protein C

Regulation of Hemostasis

July 23, 2017 Anticoagulant Therapy, Cardiology, Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , ,

Key events that initiate and propagate coagulation are the redistribution of negatively charged phospholipids to the cell surface and the exposure of tissue factor to the blood. The appropriate negatively charged phospholipids, primarily phosphatidylserine, can arise as a result of either cellular activation with strong agonists like thrombin together with collagen in the case of platelets or tissue damage or death. The negatively charged phospholipids promote the activation of factor X and IX by the tissue factor-factor VIIa pathway, the activation of prothrombin by factors Va and Xa, and the activation of factor X by factors VIIIa and IXa. In addition, it has been suggested that oxidation of a specific disulfide in tissue factor is important for expression of its procoagulant activity, a process that may be regulated by protein disulfide isomerase, although this concept remains controversial.

Normally tissue factor is not present on cells in contact with blood. Tissue factor is found on extravascular cells surrounding the blood vessel so that a potent procoagulant surface is exposed when the endothelium is breached. This helps to seal the breach. Intravascularly, inflammatory stimuli can induce tissue factor synthesis and expression on leukocytes, particularly monocytes, providing a mechanism for the initiation of coagulation. This response likely contributes to disseminated intravascular coagulation (DIC). Animal studies suggest that this coagulation response plays a role in innate immunity and prevents the dissemination of infectious agents.

Other key events that can play a major role in the pathogenesis of thrombosis, at least in animal models, involve the release of intracellular components, including ribonucleic acid (RNA) and polyphosphates. These can trigger activation of factor XII, which initiates coagulation via the contact pathway. Although factor XII does not appear to contribute to hemostasis, it drives several models of thrombosis, including pulmonary embolism and myocardial infarction.

Polyphosphates are stored in the dense granules of platelets and when released, contribute to the procoagulant potential of the platelet. In regions of cellular death or severe inflammation, histones released from the tissue can bind to these polyphosphates, increasing the procoagulant activity more than 20-fold. Heparin analogues can block polyphosphate stimulation of coagulation by histones. In addition, neutrophils in hyperinflammatory environments can release their nuclear contents to form neutrophil extracellular traps (NETs). These NETs are effective in killing bacteria and other pathogens and also provide a potent agonist for the activation of platelets and the development of thrombi. Interestingly, heparin, an anticoagulant often used in the setting of inflammation, can disrupt the NETs and diminish the activation of platelets by histones and NETs.

Inhibition of Coagulation

There are three major natural anticoagulant mechanisms that serve to limit the coagulation process. These are tissue factor pathway inhibitor (TFPI), which blocks the initiation of coagulation by tissue factor-factor VIIa; the antithrombin-heparin mechanism, which inhibits thrombin and factors VIIa, Xa, and IXa; and the protein C pathway, which inactivates factors Va and VIIIa.

  1. Tissue factor pathway inhibitor (TFPI)
  2. Antithrombin-heparin mechanism
  3. Protein C pathway

Tissue Factor Pathway Inhibitor

TFPI is a complex molecule composed of three similar domains related to a protease inhibitor type known as Kunitz inhibitor. To inhibitor the tissue factor-factor VIIa complex, the Kunitz-1 domain of TFPI binds factor VIIa, whereas the Kunitz-2 domain binds factor Xa, either because the factor X is activated on tissue factor or (probably less effectively) by first reacting with factor Xa in solution and then inhibiting factor VIIa bound to tissue factor, thus blocking the initiation of coagulation. The carboxy terminal portion of TFPI is very basic, potentially facilitating interaction with the endothelium. Protein S can augment the activity of TFPI by increasing the rate of TFPI inactivation of factor Xa. The physiologic importance of TFPI is highlighted by the fact that gene deletion results in embryonic lethality apparently because of thrombosis and subsequent hemorrhage.

  1. Inhibiting Xa (Xa-TFPI complex)
  2. Xa-TFPI complex Inhibits FVIIa-TF complex
  3. TFPI is necessary for life

Antithrombin and Heparin

Antithrombin is the major inhibitor of the coagulation proteases thrombin, factor VIIa, factor Xa, and factor IXa. Antithrombin is a member of a large class of protease inhibitors referred to as serine protease inhibitors and abbreviated sermons. Antithrombin forms a very tight complex with these proteases. This reaction is rather slow with a half-life in the order of 30 seconds in plasma. Heparin markedly accelerates the reaction, thus accounting for most of its anticoagulant activity. There is a “bait” region in antithrombin that is involved in its interaction with the proteases. In the absence of heparin, this bait region is only partially available to the protease, resulting in the slow inhibition. Heparin binding to antithrombin induces a conformational change in antithrombin that enhances protease access to the bait loop.

In addition to the conformational change in antithrombin, heparin has additional roles in the inhibition of coagulation proteases. High-molecular-weight heparin can bind to both antithrombin and the protease, creating a situation where both reactants are brought into close proximity, thus increasing the reaction rate. This function is of variable importance with different proteases of the cascade. It is essential for thrombin inhibition, but plays a less important role in the inhibition of factor Xa. High-molecular-weight heparins accelerate factor Xa inhibition somewhat better than low-molecular-weight forms, but in contrast to most of the other heparin-mediated inhibitory functions, the additional acceleration gained by the high-molecular-weight forms is dependent on calcium ions. It is of interest that as heparins become processed to generate lower-molecular-weight fractions, such as enoxaparin, the capacity to promote thrombin inhibition versus factor Xa inhibition decreases. With the smallest functional form of heparin, the synthetic pentasaccharide fondaparinux, the ability to augment thrombin inhibition is almost completely lost, while good inhibition of factor Xa is maintained. Although heparin is a potent antithrombin-dependent anticoagulant, it is not effective against clot-bound thrombin or the factor Va-factor Xa complex assembled on membrane surfaces.

The importance of antithrombin is documented by the clinical observation that antithrombin deficiency, even to 50% of normal, is associated with significant thrombotic problems in humans and mice. Deletion of the gene in mice causes embryonic lethality, apparently in a mechanism that involves thrombosis followed by hemorrhage. Although heparin-like proteoglycans have been proposed to be important in modulating antithrombin function, immunohistochemical analysis indicates that most of these are localized to the basolateral side of the endothelium. No definitive deletion of heparin sulfate proteoglycans has been reported, possibly because there are alternative mechanisms for its biosynthesis. However, deletion of ryudocan, another heparin-like proteoglycan, is associated with a thrombotic phenotype.

  1. Antithrombin is ineffective against clot-bound thrombin or the factor Va-factor Xa complex assembled on membrane surfaces
  2. Heparin itself has no anticoagulation effect
  3. Heparin accelerates the anticoagulation capacity of antithrombin
  4. Deficiency of heparin-like proteoglycan potentiates patients at risk of thrombosis

The Thrombomodulin and Protein C Anticoagulation Pathway

The protein C pathway serves many roles, probably the primary role being to work to generate activated protein C (APC) that in turn inactivates factors Va and VIIIa to inhibit coagulation. The importance of this anticoagulant activity is readily apparent because patients born without protein C die in infancy of massive thrombotic complication (purport fulminans) unless provided a protein concentrate.

The protein C anticoagulant pathway serves to alter the function of thrombin, converting it from a procoagulant enzyme into the initiator of an anticoagulant response. This occurs when thrombin binds to thrombomodulin, a proteoglycan receptor primarily on the surface of the endothelium. Thrombomodulin binds thrombin with high affinity, depending on the posttranslational modifications of thrombomodulin. Binding thrombin to thrombomodulin blocks most of thrombin’s procoagulant activity such as the ability to clot fibrinogen, activate platelets, and activate factor V but does not prevent thrombin inhibition by antithrombin. The thrombin-thrombomodulin complex gains the ability to rapidly activate protein C. In the microcirculation, where there is high ratio of endothelial surface to blood volume, it has been estimated that the thrombomodulin concentration is in the range of 100 to 500 nM. Thus a single pass through the microcirculation effectively strips thrombin from the blood, initiates protein C activation, and holds a coagulantly inactive thrombin molecule in place for inhibition by antithrombin or protein C inhibitor. Activation of protein C is augmented approximately 20-fold in vivo by the endothelial cell protein C receptor (EPCR). EPCR binds both protein C and APC with similar affinity. The EPCR-APC complex is capable of cytoprotective functions, but at least with soluble EPCR, this complex is not an effective anticoagulant. Instead, when APC dissociates from EPCR, it can interact with protein S to inactivate factors Va and VIIIa and thus inhibit coagulation. Factor V increases the rate at which the APC-protein S complex inactivates factor VIIIa.

Thrombomodulin also increases the rate at which thrombin activates thrombin-activatable fibrinolysis inhibitor (TAFI) to a similar extent as it dose protein C. Once activated, TAFI releases C-terminal lysine and arginine residues of fibrin chains. C-terminal lysine residues on fibrin enhance fibrin degradation by serving as plasminogen- and tissue plasminogen activator-binding sites. Consequently, their removal by activated TAFI (TAFIa) attenuates clot lysis.

More than any other regulatory pathway except tissue factor, the protein C pathway is sensitive to regulation by inflammatory mediators. Tumor necrosis factor-alpha and IL-1beta downregulate thrombomodulin both in cell culture and in at least some patients with sepsis. Downregulation of thrombomodulin has been observed in animal models of diabetes, inflammatory bowel disease, reperfusion injury in the heart, over human atheroma, and in villitis, in addition to sepsis.

  1. Protein C, thrombomodulin, and EPCR are essential for life
  2. Protein C targets against factors Va and VIIIa
  3. EPCR increases the protein C activation by thrombin-thormbomodulin complex by about 20-fold
  4. Protein C’s anticoagulation function needs the help of protein S
  5. Thrombin-thrombomodulin complex removes the C-terminal lysine residues of fibrin, which attenuates clot fibrinolysis.

Regulation of Fibrinolysis

Fibrinolysis is the process by which fibrin clots are dissolved, either through natural mechanisms or with the aid of pharmaceutical interventions. Plasmin is the serine protease that solubilizes fibrin. Plasmin is generated by the activation of its precursor plasminogen either by natural activators, tissue plasminogen activator (tPA), the major activator in the circulation, or urokinase plasminogen activator (uPA), or by administration of recombinant tPA or streptokinase, a bacterial protein that promotes plasmin generation and aids in dissemination of the bacteria. Staphylokinase, a protein from Staphylococcus aureus, is similar to streptokinase and also has been examined as a potential therapeutic.

Fibrin plays an active role in its own degradation because it binds plasminogen and tPA, thereby concentrating them on the fibrin surface and promoting their interaction. The resultant plasmin cleaves fibrin and exposes C-terminal lysine residues that serve as additional plasminogen and tPA binding sites. TAFIa attenuates fibrinolysis by releasing these C-terminal lysine residues. Furthermore, lysine analogues, such as ε-aminocaproic acid or tranexamic acid, bind to tPA and plasminogen. Consequently, these lysine analogues can be used to treat patients with hyperfibrinolysis and can reduce bleeding complications.

Plasmin is not specific for fibrin and degrades a variety of other proteins. Consequently, plasmin is tightly controlled. There are two major inhibitors of fibrinolysis: α2-antiplasmin, and serpin that inactivates plasmin, and plasminogen activator inhibitor 1 (PAI-1), which inhibits tPA and uPA. α2-Antiplasmin is cross-linked to fibrin by activator factor XIII. PAI-1 is an intrinsically unstable serpin that can be stabilized by interaction with vitronectin. Because of its instability, the antigen and functional levels of PAI-1 are often quite different and can vary among individuals. PAI-1 is upregulated by lipopolysaccharide, tumor necrosis factor, and other inflammatory cytokines. PAI-1 levels are increased in vascular diseases such as atherosclerosis and in metabolic syndrome, sepsis, and obesity. Increased levels of PAI-1 result in decreased fibrinolytic activity, which increases the risk for thrombosis. PAI-1 is also stored in platelets and is released with platelet activation. Platelet-derived PAI-1 may limit the degradation of platelet-rich thrombi, such as those that trigger acute coronary syndromes.

Overall the fibrinolytic system is dynamic and responsive to local phenomena, such as platelet-derived PAI-1, as well as to systemic alterations like obesity and inflammation. In this sense, the fibrinolytic control mechanisms share many similarities with the control of coagulation, in which similar considerations affect their function.

[Physiology][Hematology] Coagulation Factors, Anticoagulation Factors, and Pathways of Hemostasis and Thrombosis

July 14, 2016 Hematology, Molecular Biology No comments , , , , , , , , , , , , , , , , , ,

The role of surfaces in coagulation and coagulation inhibition and fibrinolysis

Functionally, the relationship between clotting and surface is striking. For a process that is typically termed humoral, most of the coagulation reactions take place on biologic surfaces.

  • The presence of a phospholipid surface increases the rate of activation of prothrombin by several orders of magnitude.
  • The presence of a phospholipid surface also localizes the reaction to the site of injury and may protect the reaction from inhibitors.
  • The activation of protein C by thrombin is a reaction that occurs on a cellular surface.
  • Coagulation inhibitors such as antithrombin and heparin cofactor II are also more efficient when the reactions occur on surfaces. The typical surface in these reactions is glycosaminoglycans like heparin, heparin sulfate, and derma tan sulfate.

The Vitamin K-dependent Zymogens


Tenase/Intrinsic tenase complex: factor VIIIa-factor IXa complex

Extrinsic tenase complex: tissue factor-factor VIIa complex

Prothrombinase compelx: factor Va-factor Xa complex

These zymogens all a similar domain structure of a C-terminal serine protease domain and an N-terminal γ-carboxy glutamic acid (Gla) domain, which are connected by two epidermal growth factor (EGF)-like domains or kringle domains. The Gla domain mediates the binding of zymogens to a negatively charged lipid surface (in a calcium-dependent manner), a domain that is characteristic to the vitamin K–dependent proteins.

Screen Shot 2016-08-11 at 7.36.50 PMThe Gla domain refers to the 42-residue region located in the N-terminus of the mature protein that comprises 9 to 12 glutamic acid residues that are posttranslationally γ-carboxylated into Gla residues by a specific γ-glutamyl carboxylase in the endoplasmatic reticulum of hepatocytes. This γ-carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action, hence the name vitamin K–dependent proteins. For each Glu residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form. Warfarin inhibits the activity of vitamin K epoxide reductase, thereby preventing vitamin K recycling and hinibiting γ-carboxylation, which results in a heterogeneous population of circulaing undercarboxylated forms of the vitamin K-dependent proteins with reduced activity. Recognition by and interaction with γ-carboxylase is facilitated by the propeptide sequence that is located C-terminal to the signal peptide.

The serine protease domains of the vitamin K-dependent proteins are highly homologous, as they bear a chymotrypsin-like fold and display trypsin-like activity.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Activates TAFI, platelet, fibrinogen, FV, FVIII, FXI, FXIII, and protein C

Activated by prothrombinase complex

Inhibited by serpins (enhanced by glycosaminoglycans like heparin)

Screen Shot 2016-05-20 at 2.19.12 PMProthrombin is composed of fragment 1 (F1: Gla and kringle 1), fragment 2 (F2: kringle 2), and the serine protease domain. The primary function of kringle 1 and kringle 2 domain is to be bound by prothrombinase complex. PS: Gla and kringle, kringle 2, and serine protease.

Prothrombin is proteolytically activated by the prothrombinase complex that cleaves at Arg271 and Arg320, both of which are necessary to generate procoagulant α-thrombin (IIa). Thrombin's main function is to induce the formation of a fibrin clot by removing fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize. The dynamic structural conformation of thrombin allows for binding to diverse ligands, and the subsequent ligand-indued conformational stabilization, known as thrombin allostery, regulates and controls thrombin activity. Thrombin also is able to cleave a wide variety of substrates with high specificity (TAFI, platelet, FV, FVIII, FXI, FXIII, protein C).

The physiologic inhibitors of thrombin are the serine protease inhibitors (serpins) antithrombin, heparin cofactor II, protein C inhibitor, and protease nexin 1, with antithrombin being the primary plasma inhibitor. For all four serpins, the rate of thrombin inhibition can be accelerated by glycosaminoglycans, such as heparin, through mutual binding to the serpin and thrombin, which ensures rapid inhibition of thrombin at the intact endothelial cell surface where heparin-like glycosaminoglycans are found.


Activates FIX, FX (in the form of extrinsic tenase)

Activated by Xa, thrombin, IXa, and XIIa

Inhibited by TFPI; antithrombin (only in the presence of heparin)

Screen Shot 2016-05-20 at 2.38.51 PMFactor VII consists of a Gla domain with 10 Gla residues, two EGF-like domains, a connecting region, and the serine protease domain.

Factor VII is proteolytically activated once it has formed a high-affinity complex with its cofactor tissue factor (there are small amount of VIIa in the circulation by unknown mechanism). A number of coagulation proteases including factor Xa, thrombin, IXa, and XIIa are capable of cleaving factor VII at Arg152 to generate factor VIIa, with factor Xa being considered the most potent and physiologically relevant activator of factor VII. Autoactivation can also occur, which is initiated by minute amounts of preexisting factor VIIa.

The extrinsic tenase complex activates both FIX and X.

The extrinsic tenase complex is inhibited by the tissue factor pathway inhibitor (TFPI). Antihrombin (only in the presence of heparin) also can inhibits the extrinsic tenase complex.


Aactivates FX in the form of intrinsic tenase

Activated by extrinsic tenase, factor XIa

Inhibited by antithrombin (enhanced by heparin)

Screen Shot 2016-05-20 at 3.08.29 PMFactor IX consists of a Gla domain, two EGF-like domains, a 35-residue activation peptide, and the serine protease domain. The Gla domain contains 12 Gla residues, of which the 11th and 12th Gla (Glu36 and Glu40) are not evolutionary conserved in other vitamin K-dependent proteins and are not essential for normal factor IX function.

Limited proteolysis of factor IX at both Arg145 and Arg180 by either the extrinsic tenase or factor XIa results in the release of the activation peptide and generation of factor IXa.

Factor IXa has a low catalytic efficiency as a result of impaired access of substrates to the active site that results from steric and repulsion. Reversible interaction with the cofactor VIIIa on anionic membranes and subsequent factor  X binding leads to rearrangement of the regions surrounding the active site and proteolytic factor X activation.

The primary plasma inhibitor of factor IXa is the serpin antihrombin, and this inhibition is enhanced by heparin, which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor IXa.


Activates prothrombin (prothrombinase complex); FV, VII, and VIII

Activated by extrinsic tenase; intrinsic tenase

Inhibited by antithrombin (enhanced by heparin); TFPI

Factor X is a two-chain zymogen consisting of a light chain which comprises the Gla domain with 11 Gla residues and the EGF domain, and a heavy chain that consists of a 52-residue activation peptide and the serine protease domain. The two chains are linked via a disulfide bond.

Factor X is activated by intrinsic tenase or extrinsic tenase, following cleavage at Arg194 in the heavy chain. After activated, Xa reversibly associates with its cofactor Va on an anionic membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Factor Xa is also involved in the proteolytic activation of FV, FVII, and VIII.

Further autocatalytic cleavage at Arg429 near the C-terminus of the factor Xa heavy chain leads to release of a 19-residue peptide, yielding the enzymatically active factor Xaβ. Plasmin-mediated cleavage of factor Xa at adjacent C-terminal Arg or Lys residues also results in the generation of factor Xaβ and factor Xaβ derivatives. While the coagulation activity is eliminated in the factor Xaβ derivatives, they are capable of interacting with the zymogen plasminogen and enhance its tissue plasminogen activator-mediated conversion to plasmin, thereby promoting fibrinolysis.

A primary plasma inhibitor of factor Xa is the serpin antithrombin, and this inhibition is enhanced by heparin. Another potent factor Xa inhibitor is TFPI, which inhibits both the extrinsic tenase-factor Xa complex as well as free factor Xa, for which protein S function as a cofactor.

Protein C

Inactivates FVa, FVIIIa (cofactor protein S, with calcium and surface) 

Activated by thrombin-thrombomodulin complex (enhanced by EPCR and PF4)

Inhibited by heparin-dependent serpin protein C inhibitor and by PAI-1

Protein C is synthesized as a single-chain precursor and during intracellular processing amino acids Lys146-Arg147 are excised. The zymogen consists of a light chain comprising the Gla domain and the EGF domains, which is linked via a disulfide bond to the heavy chain that consists of the activation peptide and the serine protease domain.

Screen Shot 2016-05-24 at 8.45.18 PMProtein C is proteolytically activated by alpha-thrombin in complex with the endothelial cell surface protein thrombomodulin following cleavage at Arg169. The activation peptide is released and the mature serine protease activated protein C (APC) is formed. Activation of protein C is enhanced by its localization on the endothelial surface through association with the endothelial cell protein C receptor (EPCR). Also, protein C activation is accelerated by platelet factor 4 (PF4), which is secreted by activated platelets. Upon interaction with the Gla domain of protein C, PF4 modifies the conformation of protein C, thereby enhancing its affinity for the thrombomodulin-thrombin complex.

APC consists of the disulfide-linked light chain comprising the Gla and EGF domains and the catalytic heavy chain. In complex with its cofactor protein S, APC proteolytically inactivates factors Va and VIIIa in a calcium- and membrane-dependent manner. Intact factor V has been reported to function as a cofactor for the inactivation of factor VIIIa in the presence of protein S.

APC is primarily inhibited by the the heparin-dependent serpin protein C inhibitor and by plasminogen activator inhibitor-1 (PAI-1).

The Procoagulant Cofactors V and VIII

Factors V and VIII both function as cofactors in coagulation and dramatically enhance the catalytic rate of their macromolecular enzyme complexes, resulting in the generation of thrombin (via prothrombinase) and factor Xa (via intrinsic tenase), respectively. Apart from their functional equivalence, they also share similar gene structure, amino aacid sequences, and protein domain structures.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Accelerates the ability of FXa to rapidly convert prothrombin to thrombin

Activated by thrombin (principal activator), FXa (primarily in initiation phase)

Inhibited by APC

FV and factor V-short interact with full-length TFPI

Approximately 20% percent of the total factor V in blood is stored in the alpha-granules of platelets. Although it was originally thought that megakaryocytes synthesize factor V, studies in humans indicate that platelet factor V originates from plasma through endocytic uptake. Platelet factor V is modified intracellularly such that it is functionally unique compared to its plasma-derived counterpart. It is partially activated, more resistant to inactivation by APC, and has several different posttranslational modifications.

Screen Shot 2016-05-29 at 8.22.28 PMFactor V has an A1-A2-B-A3-C1-C2 domain structure. The two C-type domains belong to the family of discoidin domains, which are generally involved in cell adhesion, and share approximately 55 percent sequence identity with the factor VIII C domains. The C domain mediate binding to the anionic phospholipid surface, thereby localizing factor V to the site of injury and facilitating interaction with factor Xa and prothrombin. In contrast, large central B domain of factor V shows weak homology to the factor VIII B domain or to any other known protein domain.

Factor V undergoes extensive postranslational modifications, including sulfation, phosphorylation, and N-linked glycoslation. Sulfation at sites in teh A2 and B domain are involved in the thrombin mediated proteolytic activation of factor V. Phosphorylation at Ser692 in the A2 domain enhances the APC-dependent inactivation of the cofactor Va.

Sequential proteolytic cleavage of the procofactor factor V at Arg709, Arg1018, and Arg1545 in the B domain results in release of the inhibitory constraints exerted by the B domain and in the generation of the heterodimeric cofactor Va, where maximal cofactor activity correlates with cleavage at Arg1545. Thrombin has generally been recognized to be the principal activator of factor V. However, recent findings suggest that in the initiation phase of coagulation factor V is primarily activated by factor Xa. Factor Xa initially cleaves factor V at Arg1018, followed by proteolysis at Arg709 and Arg1545.

Factor Va is composed of a heavy chain comprising the A1-A2 domains and the A3-C1-C2 light chain, which are noncovalently associated via calcium ions. Factor Va is a nonenzymatic cofactor within the prothrombinase complex that greatly accelerates the ability of factor Xa to rapidly convert prothrombin to thrombin.

APC catalyzes the inactivation of factor Va by cleavage at the main sites Arg306 and Arg506, upon which the cleaved A2 fragment dissociates and factor Va can no longer associated with factor Xa.

Both factor V and an alternativel spliced isoform of factor V (factor V-short), which lacks the major part of the B domain (residues 756 to 1458) and normally circulates in low abundance, interact with full-length TFPI (TFPIalpha), most likely through the acidic B domain region. The linkage of factor V and TFPIalpha is considered to attenuate the bleeding phenotype in factor V-deficient patients, as the low TFPIalpha levels in these patients allow the residual platelet factor to be sufficient for coagulation. Conversely, increased factor V-short expression caused by an A2440G mutation in the factor V gene leads to a dramatic increase in plasma TFPIalpha, resulting in a bleeding disorder.


Accelerates the ability of FIXa to rapidly convert FX to FXa

Activated by thrombin (principal) and FXa (also principal)

Activity downregulated spontaneously, by APC, FXa or FIXa

Factor VIII (antihemophilic factor) was first discovered in 1937, but it was not until 1979 that its purification by Tuddenham and coworkers led to the molecular identification of the protein. The mature factor VIII procofactor comprises 2332 amino acids and circulates in a high-affinity complex with its carrier protein VWF at a concentration of approximately 0.7 nM and a circulatory half-life of 8 to 12 hours. Complex formation with VWF protects factor VIII from proteolytic degradation, premature ligand binding, and rapid clearance from the circulation.

The primary source of factor VIII is the liver, but extrahepatic synthesis of factor VIII also occurs. While contradictory evidence exists on the cellular origin of both hepatic and extrahepatic factor VIII synthesis, recent studies in mice support that endothelial cells from many tissues and vascular beds synthesize factor VIII, with a large contribution from hepatic sinusoidal endothelial cells. This is consistent with observations on factor VIII expression in human endothelial cells from the liver and lung.

Screen Shot 2016-05-29 at 8.22.42 PMThe A1-A2-B-A3-C1-C2 domain structure of factor VIII shares significant homology with factor V except in the B domain region. In contrast to factor V, factor VIII B domain is dispensable for procoagulant activity. The C-terminal regions of the A1 and A2 domians and the N-terminal portion of the A3 domain contain short segments of 30 to 40 negatively charged residues known as the a1, a2, and a3 regions. Interaction with VWF is faciliated by the a3 region and C1 domain. The C domains mediate binding to the anionic phospholipid surface, thereby localizing factor VIII to the site of injury and facilitating interaction with factor IXa and factor X.

Factor VIII is heavily glycosylated and the majority of the N-linked glycosylation sites are found in the B domain, which mediate interaction with the chaperons calnexin and calreticulin and, in part, with the LMAN1-MCDF2 receptor complex. Sulfation of tyrosin residues is required for optimal activation by thrombin, maximal activity in complex with factor IXa, and maximal affinity of factor VIIIa for VWF.

Thrombin and factor Xa are the principal activators of the procofactor VIII and generate the cofactor VIIIa through sequential proteolysis at Arg740, Arg372, and Arg1689. The heterotrimeric factor VIIIa is composed of the A1, A2, and the A3-C1-C2 light chain subunits. The A1 and A3-C1-C2 subunites are noncovalently linked through calcium ions, whereas A2 is associated with weak affinity primarily by electrostatic interactions. Once activated, factor VIIIa functions as a cofactor for factor IXa in the phospholipid-dependent conversion of factor X to factor Xa. The rapid and spontaneous loss of factor VIIIa activity is attributed to A2 domain dissociation from the heterotrimer. Additional proteolysis by APC, factor Xa, or factor IXa also results in the downregulation of factor VIIIa cofactor activity.

The Soluble Cofactors Protein S and Von Willebrand Factor

Protein S

Protein S is a vitamin K-dependent single-chain GP of 635 amino acids that circulates with a plasma half-life of 42 hours. Part of the total protein S pool circulates in a free form at a concentration of 150 nM, whereas the majority (~60%; 200 nM) circulates bound to the complement regulatory protein C4b-binding protein (C4BP). Protein S is primarily synthesized in the liver by hepatocytes, in addition to endothelial cells, megakaryocytes, testicular Leydig cells, and osteoblasts.

The protein structure of protein S differs from the other vitamin K-dependent proteins as it lacks a serine protease domain and, consequently, is not capable of catalytic activity. Protein S is composed of a Gla domain comprising 11 Gla residues, a thrombin-sensitive region (TSR), four EGF domains, and a C-terminal sex hormone-binding globulin (SHBG)-like region that consists of two laminin G-type domains. The SHBG-like domain is involved in the interaction with beta-subunit of C4BP.

Apart from gamma-carboxylation of Glu residues, protein S is posttranslationally modified via N-glycosylation in the second laminin G-type domain of the SHBG-like region. beta-Hydroxylation of Asp95 or Asn residues in each EGF domain allows for calcium binding that orients the four EGF domains relative to each other.

Free protein S serves as a cofactor for APC in the proteolytic inactivation of FVa and FVIIIa. Interaction of protein S with APC on a negatively charged membrane surface alters the location of the APC active site relative to FVa, which accounts for the selective protein S-dependent rate enhancement of APC cleavage at Arg306 in FVa. C4BP-bound protein S also exerts a similar stimulatory effect on Arg306 cleavage, albeit to lower extent, whereas it inhibits the initial APC-mediated FVa cleavage at Arg506, resulting in an overall inhibition of FVa inactivation. Cleavage of the TSR by thrombin and/or FXa results in a loss of APC-cofactor activity. Protein S also functions as a cofactor for TFPIalpha in the inhibition of factor Xa, which is mediated by the SHBG-like region in protein S.


VWF is a large multimeric GP that is required for normal platelet adhesion to components of the vessel wall and that serves as a carrier for factor VIII. It is exclusively synthesized in megakaryocytes and endothelial cells and stored in specialized organelles in platelets and endothelial cells. VWF multimers circulate at a concentration of 10 nM with a half-life of 8 to 12 hours. Clearance of VWF multimers is mainly mediated by macrophages from the liver and spleen.

Large VWF multimers are cleaved by the plasma protease ADAMTS-13. This cleavage produces the smaller size VWF multimers that circulate in plasma. Reduced ADAMTS-13 activity is linked to various microangiopathies with increased platelet activity.

The precursor protein of VWF is composed of a 22-residue signal peptide and of a proVWF protein comprising 2791 amino acids that has 14 distinct domains. Upon translocation to the ER, the signal peptide is cleaved off, and the proVWF dimerizes in a tail-to-tail fashion through cysteines in its cysteine knot (CK) domain. During transit through the Golgi apparatus, proVWF dimers multimerize in a head-to-head fashion through the formation of disulfide bonds between cysteine residues in the D3 domain. At the same time, D1 and D2 domains are cleaved off as a single fragment to form the VWF propeptide (741 amino acids), while the remaining domains comprising 2050 amino acid residues and up to 22 carbohydrate chains form mature VWF. In the trans-Golgi network, the VWF propeptide promotes mature VWF to assemble into high-molecular-weight multimers. These multimers subsequently aggregate into tubular structures that are packaged into alpha-granules in megakaryocytes and into Weibel-Palade bodies in endothelial cells.

Upon exocytosis from Weibel-Palade bodies and at high shear rates, multimeric VWF unrolls from a globular to a filamental conformation, up to many microns long, which becomes a high-affinity surface for the platelet GPIb-V-IX complex. Large VWF multimers are more active than smaller multimers, which is explained by the fact that the former contain multiple domains that support the interactions between platelets, endothelial cells, and subendothelial collagen. VWF binds to matrix collagens via its A1 and A3 domains. The A1 domain also mediates binding to platelet GPIb, which is required for the fast capture of platelets. Platelet adhesion to VWF is further supported by VWF immobilization on surface and by high shear stress.

VWF complexes with factor VIII through the first 272 residues in the N-terminal region of the mature VWF protein subunit, thereby protecting factor VIII from proteolytic degradation, premature ligand binding, and rapid dlearance from the circulation.

Factor XI and The Contact System


Activates FIX with cofactor HK (calicum-dependent, phospholipid-independent)

Activated by FXIIa, thrombin, and FXIa

Inhibited by nexin 1, nexin 2, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin

Screen Shot 2016-06-11 at 2.57.05 PMFactor XI is synthesized in the liver and secreted as a single-chain zymogen of 607 amino acids. In the circulation, FXI is found as a homodimer at a concentration of 30 nM with a plasma half-life of 60 to 80 hours. All FXI homodimers circulate in complex with high-molecular-weight kininogen (HK). HK is thought to mediate binding of factor XI to negatively charged surfaces, thereby facilitating factor XI activation.

Each FXI subunit comprises four apple domians and a serine protease domain. The apple domains are structured by three disulfide bonds and form a disk-like platform on which the serine protease domain rests. The dimerization of two FXI subunits is mediated by interactions between the two apple 4 domains that involve a disulfide bond between the Cys321 residues, hydrophobic interactions, and a salt bridge, of which only the latter two are required for dimerization. The domain structure of FXI is highly similar to that of the monomer prekallikrein (PK), the zymogen of the protease kallikrein, which also circulates in complex with HK.

FXI does not bear a Gla domain and thus does not require gamma-carboxylation to exert its procoagulant activity.

Activation of a FXI subunit to FXIa proceeds through proteolysis at Arg369 in the N-terminal region of the serine protease domain and yields two-chain activated factor XIa. There are several catalysts capable of FXI activation, which include the contact factor XIIa, thrombin, or factor XIa itself in the presence of negatively charged surfaces. FXI must be a dimer to be activated by FXIIa, whereas thrombin and factor XIa lack this requirement.

Following activation of FXI, binding sites for the substrate FIX become available in the apple 3 domain and serine protease domian of factor XIa. FXIa proteolytically activates FIX to factor FIXa in a calcium-dependent but phospholipid-independent manner. Both forms of the FXIa dimer as well as monomeric FXIa activate FIX in a similar manner.

Accumulating evidence supports the notion that FXIIa-dependent activation of FXI is not essential to normal hemostasis, but is important in pathologic thrombus formation. Thrombin-mediated activation of FXI, on the other hand, seems most significant under conditions of low tissue factor and is assumed to enhance clot stability thorugh thrombin-activation of TAFI.

FXIa function is regulated by the serpins protease nexin 1, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin. Platelets also contain a FXIa inhibitor, the Kunitz-type inhibitor protease nexin 2.

The Contact System: FXII, Prekallikrein, and High-Molecular Weight Kininogen

FXII, HK, and PK are part of the contact system in blood coagulation, which is triggered following contact activation of FXII mediated via negatively charged surface.


Activates FXII

Activated by FXIIa

PK is synthesized in the liver, circulates as a zymogen, and is highly homologous to FXI. Conversion into the serine protease proceeds through limited proteolysis by FXIIa, and the generated kallikrein reciprocally activates more FXII.


HK, which is also synthesized in the liver, is a nonenzumatic cofactor that circulates in complex with FXI, which then activates FIX.

Contact system

The contact system is at the basis of the activated partial thromboplastin time (APTT) assay that is widely used in clinical practice. In this clinical laboratory test, the negatively charged surface is provided by reagents. FXIIa activates FXI, which then activates FIX. Despite HK and PK being required for a normal APTT, they appear to be dispensable for coagulation in vivo. Individuals who are deficient in any of these factors do not have a bleeding tendency, even after significant trauma or surgery.


Activates FXI, PK

Activated by negatively charged surface (platelet polyphosphate, microparticles derived from platelets and erythrocytes, RNA, and colalgen), kallikrein

Inhibited by serpin C1 inhibitor, antithrombin, and PAI-1

FXII, which is homologous to plasminogen activators, consists of an N-terminal fibronectin type I domain, an EGF-like domain, a fibronectin type II domain, a second EGF-like domain, a kringle domain, a proline-rich region, and a C-terminal serine protease domain. The proline-rich region is unique to FXII, as it is not found in any of the other serine proteases.

Limited proteolysis by kallikrein at Arg353 in FXII yields the activated two-chain alpha-FXIIa. Once activated, alpha-FXIIa activates FXI to FXIa. Furthermore, alpha-FXIIa activates PK, thereby contributing to its own feedback activation. Also FXI is known to acquire alpha-FXIIa activity upon contact with a negatively charged surface, the latter inducing a conformation change in FXII. This conformational change induces a limited amount of proteolytic activity in FXII, known as auto-activation. Furthermore, the surface-induced active conformation of FXII is suggested to enhance the proteolytic conversion to alpha-FXIIa. The fibronectin type I and II domains, EGF-2, the kringle domain, and the proline-rich region are reported to conribute to interaction with a negatively charged surface.

Further cleavage of alpha-FXIIa by kallikrein at Arg334 and Arg343 in the light chain results in the generation of beta-FXIIa, which comprises a nine-residue heavy-chain fragment that is disulfide-linked to the light chain. Given the absence of the heavy chain, beta-FXIIa does not interact with anionic surfaces. Even though beta-FXIIa is still capable of activating PK, it is no longer activates FXI.

The serpin C1 inhibitor is the main plasm inhibitor of alpha-FXIIa and beta-FXIIa. In addition, antithrombin (AT) and PAI-1 also inhibit FXIIa activity.

Revised Model of Coagulation

Screen Shot 2016-07-14 at 6.11.20 PM


Hemostasis is the process through which bleeding is controlled at a site of damaged vascular endothelium and represents a dynamic interplay between the subendothelium, endotheliumcirculating cells, and plasma proteins. The hemostatic process often is divided into three phases: the vascular, platelet, and plasma phases. Although it is helpful to divide coagulation into these phases for didactic purposes, in vivo, they are intimately linked and occur in a continuum.

The vascular phase is mediated by the release of locally active vasoactive agents that result in vasoconstriction at the site of injury and reduced blood flow. Vascular injury exposes the underlying subendothelium and procoagulant proteins, including von Willebrand factor (vWF), collagenn and tissue factor (TF) that then come into contact with blood.

During the platelet phase, platelets bind to vWF incorporated into the subendothelial matrix thorugh their expression of glycoprotein Iba (GPIbalpha). Platelets bound to vWF form a layer across the exposed subendothelium, a process termed platelet adhesion, and subsequently are activated via receptors, such as collagen receptors integrin alpha2beta1 and glycoprotein (GPVI), resulting in calcium mobilization, granule release, activation of the fibrinogen receptor, integrin alpha(IIb)b3, and subsequent platelet aggregation.

The plasma phase of coagulation can be further subdivided into initiation, priming, and propagation. Initiation begins when vascular injury also leads to exposure of TF in the subendothelium and on damaged endothelial cells. TF binds to the small amounts of circulating activated factor VII (FVIIa), resulting in formation of teh TF:FVIIa complex (extrinsic tenase complex); this complex binds to and activates factor X (FX) to activated FX (FXa). The TF:FVIIa:FXa complex converts a small amount of prothrombin (factor II/FII) to thrombin (activated factor II/FIIa). This small amount of thrombin is able to initiate coagulation and generate an amplification loop by cleaving factor VIII (FVIII) from vWF, activating clotting factors FVIII, XI (FXI), and platelets, which result in exposure of membrane phospholipids and release of partially activated factor V (FVa). At the end of the initiation and priming phases, the platelet is primed with an exposed phospholipid surface with bound activated cofactors (FVa and FVIIIa).

During the propagation phase, FIXa, generated either by the action of FXIa on the platelet surface or TF-VIIa complex on the TF bearing cell, bind to its cofactor, FVIIIa, to form the potent intrinsic tenase complex. FX is then bound and cleaved by the intrinsic tenase complex (FIXa:FVIIIa) leading to large amounts of FXa, which in association with its cofactor, FVa, forms the prothrombinase complex on the activated platelet surface. The prothrombinase complex (FXa:FVa) then binds and cleaves prothrombin leading to an ultimate burst of thrombin sufficient to convert fibrinogen to fibrin and to result in subsequent clot formation. The formed clot is stabilized by the thrombin-mediated activation of factor XIII (FXIII), which acts to cross-link fibrin, and thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to remove lysine residues from the fibrin clot, thereby limiting plasmin binding. Utimately, the clot undergoes fibrinolysis, resulting in the restoration of normal blood vessel architecture. The fibrinolytic process is initiated by the release of tissue plasminogen activator (tPA) near the site of injury. tPA converts plasminogen to plasmin, which (via interactions with lysine and arginine residues on fibrin) cleaves the fibrin into dissolvable fragments.


Hemostasis – Plasma Phase – Initiation

  • FX > FXa via TF:FVIIa complex
  • FII > FIIa (small amount) via TF:FVIIa:FXa complex

Hemostasis – Plasma Phase – Priming

  • FVIII:vWF complex > FVIII + vWF via FIIa (small amount)
  • FVIII > FVIIIa via FIIa (small amount)
  • FXI > FXIa via FIIa (small amount)
  • Platelet > activated platelet via FIIa (small amount)
  • Activated platelet > partially activated FV via release

Hemostasis – Plasma Phase – Propagation

  • FIX > FIXa via FXIa or TF:VII complex
  • FX > FXa (large amount) via FIXa:FVIIIa complex
  • FII > FIIa (large amount) via FXa:FVa complex
  • FXIII > FXIIIa via FIIa (large amount)

Regulation of Hemostasis

Both the hemostatic and fibrinoglytic processes are regulated by inhibitors that limit coagulation at the site of injury and quench the reactions, thereby preventing systemic activation and pathologic propagation of coagulation. The hemostatic system has three main inhibitory pathways: antihrombin (AT), the protein C:protein S complex, and tissue factor pathway inhibitor (TFPI).

AT/Antithrombin target at thrombin and FXa

AT (antithrombin) released at the margins of endothelial injury binds in a 1:1 complex with thrombin, inactivating thrombin not bound by the developing clot. Antithrombin also rapidly inactivates FXa; thus, any excess FXa generated by the TF:VIIa complex during initiation is inactivated and unable to migrate to the activated platelet surface.

Thrombomodulin, protein C and S, target at FVa and FVIIIa

Excess free thrombin at the clot margins binds to thrombomodulin, a receptor expressed on the surface of intact endothelial cells that when complexed with thrombin activates protein C; activated protein C complexes with its cofactor protein S and inactivates FVa and FVIIIa.

Tissue factor pathway inhibitor/TFPI, target at TF:FVIIa and FXa

TFPI is a protein produced by endothelial cells that inhibits the TF:FVIIa complex and FXa. Binding to FXa is required for the inhibitory effect on TF:FVIIa. This negative feedback results in reduced subsequent thrombin generation and quenching of fibrin generation. The action of both AT and TFPI inhibits FXa during the initiation phase leading to dependence of platelet-surface FXa generation during the propagation phase for adequate hemostasis.


The fibrinolytic system also includes two inhibitors, principally plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin (alpha2AP), which inhibit tPA and plasmin, respectively.