Journal of Thrombosis and Haemostasis, 1: 1504–1514
`
`R E V I E W A R T I C L E
`
`What is all that thrombin for?
`
`K . G . M A N N , K . B R U M M E L and S . B U T E N A S
`Department of Biochemistry, University of Vermont, College of Medicine, Burlington, VT, USA
`
`To cite this article: Mann KG, Brummel K, Butenas S. What is all that thrombin for? J Thromb Haemost 2003; 1: 1504–14.
`
`Summary. The hemostatic process initiated by the exposure of
`tissue factor to blood is a threshold limited reaction which
`occurs in two distinct phases. During an initiationphase, small
`amounts of factor (F)Xa, FIXa and thrombin are generated. The
`latter activates the procofactors FV and FVIII to the activated
`cofactors which together with their companion serine proteases
`form the intrinsic FX activator (FVIIIa-FIXa) and prothrombi-
`nase (FVa-FXa) which generate the bulk of FXa and thrombin
`during a propagation phase. The clotting process (fibrin for-
`mation) occurs at the inception of the propagation phase when
`only 5-10 nM thrombin has been produced. Consequently, the
`vast majority (greater than 95%) of thrombin is produced after
`clotting during the propagation phase of thrombin generation.
`The blood of individuals with either hemophilia A or hemo-
`philia B has no ability to generate the intrinsic FXase, and hence
`is unable to support the propagation phase of the reaction. Since
`clot based assays conclude before the propagation phase they
`are not sensitive to hemophilia A and B. The inception and
`magnitude of the propagation phase of thrombin generation is
`influenced by genetic polymorphisms associated with throm-
`botic and hemorrhagic disease, by the natural abundance of pro-
`and anticoagulants in healthy individuals and by pharmacologic
`interventions which influence thrombotic pathology. Therefore,
`it is our suspicion that the performance of the entire process of
`thrombin generation from initiation through propagation and
`termination phases of the reaction are relevant with respect to
`both hemorrhagic and thrombotic pathology.
`
`Keywords: fibrin, hemostasis, thrombosis, thrombin, tissue
`factor.
`
`Introduction
`
`Blood loss through lack of hemorrhage control has captured the
`attention of individuals from all walks of life and throughout
`history. The significance of hemorrhage is discussed in the
`
`Correspondence: Kenneth G. Mann, Department of Biochemistry, 89
`Beaumont Avenue Given Building, Room C401, University of Vermont,
`College of Medicine, Burlington, Vermont 05405, USA.
`Tel.: þ1 802 656 0335; fax: þ1 802 862 8229; e-mail: kmann@zoo.
`uvm.edu
`
`earliest known literature [1]. In the mid-19th century the
`clotting of fibrinogen by thrombin was accurately described
`by Schmitt [2], and since that time hemostasis, defined by
`Stedman’s Dictionary [3] as the control of hemorrhage, has
`been synonymous with blood clotting.
`The pursuit of knowledge of natural events can be divided
`into the categories of inventory, connectivity and dynamics. The
`development of plasma clot-based assays in vitro, in which the
`observation of fibrin strands is the endpoint has played a central
`role both in describing the inventory and in establishing the
`logical connectivity between the reactants and reactions rele-
`vant to the hemostatic process. Morowitz [4] formalized the
`relationship between the introduction of tissue components into
`plasma and the formation of a clot. This reaction was exploited
`quantitatively by Quick [5] in the development of the pro-
`thrombin time assay. In its current state, this series of reactions
`(represented in Fig. 1) includes additional components now
`known to be required to produce the fibrin endpoint effectively.
`Langdell and colleagues [6] subsequently explored the sponta-
`neous clotting of recalcified citrate plasma and developed the
`partial thromboplastin time, thus expanding the inventory cat-
`alog and further defining the connectivity of the intrinsic
`pathway of coagulation [Fig. 1]. The connectivity of this ex-
`panded system was defined by the cascade/waterfall paradigms
`of MacFarlane [7], and Davie and Ratnoff [8]. Intersections
`connecting these pathways were extended by the work of
`O¨ sterud and Rapaport [9], and Galiani and Broze [10], who
`identified transactions that explicitly linked the two classical
`pathways leading to thrombin generation and a fibrin clot. The
`apparent absence of kinetic efficiency in the tissue factor (TF)
`activation of factor (F) VIIa was solved by Lawson et al. [11],
`who showed that FXa–membrane could cooperate in the FVIIa–
`TF activation of FIX.
`The title selected by the organizers for this presentation is
`based upon activities championed by our laboratory and Hem-
`ker’s, which have explored the dynamics of the total generation
`of thrombin and the significance of this process in maintaining
`hemorrhagic and antihemorrhagic qualities from the physiolo-
`gic and pathologic perspectives [12,13]. In vitro, blood and
`plasma clot when only a tiny fraction of prothrombin is con-
`verted to thrombin, and a great deal of hemostatic and throm-
`botic physiology and pathology is not captured by the fibrin
`clotting endpoints used commonly to evaluate the hemostatic
`
`# 2003 International Society on Thrombosis and Haemostasis
`
`MYLAN - EXHIBIT 1045
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`What is all that thrombin for? 1505
`
`Fig. 1. Inventory and connectivity of the reaction
`complexes that lead to the generation of
`thrombin. Each complex catalyst is illustrated
`with its participating serine protease and cofactor
`protein. For the vitamin K-dependent catalysts,
`an anionic phospholipid-like membrane is
`required for assembly and expression of activity.
`Various thrombin substrates are illustrated as
`sidebars to the complexes.
`
`process. In short, it appears that hemostasis is not synonymous
`with the endpoint of the fibrin-clotting reaction, and the latter is
`not a sufficient descriptor of the pathology associated with
`errors in the hemostatic process or the potential for development
`of a thrombotic occlusion.
`We begin this review with a disclaimer with respect to the
`title of this manuscript: we do not know ‘what all the thrombin
`is for’, but we do believe that the hemostatic process cannot be
`adequately evaluated by a simple clotting endpoint. In this
`review, we provide: (i) a description of the dynamics of the
`TF pathway to thrombin from a mechanistic standpoint; (ii) a
`collection of our experiences dealing in thrombin generation in
`hemorrhagic and thrombotic situations; and (iii)
`some
`potential approaches to the anticipation of hemostatic and
`thrombotic responses by using an integrated approach to labora-
`tory analysis.
`
`The central importance of thrombin
`
`Thrombin is a multifaceted protein with functions extending
`from coagulation activator and inhibitor to cellular regulator. Its
`central importance in biology, physiology, and pathology is
`underscored by studies conducted in genetically homogenous
`transgenic mice made deficient in components of TF pathway
`that are essential to the thrombin generation process and its
`regulation: TF [14], FVII [15], TF pathway inhibitor (TFPI)
`[16], FX [17], FV [18], prothrombin [19], and protein C (PC)
`/
`mouse constructs have been reported. In the mouse
`[20]
`populations studied, deletion of these key components are
`lethal, indicating that when evaluated in a homogenous genetic
`background, thrombin formation/regulation is essential to life.
`
`# 2003 International Society on Thrombosis and Haemostasis
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`/
`
`/
`
`[22] mice incur hemorrhagic risk. In
`[21] and FIX
`FVIII
`contrast, in the outbred human population, total deficiencies in
`coagulation components that are lethal in mice may yield
`consequences ranging from mild to extreme pathology. We
`hypothesize this is a consequence of human genetic hetero-
`geneity that provides alternative pathways that can achieve
`survival in spite of potentially lethal abnormalities. In this
`hypothesis we assume that the majority of embryos congenitally
`deficient in the ‘essential’ components of the TF pathway do not
`survive.
`Plasma clot-based assays have been the endpoints for the
`most commonly used tests of the hemostatic process. The
`prothrombin time (PT) and activated partial thromboplastin
`time (APTT) reflect, however, only the contributions of the
`diluted plasma component of the hemostatic system in adult-
`erated form. Both tests terminate with endpoints that occur with
`less than 5% of the reaction complete. Fibrin clotting thus
`occurs when only minimal levels of prothrombin have been
`activated. It is also somewhat paradoxical that while we use the
`terms blood coagulation and hemostasis somewhat interchange-
`ably, fibrinogen deficiency in mouse [23] is frequently only
`mildly symptomatic, in marked contrast to those deficiencies
`that negate thrombin formation. While clotting tests have
`been especially useful in identifying congenital abnormalities
`associated with hemophilia A, B and C, and in the evaluation of
`oral anticoagulant therapy, they overlook most thrombin for-
`mation, and their utility in evaluating many anticoagulant
`therapies [24] has been somewhat limited. In addition, prolon-
`gation of the endpoint in the APTT may reflect genetic defi-
`ciencies with little consequence for hemorrhagic pathology in
`the host.
`
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`1506 K. G. Mann et al
`
`The tissue factor pathway
`
`Most investigators believe that the generation of thrombin via
`the TF pathway is the biologically relevant process by which
`thrombin is elaborated and hemostasis is achieved. The reaction
`begins with the expression/exposure of TF, which is maintained
`in an inactive form either by compartmentalization or by some
`regulatory process that permits expression of this membrane-
`bound receptor protein [25–29]. The function of TF is expressed
`by binding pre-existent plasma FVIIa, which is present at
`approximately 1–2% of the total FVII zymogen concentration
`[30]. FVII competes with FVIIa for TF binding, thus serving as
`a negative regulator in the overall reaction [31]. When bound to
`TF, FVIIa function is efficiently expressed toward its macro-
`molecular substrates, FIX and FX [9,32–34], with the latter
`being the more efficient and abundant substrate. The resultant
`initial FXa with a fraction of active membranes converts
`prothrombin to thrombin, albeit inefficiently. FXa also con-
`tributes to factor IX activation by cleaving the zymogen to the
`intermediate FIX [11]. The small amounts of thrombin pro-
`duced by FXa–membrane initiates platelet activation [35], and
`activates minute amounts of plasma FV and FVIII to the
`cofactor forms FVa and FVIIIa [36]. These two cofactors
`ultimately form the receptor sites, both locating and activating
`FXa and FIXa, and forming the intrinsic FXase and prothrom-
`binase complexes on the activated platelet surface [37].
`Each membrane-bound,
`vitamin K-dependent
`enzyme
`complex [Fig. 1] is 104106 more active than the respective
`proteases towards their macromolecular substrates in solution
`[38].
`In addition to the feedback activation by thrombin to activate
`FV and FVIII, other feedback steps accentuate the overall
`process of catalyst generation. FXa–membrane can also
`activate FV and FVIII to their respective cofactors [39],
`although the biological
`relevance of
`these processes is
`suspect considering the small amounts of FXa available and
`competitive substrates. Thrombin also activates FXI to FXIa
`[10], initiating an accessory pathway that enhances FIX activa-
`tion [40].
`The hemostatic reaction is under the control of both stoichio-
`metric and dynamic inhibitory systems. TFPI is the principal
`inhibitor of the extrinsic FX activator [41,42]. TFPI is a high-
`affinity, low-abundance inhibitor present in plasma and secreted
`by vascular cells contributing to the local anticoagulant envir-
`onment of the vascular wall. The major stoichiometric inhibitor
`of thrombin and its generation is antithrombin III [45]. FXa and
`IXa in complex display decreased reactivity with antithrombin
`III [43]. In contrast, the FVIIa–TF complex shows increased
`reactivity compared with FVIIa in plasma [44,45]. In plasma,
`FVIIa is almost impervious to inhibition by this ubiquitous
`serpin, and this lack of reactivity permits the existence of FVIIa
`in the hostile, inhibitor-rich blood environment. Antithrombin
`1) of
`III is present at over twice the concentration (3.2 mmol L
`the highest potential procoagulant enzyme concentration
`1). This serpin effectively neutralizes
`(thrombin) (1.4 mmol L
`all the serine proteases associated with the hemostatic process.
`
`Other serpins, including heparin cofactor II, may also contribute
`inhibitory capacity, suppressing the procoagulant proteases
`[46,47].
`In addition to being an effective feedback procoagulant
`activator, thrombin is also an effective feedback anticoagulant.
`The enzyme binds to vascular cell-associated thrombomodulin
`[48]. The complexed thrombin effectively recognizes the vita-
`min K-dependent zymogen PC and ceases to be an activator for
`the procofactors and fibrinogen. The activated PC (APC) pro-
`duct interferes with the coagulation system by binding FVIIIa
`and FVa in competition with FXa and FIXa and proteolyzing the
`two cofactors, leading to their destruction [49,50]. The inacti-
`vated membrane-bound factor FVai and FVIIIai suppress APC
`function by continuing to effectively bind the enzyme, thus
`serving as product inhibitors.
`The overall expression of thrombin function is a tightly
`regulated, highly intercalated system, the performance of which
`cannot be anticipated with the simple ‘guesstimate’ analyses
`that investigators typically apply to simple reactant systems. It
`is the composite of qualitative and quantitative features of the
`overall system that dictates the ultimate process, including the
`rates, extents of formation, and durabilities of catalysts and
`thrombin [51]. In addition, many products occur transiently
`during the course of the reaction, leading to increased complex-
`ity. Even alterations of plasma-protein concentrations in the
`‘normal range’ observed in the human populations can have an
`extraordinary effect on the ultimate amount of procoagulant
`activity generated for a given level of TF stimulation of the
`system [52]. Even dilution of blood or plasma produces sig-
`nificant and often unexpected changes. ‘Seat of the pants’
`speculation of how these reactions will be altered by changes
`in reactant concentrations, solvent conditions or temperature
`are inherently dangerous and have contributed to misinterpreta-
`tions of the biological processes.
`
`Models of hemostasis
`
`In attempting to examine how the TF pathway works in gen-
`erating thrombin, our laboratory has evaluated four models to
`try to describe the dynamics of the thrombin generating system.
`These are: (i) synthetic plasma mixtures prepared using purified
`proteins and natural or synthetic membranes induced to react by
`the addition of lipid-reconstituted TF [52,53]; (ii) numerical
`models of the coagulation system based upon reaction-rate
`constants, concentrations and mechanisms [51,54,55]; (iii) para
`vivo studies involving whole blood induced to clot purely by a
`TF stimulus [11,56,57]; and (iv) in vivo studies in blood exuding
`from a microvascular wound [58–60].
`Each model has its advantages and disadvantages. Models
`that involve human volunteers are constrained by both ethical
`and
`technical
`considerations. Synthetic
`and
`computer
`models are used to anticipate knowledge of the true biology
`of the process and aid in design of both para vivo and in
`vivo experiments. When the models converge with in vivo
`observations,
`the appropriateness of the approximations is
`assured.
`
`# 2003 International Society on Thrombosis and Haemostasis
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`

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`What is all that thrombin for? 1507
`
`Fig. 2. Thrombin generation during para vivo whole-blood experiments.
`Thrombin–antithrombin III (TAT) complex formation is plotted as a
`function of time for sequential samples of human blood at 37 8C inhibited
`with respect to contact pathway activation and activated by the addition of
`1 phospholipid. The data ( SEM) are
`1 tissue factor, 10 pmol L
`5 pmol L
`presented for 35 individual experiments with an average clot time of
`4.7 0.2 min. The operationally defined initiation and propagation phases
`correspond to the slow and fast kinetic expressions of thrombin
`formation. The propagation phase is invisible to clot-based assays. From
`Brummel et al. [58], by permission.
`
`Thrombin generation
`
`In all models, the TF-initiated display of thrombin generation is
`approximately the same. This behavior, illustrated in Fig. 2,
`may be operationally described as occurring in two phases. At
`first, tiny (nanomolar) amounts of thrombin are produced during
`an interval (the initiation phase). The major bolus (>96%) of
`thrombin is produced secondarily during a propagation phase.
`During the initiation phase, the FVIIa–TF complex forms, and
`generates sub-picomolar amounts of FXa and FIXa. FXa, in
`collaboration with the membrane surface, activates a small
`amount of prothrombin to thrombin, which serves to generate
`the platelet membrane and cofactor components required for the
`major generation of thrombin. These autocatalytic processes
`lead to increased catalyst formation.
`Signal events occurring during the initiation phase are illu-
`strated in Fig. 3, which shows the inception points for the
`detection of thrombin products generated during the reaction
`measured in para vivo experiments [57]. These products pro-
`vide the elements of the catalysts (Fig. 1) that generate the
`majority of the thrombin produced during the propagation phase
`of the reaction.
`The cleavage of fibrinopeptide (FP) A and subsequent clot
`formation (Figs. 2 and 3) occur just prior to the propagation
`phase of the reaction. Under normal conditions, the activation of
`platelets and FV occurs rapidly to produce a surplus of FVa and
`platelet-membrane binding sites, leaving the rate-limiting re-
`agent for prothrombinase formation as the concentration of
`FXa. However, with congenital deficiencies, thrombocytopenia,
`platelet pathology or pharmacologic interventions, the reaction
`can become sensitive to FV or platelets [61].
`The endpoint utilized in evaluating hemostasis in most
`bioassays is the generation of a fibrin clot. As illustrated in
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`# 2003 International Society on Thrombosis and Haemostasis
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`Fig. 3. The onset of detection of products from thrombin substrates during
`the initiation phase for the experiments presented in Fig. 2. Active thrombin
`and thrombin–antithrombin III (TAT) concentrations are plotted on the
`vertical axis on an exponential scale vs. time. The onset of product detection
`corresponding to platelet activation (OSN) and other well-established
`thrombin substrates are depicted. The inception of propagation phase
`corresponds to the point at which there is a transition from slow to rapid
`thrombin generation. From Brummel et al. [58], by permission.
`
`Figs 2 and 3, in largely unadulterated whole blood at 37 8C, the
`1
`formation of a visible fibrin clot occurs at 10–30 nmol L
`thrombin, or 3–5% of the total amount of thrombin produced.
`This thrombin in turn is provided by only 7 pmol L
`1 pro-
`thrombinase [12,57]. Thus, most catalyst and thrombin forma-
`tion is undetected by current technology for evaluating clinical
`hemorrhagic risk or thrombosis.
`Figure 4 illustrates the time course of removal of fibrinogen
`and fibrin products from the fluid phase of blood and the
`formation of products within the insoluble clot. This figure
`should be compared with the data of Figs 2 and 3 to register the
`formation of thrombin with the cleavage of fibrinogen and the
`formation of the fibrin clot. In Fig. 4A, at the point of visual clot
`formation (CT), virtually all fibrinogen (and some product
`already crosslinked) disappears from the fluid phase of the
`reaction [62]. At this point 50% of the FPA has been cleaved,
`thus the ‘clot’ is a mixture composed of a mixture of fibrin 1 and
`fibrinogen. The insoluble material present in the fibrin clot
`(panel B) is virtually all cross-linked by FXIIIa, the activation
`of which is nearly simultaneous with FPA removal (Fig. 3). In
`purified systems it has been observed that FPB removal pre-
`cedes the cross-linking reaction, however, as seen in the a-FPB
`immunoblot in Fig. 4C, the B peptide antigen epitope is still
`detectable associated with the Bb chain.
`
`The significance of intrinsic factor Xase
`
`During the transition between the initiation and propagation
`phases, increased concentrations of the FVIIIa–FIXa complex
`are generated, contributing an increasing concentration of FXa.
`The TFPI downregulation of FXa formation by the FVIIa–TF
`
`

`

`1508 K. G. Mann et al
`
`Fig. 4. Fibrinogen and fibrin products during the tissue factor-induced clotting of whole blood. Panel A reflects the immunoblot data for sequential aliquots
`1 tissue factor–phospholipid. The label ‘CT’
`taken from the solution phase of contact pathway inhibited whole blood induced to clot with 5 pmol L
`corresponds to the clotting time observed visually for the experiment. Panel B illustrates a Coomassie Blue-stained gel of the reduced insoluble clotted
`material for the same experiment in Panel A. Panel C illustrates an immunoblot of the gel of panel B using an antibody (supplied by B. Kudryk) specific
`for the B peptide region of the Bb chain. From Brummel et al. [63], by permission.
`
`complex and the enhanced efficiency (50-fold) of the FVIIIa–
`FIXa complex effectively switch the primary path of FXa
`production to the latter catalyst. Figure 5 illustrates a numerical
`analysis of the percentage formation of FXa by the two com-
`plexes during the progress of the reaction. Shortly before the
`propagation phase of thrombin generation is observed, the
`majority of FXa begins to be produced by the intrinsic FXase.
`Operationally, the onset of the propagation phase that signals
`enhanced thrombin generation is coincident with the intrinsic
`FXase being the principal generator of FXa.
`From these observations one would conclude that in con-
`genital hemophilia A and B a significant deficit in thrombin
`generation during the propagation phase would occur. This is in
`fact observed in all models, most significantly in para vivo
`studies of individuals with hemophilia A and B [Fig. 6]. The
`blood of these individuals displays a slight prolongation of the
`time to form a clot, but the major impairment is in thrombin
`generation during the propagation phase of the reaction. The
`deficit in hemophilia C, or FXI deficiency, is also observable as
`
`Fig. 5. A numerical estimation of the percentages of FXa produced by the
`intrinsic FXase and by the extrinsic FXase. Initially 100% of FXa is
`generated by FVIIa–tissue factor. However, as the reaction progresses, the
`major FXa production is contributed by the more efficient FIXa–FVIIIa
`catalyst. The arrow indicated the approximate clotting time. Modified from
`Hockin et al. [52], by permission.
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`# 2003 International Society on Thrombosis and Haemostasis
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`What is all that thrombin for? 1509
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`yield robust and almost equivalent thrombin generation. In a
`similar fashion, TFPI and the dynamic PC–thrombomodulin–
`thrombin system cooperate to provide a threshold-limited
`synergistic inhibition of thrombin production [63]. In this
`instance, TFPI slows the initiation phase while the APC system
`interferes with the activation of the cofactors FV and FVIII.
`The activations of
`the cofactors FV and FVIII are
`multistep processes, and at high thrombomodulin concentra-
`tions the PC system is an effective neutralizer of
`the
`reaction. FV activation involves cleavages at arginines 709,
`1018 and 1545 [64]. The cleavage at Arg709 occurs first and
`produces the heavy chain (residues 1–709) of the molecule. FVa
`activity, however, requires cleavage at Arg1545 to produce
`the light chain (residues 1546–2329) of FVa. APC inactivates
`FVa (and intermediates in the activation process) principally
`by cleavage at Arg506 and Arg306. Each of these cleavages is
`in the heavy chain [50]. Thus,
`the heavy chain can be
`inactivated prior to generation of the light chain of FVa,
`eliminating its procoagulant receptor ‘activity’ prior to its
`genesis.
`The effectiveness of the synergy between TFPI and AT-III
`can be observed in the empiric experiments described in Fig. 7.
`In these experiments, performed with a synthetic plasma sys-
`tem, the reaction mixture is initiated with varying concentra-
`tions of FVIIa–TF. At the two highest concentrations, using
`1 and 25 pmol L
`1, thrombin generation is almost
`125 pmol L
`equivalent. When the activator concentration is reduced to
`1 a virtual shutdown of the reaction system occurs.
`10 pmol L
`Similar observations have been made with a combination of
`TFPI and APC.
`The consequence of the integration of the effects of procoa-
`gulant and anticoagulant systems governing thrombin genera-
`tion is the establishment of an effectively ‘digital’ system that is
`either off or on. Once a threshold has been reached, the response
`of the process is only dictated by the pro- and anti-coagulant
`reagent concentrations present
`in a reaction volume. This
`produces a teleologically desirable expectation for the hemo-
`static system, i.e. it responds effectively and rapidly to a threat
`dealing with hemorrhage but is not provoked to activation by
`inconsequential stimuli.
`
`The role of FXI
`
`The significance of FXI as an important procoagulant is estab-
`lished by the bleeding pathology frequently associated with its
`qualitative or quantitative absence [65]. This zymogen is also a
`substrate for thrombin and has been invoked in the ‘revised
`pathway of coagulation’ [10]. In studies of natural hemophilia
`C blood, antibody-acquired hemophilia C, or
`synthetic
`plasma FXI deficiency, the significance of FXI deficiency is
`only prominent at the lowest TF concentrations [56]. At mod-
`1), congenital
`erate concentrations of TF (5–10 pmol L
`FXI deficiency has little or no effect on thrombin generation
`or other procoagulant parameters. However, at low levels of
`1), which produces clotting in the range
`TF (1–2 pmol L
`of 12–15 min, the generation of thrombin and formation of
`
`Fig. 6. Thrombin generation represented as thrombin–antithrombin (TAT)
`complex during tissue factor-induced whole-blood reactions. Illustrated are
`data for normal individuals, and data for two individuals with hemophilia A.
`The clotting times for the composite and two individual experiments are
`illustrated on the horizontal axis. Modified from Cawthern et al. [57], by
`permission.
`
`a defect in the propagation phase generation of thrombin;
`however, this observation can only be made when the reaction
`is initiated by extremely low concentrations of TF with clotting
`times >10 min [56].
`The clinical diagnoses of hemophilia A, B and C involve
`functional analyses of plasma clotting using APTT, which is
`most likely a consequence of an in vitro artefact. While it is well
`established that these congenital deficiency diseases are asso-
`ciated with hemorrhagic disease, these deficiencies are not
`reflected in the TF-initiated PT assay. Since we conclude that
`the TF pathway is relevant to the hemostatic mechanism a
`rationale for the hemophilia insensitivity of PT requires some
`explanation. A typical PT assay employs  20 nmol L
`1 TF,
`which will produce a clot in 11–15 s. Since the presentation of a
`1 throm-
`clot depends only on the generation of 10–30 nmol L
`bin, at the high TF concentrations used, robust generation of the
`1 FXa by FVIIa–TF completely masks the
`required 7 pmol L
`1FIXa complex in clot end-
`contribution of the FVIIIa nmol L
`point assays. For the illustrations of Figs 2–6, a concentration of
`1 TF was used, producing a clotting time of 4–
`5 pmol L
`5 min. At these TF concentrations, the clotting time in hemo-
`philia A and B is prolonged, but the major defect is associated
`with the absence of a propagation phase [54,56].
`
`Reaction thresholds
`
`The principal stoichiometric inhibitors of the process are TFPI
`and anti-thrombin III (AT-III). TFPI is the principal regulator of
`the initiation phase of thrombin generation, while AT-III
`serves to attenuate thrombin activity and its generation. These
`two
`agents, when
`combined,
`provide
`a
`synergistic
`regulatory effect by inducing kinetic ‘thresholds’ such that
`the initiating TF stimulus must achieve a significant magnitude
`to propel thrombin generation [53]. TF concentrations below
`the threshold concentration are ineffective in promoting
`robust thrombin generation, because of the cooperative influ-
`ence of the inhibitors; concentrations in excess of the threshold
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`# 2003 International Society on Thrombosis and Haemostasis
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`

`1510 K. G. Mann et al
`
`Fig. 7. Procoagulant mixtures are induced to
`thrombin generation with the addition of
`FVIIa–tissue factor complexes at the
`concentrations listed. Reaction mixtures contain
`procoagulants and the stoichiometric
`anticoagulants. The total (AREG) of thrombin
`1 activator and
`produced at 125 pmol L
`1 activator are essentially the same.
`25 pmol L
`When the concentration of activators is reduced
`1 the reaction is largely attenuated,
`to 10 pmol L
`thus a reaction threshold exists between
`
`1 and 10 pmol L1 activator in this
`25 pmol L
`illustration. From van’t Veer et al. [54], by
`permission.
`
`fibrin are defective in FXI deficiency. The variability of
`pathology in FXI deficiency is perhaps a reflection of the
`dimension of the TF stimulus associated with the vascular
`lesion.
`
`The significance of thrombin generation
`
`The ‘initiation/propagation phase’ description of thrombin gen-
`eration is an operational concept that permits a segregated
`discussion of the heterogeneous kinetics of the thrombin gen-
`eration process. However,
`the two phases are intrinsically
`intertwined, and thrombin production during both phases is
`essential to the overall hemostatic process. Since the blood
`clotting process coincides with the transition between the
`initiation and propagation phases of reaction, most of our
`knowledge of hemorrhagic syndromes is presently confined
`to the associations of bleeding pathology with thrombin for-
`mation during the initiation phase of the reaction. However, it is
`clear that in well-established hemorrhagic syndromes only
`modest alterations of thrombin production occur during the
`initiation phase, while depression of thrombin production dur-
`ing the propagation phase is the hallmark of these congenital
`bleeding diseases.
`We have studied a variety of drug effects, pathologic
`syndromes and congenital polymorphisms using the model
`systems described in this presentation.
`In this
`section
`we describe the influences of these events on thrombin gen-
`eration.
`
`Platelets and thrombin generation
`
`An essential platelet function provides the binding sites for the
`vitamin K-dependent complexes the components of which are
`derived from plasma. We have studied the influence of throm-
`bocytopenia, thrombocytosis, and impaired platelet function by
`pharmacologic agents on the process of thrombin generation
`[61,66,67].
`
`While some platelet function is essential to all elements of
`thrombin generation in a TF-induced reaction, the principal
`impact of reduced platelet concentrations or pharmacologically
`impaired platelet function is on the propagation phase of the
`reaction. The initiation phase of the reaction, defined as the time
`1 thrombin or the clotting time of blood,
`to generate 10 nmol L
`becomes impaired when platelet concentrations fall below
`3.
`10 000 mm
`Because of the extreme sensitivity of platelets to thrombin,
`platelet activation is ordinarily not a rate-limiting step in the TF
`pathway. In fact, preactivation of platelets with the thrombin
`receptor activation peptide has no effect on thrombin generation
`either during initiation or propagation phases. However, the
`inhibition of platelet function by strong antagonists has pro-
`found effects both on the initiation and propagation phases of
`the reaction. Selective inhibitors, such as the clinically useful
`glycoprotein IIbIIIa antagonists Integrelin and Abciximab,
`suppress the propagation phase of the reaction. Perhaps this
`propagation phase inhibition contributes to the antithrombotic
`effects of these agents.
`
`Plasma protein quantitative and qualitative
`alterations
`
`The journal Blood uses the term ‘healthy’ to describe the
`properties occurring in an apparently normal population.
`Studies of severe congenital hemorrhagic and thrombotic
`disease are confined to the extremes of the ‘normal’ or
`‘healthy’ population distribution [Fig. 8]. However, significant
`concentration variations occur within the normal population.
`For many plasma proteins, variations in concentration extend
`over a range of 50–150% of the mean plasma value. These
`values are frequently used in clinical evaluations as indepen-
`dent variables.
`In studies in vitro, altering plasma protein concentrations
`within the nominal range of 50–150% can produce major
`influences on thrombin generation [52]. AT-III and prothrom