Initiation

The factor VIIa–TF–membrane complex (extrinsic factor Xase) ( Fig. 27-4, A ; also see Fig. 27-3 ) catalyzes the activation of both factor IX and factor X, the latter being the more efficient substrate. Thus the initial product formed is factor Xa. Feedback cleavage of factor IX by membrane-bound factor Xa enhances the rate of generation of factor IXa in a cooperative process with the factor VIIa–TF complex.

A, The factor Xa generated by the factor VIIa–tissue factor (TF) complex catalyzes the formation of a small amount of thrombin, which activates factors V and VIII and leads to the presentation of the intrinsic factor Xase (factor IXa-VIIIa) and prothrombinase (factor Xa-Va). At this point in the reaction, generation of factor IXa is cooperatively catalyzed by membrane-bound factor Xa and by factor VIIa–TF. The thick arrow representing generation of factor Xa by the intrinsic factor Xase illustrates the more efficient generation of factor Xa by this catalyst. B, The tissue factor pathway inhibitor (TFPI) interacts with the factor VIIa–TF–factor Xa product complex to block TF-initiated activation of both factor IX and X, thus leaving the factor IXaβ-VIIIa complex as the only viable catalyst for activation of factor X.

(Redrawn with permission from Mann KG: Dynamics of Hemostasis. Essex Junction, Vt., Haematologic Technologies, 2002.)

The initially formed, membrane-bound factor Xa activates small amounts of prothrombin to thrombin. This initial activation of prothrombin provides the thrombin essential for acceleration of the hemostatic process by serving as the activator for platelets and the procofactors factor V and factor VIII (see Fig. 27-1 ). Once factor VIIIa is formed, the factor IXa generated by the factor VIIa–TF complex combines with factor VIIIa on the activated platelet membrane to form the intrinsic factor Xase (see Figs. 27-3 and 27-4, A ), which becomes the major activator of factor X. The factor IXa–factor VIIIa complex is 10 ⁶ -fold more active as a factor X activator and 50 times more efficient than the factor VIIa–TF complex in catalyzing activation of factor X (thus the bulk of factor Xa is ultimately produced by the factor IXa–factor VIIIa complex).

Propagation

As the reaction progresses, generation of factor Xa by the more active intrinsic factor Xase exceeds that of the extrinsic factor Xase complex. In addition, the extrinsic factor Xase complex is subject to inhibition by TFPI ( Fig. 27-4, B ). As a consequence, most (>90%) of factor Xa is ultimately produced by the factor VIIIa–factor IXa complex. In hemophilia A and hemophilia B, this amplification of factor Xa generation does not occur. Factor Xa combines with factor Va on the activated platelet membrane receptors, and this factor Va–factor Xa prothrombinase catalyst (see Figs. 27-3 and 27-4 ) converts most prothrombin to thrombin. Prothrombinase is 300,000-fold more active than factor Xa alone in catalyzing activation of prothrombin.

Termination

The same hemostatic process required for preventing leaks from the vasculature may also be life threatening when responsible for intravascular occlusion. Thus equally important to the hemostatic process are the stoichiometric and dynamic inhibitory systems, which block the presentation of thrombin. The sum of inhibitory functions is far in excess of the potential coagulant response. These inhibitory processes governed by TFPI, AT, and the protein C pathway act in synergy to provide activation thresholds, for which a minimum level of TF stimulation must be achieved before significant thrombin generation can occur. The termination phase thus occurs throughout the reaction process.

Flow Regulation

Current data are consistent with the concept of a two-compartment model for regulation of the procoagulant response to a breach in a blood vessel ( Fig. 27-5 ). Regulation of clotting in the extravascular space, apart from providing the initiating TF, is primarily passive and controlled by the availability of fresh blood, whereas regulation in the intravascular compartment is dynamic. Mechanical disruption of a blood vessel results in the movement of blood to an environment where TF is exposed and platelets bind and are activated (see Fig. 27-5 , stage 1 ). These events localize the initiating procoagulant reactions to blood now outside the vasculature, with adhesion and activation of platelets in this extravascular blood providing sufficient receptors and surfaces to support the propagation phase of thrombin generation (see Fig. 27-5 , stage 2 ). This extravascular process rapidly becomes independent of TF as generation of thrombin becomes supplied by the intrinsic factor Xase and prothrombinase complexes. As long as new blood flows into this space, and fibrin formation and platelet aggregation have not achieved a sufficient barrier to stop flow, the system is open and the propagation phase of thrombin generation will continue. If blood ceases to move into the extravascular compartment, the system becomes closed, available prothrombin is consumed, and the complex enzymes are ultimately destroyed or sequestered into inactive complexes (see Fig. 27-5 , stage 3 ). However, the stability of the prothrombin–activating potential incorporated into the platelet-rich thrombus and its localization in the extravascular space ensure that if the barrier springs a leak, robust generation of thrombin will begin immediately on contact and continue until consequent platelet activation and fibrin deposition reestablish a secure barrier.

Schema of a two-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 four stages. TF–factor VIIa complex, TF-VIIa; prothrombinase complex, Xa-Va; intrinsic factor Xase, VIIIa-IXa; antithrombin (AT)–endothelial cell heparan sulfate (HS) proteoglycan complex bound to thrombin or factor Xa, HS-AT-(IIa or Xa); protein C (PC) bound to thrombomodulin-thrombin, TM-IIa-PC. Stage 1. 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, thereby initiating the process of activation of factors IX and X. Factor Xa activates small amounts of prothrombin to thrombin, which in turn activates more platelets and converts factors V and VIII to factors Va and VIIIa. Stage 2. The reaction is propagated by platelet-bound intrinsic factor Xase and prothrombinase, with the former being the principal generator of factor Xa. Initial clotting occurs and fibrin begins to fill in the void in cooperation with activated platelets. Stage 3. 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 consumption of reagent, which attenuates further generation of thrombin, but functional procoagulant enzyme complexes persist because they are protected from the dynamic inhibitory processes found on the intravascular face. Stage 4. View downstream of the perforation. Enzymes escaping from the plugged perforation are captured by AT-heparan complexes, and the protein C system is activated by binding of residual thrombin to endothelial cell thrombomodulin, thereby initiating the dynamic anticoagulant system. These intravascular processes work against occlusion of the vessel despite continuous resupply of reactants across the intravascular face of the thrombus.

(From Orfeo T, Butenas S, Brummel-Ziedens KE, Mann KG: The tissue factor requirement in blood coagulation. J Biol Chem 280:42887-42896, 2005.)

Three mechanisms act to limit clot growth into the intravascular space (see Fig. 27-5 , stage 4 ): thrombomodulin-dependent activation of protein C, the presence of abundant AT, and the actively nonthrombogenic and nonadhesive surface of the vascular endothelium. Thus thrombin generation with consequent ongoing fibrin deposition and platelet activation is suppressed at the extravascular-intravascular boundary by these processes. In contrast, complexes at the extravascular side will remain relatively protected from APC and AT by the forming barrier and are therefore ready to respond to any leakage of blood.

The general properties of proteins of the blood coagulation proteome are summarized in Table 27-1 . It has been stated that “a mouse is not a small furry human.” A corollary is that an infant is not a tiny adult. This is especially true for the average plasma proteome concentration values for the newborn. These data are presented in parentheses in Table 27-1 . Note that except for factor VIII and fibrinogen, most newborn functional protein concentrations are low relative to adult values (see Chapter 5 ).

Factor VII (Proconvertin, Convertin)

The single-chain factor VII zymogen is activated to its two-chain protease form by cleavage of a single peptide bond between Arg152 and Ile153 (see Fig. 27-6 ). The mechanism for the initial activation of this zymogen is unclear. The resulting protease consists of an NH ₂ -terminal light chain linked by a single disulfide bond to a COOH-terminal heavy chain containing the catalytic domain. Factor VIIa has very poor catalytic efficiency without the presence of its cofactor TF. Cleavage of factor VII to factor VIIa can be catalyzed by several proteases, including α-thrombin, factor IXa, factor Xa, factor XIIa, and factor VII–activating protease, as well as by autoactivation by factor VIIa. The active factor VIIa serine protease bound to TF forms the extrinsic factor Xase complex (see Fig. 27-3 ).

Factor VIIa, unlike other serine proteases of the coagulation cascade, is not readily inhibited by circulating protease inhibitors, including the AT-heparin complex, unless bound to TF. Thus regulation of TF expression is the primary means of controlling factor VIIa activity. Paradoxically, the zymogen factor VII (10 nM) is an effective competitor of factor VIIa (0.1 nM) for binding to TF. This competition downregulates the level of enzymatically active complex (factor VIIa–TF), thereby suppressing initiation of the clotting cascade. Once the extrinsic tenase complex activates factor X to factor Xa, TFPI can form a quaternary complex (factor VIIa–TF–factor Xa–TFPI) with no enzymatic activity (see Fig. 27-4 ). Thus once assembled and functioning, the extrinsic tenase, along with its substrate factor Xa, is a target for inactivation through the action of TFPI.

Factor IX (Plasma Thromboplastin Component, Christmas Factor, Hemophilia B Factor)

Factor IX is a single-chain zymogen consisting of 415 amino acids organized with a Gla domain (12 Gla residues), two tandem EGF domains, an activation peptide region, and a serine protease domain (see Fig. 27-6 ). Glycosylation in the form of O-linked and N-linked oligosaccharides makes up 17% of the factor IX protein by weight. Activation of factor IX is a two-stage process that requires cleavage at Arg145 and Arg180 and results in the release of a 35-residue activation peptide (see Fig. 27-6 ). The physiologic activators of factor IX are either TF–factor VIIa (extrinsic tenase complex) or factor XIa. The first cleavage at Arg145-Ala146 results in factor IXα. This cleavage has been shown to be important in its affinity for its cofactor factor VIIIa. The second cleavage occurs at Arg180-Val181 and results in factor IXαβ, the active form, also referred to as factor IX a. Both cleavages are required for the biologic activity of factor IXa. Plasma factor IXa is primarily inhibited by AT (see Fig. 27-2 ). In vitro experiments conducted on phospholipid vesicles and cell membranes showed that protease nexin-2/amyloid β-protein precursor (PN2/APP) can also inhibit factor IXa.

Factor X (Stuart Factor)

Factor X is synthesized in the liver and circulates as a two-chain molecule linked by a disulfide bond. The NH ₂ -terminal light chain consists of a Gla domain and two EGF-like domains (see Fig. 27-6 ). The COOH-terminal heavy chain consists of an activation peptide region and a catalytic (serine protease) domain. Factor X is activated by both the intrinsic and extrinsic factor Xases (see Figs. 27-1 and 27-3 ) by cleavage at Arg194-Ile195 (see Fig. 27-6 ). A 52–amino acid activation peptide is released. The resulting catalytic serine protease, factor Xa, is composed of a light chain and a disulfide-linked heavy chain. Factor X has to be bound to the membrane before activation. Factor Xa participates in forming the prothrombinase complex (see Fig. 27-4 ). The propagation phase of thrombin generation is catalyzed principally by factor Xa generated via the intrinsic tenase complex (see Fig. 27-3 ), which activates factor X at a 50- to 100-fold higher rate than the extrinsic tenase complex does. The factor Xa active site associates with the COOH-terminus of TFPI. The factor Xa–TFPI complex rapidly inactivates TF–factor VIIa to form a stable quaternary complex, TF–factor VIIa–TFPI–factor Xa. Factor Xa is inhibited by AT but only poorly when in the prothrombinase complex.

Factor II (Prothrombin)

Prothrombin is the single-chain precursor of factor IIa (thrombin), the key enzyme in blood coagulation. The zymogen prothrombin is the most abundant of the plasma VKDPs and circulates in plasma at a mean concentration of 1.4 µM, or 100 µg/mL (see Table 27-1 ). Prothrombin is synthesized primarily in the liver. Low levels of prothrombin expression have also been identified in other tissues, including the brain, diaphragm, stomach, kidney, spleen, intestine, uterus, placenta, and adrenal gland. Removal of the 43–amino acid propeptide generates the mature protein of 579 amino acids. An NH ₂ -terminal Gla domain is followed by two kringle domains and the serine protease precursor domain (see Fig. 27-6 ). Kringles have been identified as common motifs in many plasma proteins, including prothrombin, plasminogen, tissue plasminogen activator, urokinase, factor XII, and apolipoprotein(a). The kringle 1 domain may interact with factor Va, although kringle 2 appears to be the primary region mediating interaction with factor Va to form the prothrombinase complex. This latter interaction may initiate conformational changes that make reaction sites accessible for enzymatic cleavage of prothrombin. The NH ₂ -terminal Gla domain and the first kringle domain together are referred to as prothrombin fragment 1 , with the kringle 2 domain being located in prothrombin fragment 2. The sequence of the enzyme α-thrombin is contained within prethrombin 2.

Activation of prothrombin by prothrombinase proceeds through an initial cleavage of prothrombin at Arg320-Ile321, which gives rise to meizothrombin, a two-chain disulfide-linked molecule (see Fig. 27-6 ). Meizothrombin expresses some of the activities of α-thrombin. However, the fibrinogen clotting ability of meizothrombin is impaired in comparison to that of α-thrombin. Meizothrombin is subsequently cleaved at Arg271-Thr272 to yield the NH ₂ -terminal half of the molecule (prothrombin fragment 1.2) and α-thrombin. α-Thrombin consists of an NH ₂ -terminal 49-residue A-chain disulfide linked to a COOH-terminal 259-residue B chain containing the catalytic triad.

An alternative cleavage pathway occurs when factor Xa independently acts on prothrombin. In the presence of factor Xa and Ca ²⁺ , the initial cleavage occurs at Arg271-Thr272 and gives rise to prothrombin fragment 1.2 and prethrombin 2. Subsequent cleavage at Arg320 in prethrombin 2 yields α-thrombin. Prothrombin fragment 1.2 remains noncovalently associated with α-thrombin. In contrast to other VKDPs, the phospholipid-binding Gla region is not covalently attached to the serine protease domain after cleavage of prothrombin to thrombin.

α-Thrombin is the central enzyme in blood coagulation in that it contributes to reactions at all levels, thereby allowing overall maintenance of vascular integrity. Its premier role in hemostasis results in platelet-fibrin clot formation. However, the events that lead up to and beyond fibrin formation also involve protein cleavage by α-thrombin, as well as activating platelets, factor VII, factor V, factor VIII, factor XI, and factor XIII. The contributions of α-thrombin also extend to anticoagulant and fibrinolytic reactions, including activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI).

The stable form of human α-thrombin possesses at least five distinct binding sites for substrates, inhibitors, cofactors, apolar molecules, and sodium ions (Na ⁺ ). Exosite I, the fibrinogen binding site, is an electropositive anion binding site distinct from, but acting in concert with, the active site of the α-thrombin molecule. In addition to fibrinogen, exosite I also recognizes the COOH-terminal domain of hirudin, the hirudin-like region of PAR-1, and the fifth and sixth EGF-like domains of thrombomodulin.

Many of the effects of α-thrombin on platelets and cells are elicited through the interaction of α-thrombin with receptor molecules, with α-thrombin binding to a receptor and initiating a signal transduction mechanism. The interaction between α-thrombin and the human platelet thrombin receptor PAR-1, however, is characterized by a more unusual mechanism in which the receptor is also a substrate for α-thrombin. α-Thrombin cleaves the extracellular region of the receptor and produces a “tethered ligand.” The new NH ₂ -terminus binds back to and activates the receptor. Two additional α-thrombin receptors homologous to PAR-1, PAR-3, and PAR-4 have been identified. α-Thrombin’s role continues beyond hemostasis into the realm of tissue repair and the remodeling phase necessary to regenerate damaged vascular tissue. α-Thrombin is a potent mitogen and stimulates cell division in macrophages, smooth muscle cells, and endothelial cells.

Protein C (Autoprothrombin II-A)

Protein C circulates as a heterodimer consisting of disulfide-linked heavy and light chains (see Fig. 27-6 ). However, approximately 5% to 10% of circulating protein C is in the single-chain form. The NH ₂ -terminal light chain contains the Gla domain and a hydrophobic region that connects the Gla domain to two EGF domains. The COOH-terminal heavy chain contains the serine protease domain. Cleavage at Arg169-Leu170 releases the activation peptide from the heavy chain to generate the enzyme. Although the α-thrombin–thrombomodulin complex is probably the major physiologic activator of protein C, other potential activators include plasmin and factor Xa. The half-life of protein C (8 to 10 hours; see Table 27-1 ) is markedly shorter than the half-life of other members of the VKDP family, which is probably the basis of the transient hypercoagulable state that may occur subsequent to the administration of coumarin-based anticoagulants.

The anticoagulant role of the dynamic protein C inhibitory pathway is proteolytic inactivation of factor Va. APC inactivates factor Va via a sequential series of proteolytic cleavages and thus inhibits the generation of α-thrombin. APC also cleaves and inactivates factor VIIIa, although the spontaneous dissociation/inactivation of factor VIIIa is probably the physiologic regulator of factor Xa generation. Complete inactivation of factor Va by APC requires an anionic phospholipid surface, and rates of inactivation of factors Va and VIIIa by APC have been reported to be enhanced by protein S. APC also has profibrinolytic effects associated with decreased activation of TAFI. TAFI is activated by the α-thrombin–thrombomodulin complex and acts to prolong clot lysis. Cleavage of factor Va by APC inhibits α-thrombin generation, thus reducing α-thrombin–thrombomodulin–mediated TAFI activation.

Protein C activation and APC activity are controlled on several levels. The endothelial cell protein C receptor (EPCR) provides cell-specific binding sites for both protein C and APC. Inflammatory agents such as endotoxin, interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), and TNF-α upregulate protein C activation on endothelial cells. TNF-α downregulates thrombomodulin on the endothelial cell surface. There are multiple other factors that downregulate thrombomodulin as well. APC activity is further regulated by the protein C inhibitor, plasminogen activator inhibitor 3 (PAI-3).

Protein S

Protein S is a VDKP that is not a serine protease precursor. Approximately 40% of protein S circulates in the free form, and the remaining 60% circulates as a 1 : 1 complex with C4b-binding protein (C4bBP), a regulatory protein of the complement system. The precise function of protein S in the protein C inhibitory pathway is controversial. Protein S is thought to function as a cofactor for APC in the inactivation of factors Va and VIIIa. Protein S has also been reported to bind to factor Xa, factor VIII, and factor Va and compete for prothrombinase binding sites on the membrane surface. The C4bBP–protein S complex may inhibit factor X activation as well. Protein S may have other roles apart from anticoagulation. α-Thrombin cleavage of protein S inhibits the ability of protein S to act as a cofactor for APC. The anticoagulant activity of protein S is also neutralized when bound to C4bBP.

Protein Z

Protein Z is an enzymatically inactive homologue of factors VII, IX, and X and protein C. Protein Z circulates in plasma in a complex with protein Z–dependent protease inhibitor (ZPI). ZPI is a 72-kD member of the serpin superfamily. However, like protein S, protein Z does not function as a protease. Although the COOH-terminus contains a homologue to the catalytic domains present in serine protease zymogens, the catalytic triad is not present. The function of protein Z in vivo is unclear. Thrombosis is observed in mice when factor V Leiden is protein Z deficient.