xa activates small amounts of prothrombin to thrombin, which activates more platelets and converts factor V and factor VIII to factor Va and factor VIIIa. B, The reaction is propagated by platelet-bound intrinsic factor Xase and prothrombinase, with the former being the principle factor Xa generator. Initial clotting occurs, and fibrin begins to fill the void in cooperation with activated platelets. C, A barrier composed of activated platelets laden with procoagulant complexes and enmeshed in fibrin scaffolding is formed. The reaction in the now filled perforation is terminated by reagent consumption, which attenuates further thrombin generation, but functional procoagulant enzyme complexes persist because they are protected from the dynamic inhibitory processes found on the intravascular face. D, View downstream of the perforation. Enzymes escaping from the plugged perforation are captured by antithrombin-heparan complexes, and the protein C system is activated by residual thrombin binding to endothelial cell thrombomodulin, which initiates the dynamic anticoagulant system. These intravascular processes work against occlusion of the vessel despite the continuous resupply of reactants across the intravascular face of the thrombus. Reprinted from Orfeo et al30 with permission of the publisher. Copyright © 2005, American Society for Biochemistry and Molecular BiologyThe procoagulant reaction is multiphasic and can be divided operationally into 3 segments (initiation, propagation, and termination), which are presented as an animated cartoon here, as well as on YouTube.48,49 The latter occurs throughout the entire process. The events that occur during the initiation phase include activation of platelets, cofactors, and serine proteases that form the membrane assembled complexes that drive most thrombin generation during the propagation phase.The initiation-phase activation events are illustrated by a numerically modeled time course50 (Figure 3A) that shows the dynamics of initial thrombin formation and the activation of platelets, FV, and FVIII. These processes involve thrombin cleavage of protease-activated receptors 1 and 4,36,51 which activate platelets, and cleavage of the procofactors FV and FVIII, which release activation peptides and provide the cofactors, ie, FVa and FVIIIa. This interval of the reaction is driven by the tiny amounts (10−17 to 10−14 mol/L) of thrombin (Figure 3A) produced by FXa during the initiation phase. The activated platelets provide specific (and presently uncharacterized) receptor sites for the FVIIIa-FIXa and FVa-FXa complexes. The overall time course for presentation of the procoagulant catalysts is presented in Figure 3B. Enhanced activation occurs because of accelerated thrombin formation by prothrombinase, which is >105 times more active than unbound FXa, with amplification of FXa generation via FVIIIa-FIXa that results in accelerated thrombin formation. Furthermore, when bound in complex with FVIIIa and FVa, FIXa and FXa are not accessible to antithrombin.Download figureDownload PowerPointFigure 3. A, Numeric simulations of the earliest events leading to catalyst formation with a tissue factor insult. The scale is logarithmic and illustrates the initial activation over time of factor (F) V, FVIII, and platelets by the thrombin initially produced (by FXa) in the reaction. B, The generation of thrombin, FVa, and FVIIIa and platelet activation over the entire course of the reaction. The thrombin-catalyzed feedback amplifications that resulted in enhanced platelets and FVIII activation associated with the generation of increased amounts of thrombin by the prothrombinase complex are shown in this numeric simulation.In both whole blood and plasma, fibrinogen clotting43,52 occurs at the intersection between the initiation and propagation phases, when ≤5% of the prothrombin is converted to thrombin. Thus, clot-based assays, ie, the activated clotting time, the prothrombin time, and the partial thromboplastin time, exclude 95% of the reaction.Most clotting analyses are conducted in a closed system in which the reactants present at the beginning of the reaction are largely consumed in the resulting procoagulant and anticoagulant processes. In contrast, during hemorrhage, flow continues until a coagulant platelet-fibrin "dam" is established. Insights into the continuity of the process with flow have been provided by experiments in which new supplies of blood were made available to an apparently quiescent TF-initiated reaction after no additional thrombin-antithrombin was accumulating, ie, in resupply experiments.53 The addition of a new blood supply/reactants to a quiescent thrombus results in rapid and more extensive thrombin generation. This overresponse is a consequence of the platelet-bound procoagulant complex enzymes resident in the original thrombus generating more products from the substrates present in the newly added blood. In hemophilia, the absence of intrinsic factor Xase (FVIIIa-FIXa) prevents this accelerated FXa generation, and lesser amounts of thrombin are produced at a slower rate. As a consequence, an imperfect cross-linked fibrin clot is formed with inadequate strength to block blood flow.54,55A numeric simulation of consecutive resupplies of electronic blood is illustrated in Figure 4.30 In this case, total thrombin is plotted versus time for an experiment in which fresh aliquots of plasma are added after the initial TF reaction becomes quiescent with respect to active thrombin. Each new resupply produces a faster rise of thrombin activity to a higher level as a consequence of the increased catalyst concentrations generated. The data show that when provided with more substrate, the catalysts present in the quiescent thrombus will generate more catalysts that provide more intense thrombin generation, a process that will continue to expand until the leak is sealed. Notably, no additional TF is required, and the reaction is no longer dependent on the initial TF trigger that began the thrombin-generating process.53Download figureDownload PowerPointFigure 4. A numeric simulation of consecutive resupply of electronic plasma reactants to a quiescent thrombus at 790, 1200, and 1800 seconds, which results in a more intense thrombin generation and provides higher concentrations of prothrombinase (Xa=Va). Reprinted from Orfeo et al30 with permission of the publisher. Copyright © 2005, American Society for Biochemistry and Molecular BiologyThus, hemorrhage control can be described as a 2-compartment model for TF-initiated blood coagulation. Vascular perforation and blood extravascular flow lead to platelet adhesion, secretion, and aggregation. Plasma FVIIa binds extravascular TF, which leads to the generation of the procoagulant complexes and thrombin. In an open system, flow will continue to supply substrates to the reactive and growing thrombus until the vascular leak is plugged or the blood supply is exhausted. The reaction system is then deprived of additional substrates, and the proteases are ultimately neutralized by antithrombin. Any reactive enzyme components that escape downstream are inactivated by the excess of stoichiometric inhibitors and vascular thrombomodulin, which combines with surplus thrombin to activate PC to activated protein C, which inactivates FVa and FVIIIa.Bad Thrombin Generation: Venous ThrombosisVirchow postulated elements that are associated with the formation of occlusive venous clots, including hypercoagulable elements within the blood, endothelial injury or dysfunction, and alterations of blood flow.2 Clinical experience with venous thrombosis endorses the use of the anticoagulants heparin and warfarin, which serve to enhance the antithrombin neutralization process15 or impair the biosynthesis of the procoagulant zymogens.56 In general, platelet inhibitors are ineffective.Congenitally acquired plasma risk factors for venous thrombosis are FVLeiden and PC and protein S deficiencies.57–59 These deficiency states are not ordinarily associated with arterial events. Thus, the venous occlusive pathologies associated with these 3 congenital deficiencies clearly suggest that the impaired inhibition of thrombin production by elements associated with the thrombomodulin-EPCR-PC system is a contributor to venous thrombosis at the level of the vascular wall.19,20,22The endothelial cells that line the vascular wall are heterogeneous with respect to their display of anticoagulant and fibrinolytic properties.47,60–62 Unfortunately, insufficient quantitative data are available to define the concentrations of the vascular wall anticoagulants thrombomodulin, EPCR, heparan-sulfate proteoglycan, and TFPI, all of which are major regulators of the blood clotting pathway.It has been suggested that the endothelial cell surface area per unit of blood volume would permit a first-pass estimate of the anticoagulant concentrations provided by the vasculature.63 We have used this approach to estimate the effective thrombomodulin concentrations in different vessels. These data are shown in Figure 5A. Numeric simulations for the thrombin generation that would occur in a closed system (no flow) with these vessels with identical TF insults are presented in Figure 5B. The high concentrations of thrombomodulin present in arterioles and capillaries suggest that these vessels are protected from a TF insult even under relatively static conditions of blood flow unless impaired with respect to thrombomodulin presentation or other defects in the PC regulatory pathway. PC and protein S congenital deficiencies represent the extremes of such pathologies (lethality).58,59 The result is purpura fulminans, a consequence of systemic microvascular thrombosis. FVLeiden represents a better tolerated and more common venous thrombosis risk factor that is also an impairment in this system (but at the substrate level). Therefore, it is reasonable to speculate that among the constellation of candidate events that likely occur in venous thrombosis are impairments of the thrombin-PC-EPCR-thrombomodulin system. A reduction in anticoagulation potency coupled with low-flow or stasis and a blood-borne or vascular cell source of TF would result in an intravascular triggering of the coagulation mechanism.Download figureDownload PowerPointFigure 5. A, A hypothetical construct displaying the potential molar concentrations of thrombomodulin in various regions of the vasculature. An underlying assumption is that the thrombomodulin concentration of each endothelial cell is independent of the vascular source. B, Numeric simulation of thrombin generation versus time in vessels displaying the thrombomodulin concentrations estimated in Figure 5A after a tissue factor insult. Tm indicates thrombomodulin.Paradoxically, thrombin is not only a procoagulant, but also an anticoagulant. Prothrombin activation (Figure 6) involves 2 peptide bond cleavages, with the predominant reaction pathway proceeding through meizothrombin, which is subsequently cleaved to yield α-thrombin. The activity of meizothrombin is preferentially anticoagulant in character (Table65). In closed systems, meizothrombin is short-lived and rapidly converted to α-thrombin; however, under conditions of flow, significant amounts of meizothrombin accumulate. This significant fraction (≈50%) of thrombin formed at the vessel wall under conditions of flow is primarily anticoagulant in nature.66Download figureDownload PowerPointFigure 6. Pathways of prothrombin activation. The order of the 2 bond cleavages yield either the thrombin precursor prethrombin-2 or the enzyme, meizothrombin, as intermediates. Reprinted from Krishnaswamy et al64 with permission of the publisher. Copyright © 1987, the American Society for Biochemistry and Molecular Biology.Table. Meizothrombin ActivitySubstrateRelative Activity (% α-IIa)*Fibrinogen765Platelets265Factor V4134Thrombomodulin plus protein C9365Thrombomodulin plus TAFI1065Antithrombin2565TAFI indicates thrombin-activatable fibrinolysis inhibitor.*Based on comparison between r-wt-α-IIa and r(R155A, R271A, R284A)mIIa.Virchow postulated a circulating source of hypercoagulability that may be attributed to a source of TF within the blood. Potential mobile targets include inflammatory blood cells that express TF on their surfaces or cell-derived microparticles that bear TF.67–70 In addition to blood-borne procoagulants, the endothelium itself may contribute. The unperturbed endothelial cell in vitro does not appear to express TF at concentration levels that would likely provoke a coagulant response67,71,72; however, with inflammatory cytokine (tumor necrosis factor-α) stimulation, a variety of observations suggest that relevant levels of TF may be expressed by endothelial cells in vitro.73 Conversely, there is no question that the leukocyte populations in blood, in particular the monocyte, when subjected to inflammatory cytokines, translocate internally stored, highly procoagulant TF to their surface. These cells are likely candidates for Virchow's mobile source of systemic hypercoagulability. Similarly, cell-derived microparticles in circulation may provide a mobile source of TF that would produce a venous clot under conditions of low flow and impaired endothelium.74–77 A TF trigger with low flow and impaired local regulation would produce explosive thrombin generation and occlusive clot formation within the vessel.Ugly Clots: Arterial OcclusionIn contrast to the 3 centuries of experience with clotting in hemorrhage control and 2 centuries of experience with venous thrombosis, clotting in the arterial circuit has been investigated intensively only in more recent times. Arterial clots differ from venous clots in a number of respects. They are highly platelet dependent (white clots), occur in vessels of high shear, and occur in response to a local intravascular presentation of TF associated with rupture of an atherosclerotic lesion.6,78 Clinical anticoagulation in venous and arterial environments is routinely provided by warfarin anticoagulation. Although systemic antiplatelet agents are largely ineffective in venous thrombosis, they are, in contrast, highly effective for arterial thrombosis. Prophylaxis with systemic antiplatelet therapy has proven quite effective.79,80 The basis for the higher quantities of accumulated platelets in arterial thrombi is most likely a consequence of the superposition of a high shear environment, with other aspects governing thrombin generation.Under conditions of flow, thrombin generation from prothrombinase is highly shear dependent.81–83 When prothrombin is activated under conditions of flow with a vessel wall–bound prothrombinase catalyst, the substrate, prothrombin, arrives at the wall-bound prothrombinase catalyst primarily by diffusion only from the solvent in close proximity to the vessel wall, and the reaction product is limited to the fluid volume immediately adjacent to the vessel wall. As the flow rate is increased, new solvent entering the vessel segment dilutes the thrombin that is produced at the vessel wall. In blood, that thrombin would be surrounded by high concentrations of antithrombin and blocked from function. As a consequence, as flow rates increase, decreasing concentrations of active thrombin are found in regions proceeding from the vessel wall into the vessel lumen downstream from the point of its generation.From a biomechanical standpoint, thrombin generation by immobilized prothrombinase under conditions of flow is largely under dilutional control. As flow rates increase, thrombin produced in a local region of a vessel is largely washed out by the incoming blood supply, which leads to greatly reduced thrombin concentrations in regions extending from the vessel wall site of its production. These boundary-layer concentrations can be predicted by computational fluid dynamics model data for concentrations downstream from the point of formation (Figure 7). At venous flow rates (100·s−1), significant thrombin concentrations extend throughout the vessel. In contrast, at arterial flow rates, because of dilution effects by the incoming stream of fluid over the reaction site, thrombin is diluted rapidly, and high concentrations are only generated in very close proximity to the vessel wall. As a consequence, abnormalities that reduce flow serve to effectively reduce vessel aperture to dimensions essential to generate clot-effective thrombin concentrations in larger vessels at arterial shear rates. Flow restrictions by the vascular architecture and alterations associated with plaque and platelet accumulation are thus essential components that alter flow parameters to permit a concentration of thrombin to be produced throughout the vessel to achieve a stable platelet-fibrin clot.Download figureDownload PowerPointFigure 7. Computational flow dynamic model of thrombin concentrations extending from the wall site of formation at venous (100 s−1) and arterial (1000 s−1) shear rates. Figured compiled from data from Haynes et al.66Platelets are essential components for hemorrhage control, as evidenced by the sensitivity of the bleeding time to their function and number.84 In vitro, in static blood, clotting becomes sensitive to platelets at concentrations below 10 000/μL, whereas effective thrombin generation during the propagation phase requires platelet counts approaching 200 000/μL.85 Impairment of platelet function with the platelet IIb/IIIa antagonists eptifibatide and abciximab and with aspirin has a significant influence on the rate of thrombin generation during the propagation phase of the reaction in blood, and these drugs are recognized antiocclusive pharmaceuticals86–90 in the arterial circuit.Platelet function is required to provide the binding sites on which coagulation complexes are formed,41,91 but in arterial flow, platelets also provide the anchor for clot location and probably contribute to flow impairment that permits extension of the clot into the lumen to produce an occlusion of the vessel. In whole blood activated with TF, the initial platelet-fibrin clot forms at thrombin concentrations of approximately 2 nmol/L in a closed (no-flow) system. Thus, 2 nmol/L thrombin appears to be a reasonable concentration that must be developed locally in a flowing system to produce a fibrin clot.If we use 2 nmol/L thrombin as the platelet-fibrin clot threshold,92 we can estimate the vertical height intruding into the vessel lumen that a clot might extend from the vessel wall as a function of shear (Figure 8). This analysis suggests that at (healthy) arterial flow rates, clot penetration into the vessel lumen would be significantly reduced by flow dilution and inhibition as the shear rate increased. Vessel diameter thus would also play a role. Therefore, on the basis of fluid mechanics and geometry, at high shear rates, a vascular obstruction or irregularity must be present to permit development of a
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