Phosphatidylserine and FVa regulate FXa structure.

Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.

An update on the coagulopathy of trauma.

Trauma remains the leading cause of death with bleeding as the primary cause of preventable mortality during the first 24 h following trauma. When death occurs, it happens quickly, typically within the first 6 h after injury. One of four patients to arrive in the emergency department after trauma is already in the state of acute traumatic coagulopathy and shock. The principal drivers of acute traumatic coagulopathy have been characterized by tissue hypoperfusion, inflammation, and the acute activation of the neurohumoral system. Hypoperfusion leads to an activation of protein C with cleavage of activated factors V and VIII and the inhibition of plasminogen activator inhibitor 1 with subsequent hyperfibrinolysis. Endothelial damage and activation result in Weibel-Palade body degradation and glycocalyx shedding associated with autoheparinization. In contrast, there is an iatrogenic coagulopathy that occurs secondary to uncritical volume therapy leading to acidosis, hypothermia, and hemodilution. This coagulopathy then may be an integral part of the "vicious cycle" when combined with acidosis and hypothermia. The present article summarizes an update on the principal mechanisms and triggers of the coagulopathy of trauma including traumatic brain injury.

Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va.

Activated protein C inhibits the procoagulant function of activated factor V (FVa) through proteolytic cleavages at Arg-306, Arg-506, and Arg-679. The cleavage at Arg-506 is kinetically favored but protected by factor Xa (FXa). Protein S has been suggested to annihilate the inhibitory effect of FXa, a proposal that has been challenged. To elucidate the effects of FXa and protein S on the individual cleavage sites of FVa, we used recombinant FVa:Q306/Q679 and FVa:Q506/Q679 variants, which can only be cleaved at Arg-506 and Arg-306, respectively. In the presence of active site blocked FXa (FXa-1.5-dansyl-Glu-Gly-Arg), the FVa inactivation was followed over time, and apparent second order rate constants were calculated. Consistent with results on record, we observed that FXa-1.5-dansyl-Glu-Gly-Arg decreased the Arg-506 cleavage by 20-fold, with a half-maximum inhibition of approximately 2 nM. Interestingly and in contrast to the inhibitory effect of FXa on the 506 cleavage, FXa stimulated the Arg-306 cleavage. Protein S counteracted the inhibition by FXa of the Arg-506 cleavage, whereas protein S and FXa yielded additive stimulatory effect of the cleavage at Arg-306. This suggests that FXa and protein S interact with distinct sites on FVa, which is consistent with the observed lack of inhibitory effect on FXa binding to FVa by protein S. We propose that the apparent annihilation of the FXa protection of the Arg-506 cleavage by protein S is due to an enhanced rate of Arg-506 cleavage of FVa not bound to FXa, resulting in depletion of free FVa and dissociation of FXa-FVa complexes.

Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation.

The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent-exposed amino acids, Tyr(1956)/Leu(1957) in the C1 domain and Trp(2063)/Trp(2064) in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp(2063)/Trp(2064) and/or Tyr(1956)/Leu(1957); (ii) combined mutation of Trp(2063)/Trp(2064) and Tyr(1956)/Leu(1957) results in only a modest 4-fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3-C1-C2).

Factor V Leiden: association with venous thromboembolism in pregnancy and screening issues.

Disturbances of the natural balance between procoagulant and anticoagulant mechanisms can result in bleeding or thrombotic tendencies. Factor V, on activation by thrombin to factor Va, forms an essential component of the prothrombinase complex, in which it demonstrates its cofactor activity for factor Xa. Down-regulation of factor Va by activated protein C (APC) occurs through cleavage of specific peptide bonds in the heavy chain of the molecule. Factor V Leiden (FV Leiden) is a mutation of factor V that renders factor Va resistant to APC, due to loss of one of these cleavage sites. This mutation predisposes the patient to thrombosis. Prevalence of FV Leiden varies; however, heterozygosity for the FV Leiden mutation is recognised as the most common heritable thrombophilic defect in Caucasian populations. The association this inherited thrombophilia has with venous thromboembolism (VTE) is well established. Pregnancy is notably an acquired hypercoagulable state, due in part to physiological changes that occur in the coagulation system. This seems to have potential for interaction with FV Leiden to cause adverse experiences. A role has been suggested for FV Leiden in VTE events during pregnancy. At present only selected women are screened for FV Leiden. Pregnant women with a history of VTE or with a family history of the mutation are investigated. Whether or not the introduction of a routine screening plan for this mutation is justified remains a matter for debate.

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function.

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1.A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

Coagulation factor Va Glu-96-Asp-111: a chelator-sensitive site involved in function and subunit association.

Coagulation FVa (factor Va) accelerates the essential generation of thrombin by FXa (factor Xa). Although the noncovalent Ca2+-dependent association between the FVa light and heavy subunits (FVaL and FVaH) is required for function, little is known about the specific residues involved. Previous fragmentation studies and homology modelling led us to investigate the contribution of Leu-94-Asp-112. Including prospective divalent cation-binding acidic amino acids, nine conserved residues were individually replaced with Ala in the recombinant B-domainless FVa precursor (DeltaFV). While mutation of Thr-104, Glu-108, Asp-112 or Tyr-100 resulted in only minor changes to FXa-mediated thrombin generation, the functions of E96A (81%), D111A (70%) and D102A (60%) mutants (where the single-letter amino acid code is used) were notably reduced. The mutants targeting neighbouring acidic residues, Asp-79 and Glu-119, had activity comparable with DeltaFV, supporting the specific involvement of select residues. Providing a basis for reduced activity, thrombin treatment of D111A resulted in spontaneous dissociation of subunits. Since FVaH and FVaL derived from E96A or D102A remained associated in the presence of Ca2+, like the wild type, but conversely dissociated rapidly upon chelation, a subtle difference in divalent cation co-ordination is implied. Subunit interactions for all other single-point mutants resembled the wild type. These data, along with corroborating multipoint mutants, reveal Asp-111 as essential for FVa subunit association. Although Glu-96 and Asp-102 can be mutated without gross changes to divalent cation-dependent FVaH-FVaL interactions, they too are required for optimal function. Thus Glu-96-Asp-111 imparts at least two discernible effects on FVa coagulation activity.

Exposure of platelet membrane phosphatidylserine regulates blood coagulation.

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.

Structural requirements for expression of factor Va activity.

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.

Factor V and thrombotic disease: description of a janus-faced protein.

The generation of thrombin by the prothrombinase complex constitutes an essential step in hemostasis, with thrombin being crucial for the amplification of blood coagulation, fibrin formation, and platelet activation. In the prothrombinase complex, the activated form of coagulation factor V (FVa) is an essential cofactor to the enzyme-activated factor X (FXa), FXa being virtually ineffective in the absence of its cofactor. Besides its procoagulant potential, intact factor V (FV) has an anticoagulant cofactor capacity functioning in synergy with protein S and activated protein C (APC) in APC-catalyzed inactivation of the activated form of factor VIII. The expression of anticoagulant cofactor function of FV is dependent on APC-mediated proteolysis of intact FV. Thus, FV has the potential to function in procoagulant and anticoagulant pathways, with its functional properties being modulated by proteolysis exerted by procoagulant and anticoagulant enzymes. The procoagulant enzymes factor Xa and thrombin are both able to activate circulating FV to FVa. The activity of FVa is, in turn, regulated by APC together with its cofactor protein S. In fact, the regulation of thrombin formation proceeds primarily through the upregulation and downregulation of FVa cofactor activity, and failure to control FVa activity may result in either bleeding or thrombotic complications. A prime example is APC resistance, which is the most common genetic risk factor for thrombosis. It is caused by a single point mutation in the FV gene (factor V(Leiden)) that not only renders FVa less susceptible to the proteolytic inactivation by APC but also impairs the anticoagulant properties of FV. This review gives a description of the dualistic character of FV and describes the gene-gene and gene-environment interactions that are important for the involvement of FV in the etiology of venous thromboembolism.

Functional properties of factor V and factor Va encoded by the R2-gene.

Carriership of the factor V (FV) gene marked by the R2-haplotype, a series of linked polymorphisms encoding several amino acid changes in FV, is associated with mild resistance to activated protein C (APC) and with an increased risk of thrombosis. We compared the functional properties of normal FV(a) and R2-FV(a) in model systems and in plasma. FV and R2-FV were equally well activated by thrombin and expressed identical cofactor activities in prothrombin activation. Rate constants of APC-catalyzed inactivation of FVa and R2-FVa were similar both with and without protein S. However, significant differences were observed between haemostatic parameters determined in plasma from homozygous carriers of the R2-gene (n = 5) and age-matched non-carriers (n = 19). Plasma from R2-carriers contained significantly lower FV levels and the ratio of the two FV isoforms (FV1 and FV2) was shifted in favor of FV1. The FV2/FV1 ratio was 1.4 (95% CI = 1.3-1.5) in homozygous carriers of R2 and 2.8 (95% CI = 2.5-3.1) in controls (p < 0.00001). In an APC resistance test which quantifies the cofactor activity of FV in APC-catalyzed FVIII(a) inactivation, homozygous R2-carriers had significantly lower (p < 0.00001) APC sensitivity ratios (APCsr = 1.54, 95% CI = 1.48-1.60) than controls (APCsr = 2.17, 95% CI = 2.05-2.28). This indicates that R2-FV has reduced cofactor activity in APC-catalyzed FVIII(a) inactivation. The changes of the relative amounts of FV1 and FV2 in carriers of the R2-gene will result in increased thrombin formation in the presence of APC and may provide a mechanistic explanation for the increased thrombotic risk associated with the R2-haplotype.

Coagulation factor V: an old star shines again.

Blood coagulation factor V plays an important role in the regulation of thrombin formation. Activation of factor V by traces of activated coagulation factors (thrombin, factor Xa or meizothrombin) yields factor Va, the non-enzymatic cofactor of the prothrombinase complex. Since factor Va accelerates prothrombin activation under physiological conditions more than 10(4)-fold it is not surprising that down-regulation of factor Va cofactor activity by the protein C pathway is a very effective way for maintaining the hemostatic balance. In this paper we have reviewed the present status of structural knowledge of factor V and Va, the molecular changes in factor V that occur during factor V activation, the function of factor Va in prothrombin activation and the molecular mechanism of inactivation of factor Va by APC. Although considerable insight in the structure-function relationship of factor V and Va has been achieved, the study of mutated factor V molecules obtained by recombinant DNA technology will undoubtedly resolve remaining questions. The latter is illustrated by the fact that the discovery of factor VaLeiden has significantly contributed to our present knowledge on the regulation of the cofactor activity of factor Va via the protein C pathway. It appears that modulation of the activity of APC by protein S and factor Xa will strongly affect the in vivo activity of this pathway. Factor V not only plays an important role in the regulation of the activity of the prothrombinase complex but also acts as cofactor in APC-mediated inactivation of factor VIIIa. This gives rise to a rather intricate mechanism of regulation of thrombin formation by APC that thus far has been mainly studied in model systems containing purified proteins. Thus, extensive studies in plasma will be required in order to get more insight in the in vivo regulation of thrombin formation via the protein C pathway.

Factor V.

Factor V is a single chain glycoprotein that plays an essential role in the regulation of blood coagulation. After initiation of coagulation, factor V is converted into factor Va through limited proteolysis. Factor Va acts as protein cofactor in the prothrombin-activating complex, which is comprised of the serine protease factor Xa, Ca2+ ions and a procoagulant membrane surface. Factor Va accelerates factor Xa-catalysed conversion of prothrombin into thrombin more than 10(4)-fold. The cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C (APC). The physiological importance of this regulatory pathway is demonstrated by the occurrence of hereditary thrombophilia in individuals with a genetic defect that makes factor Va less sensitive to proteolytic inactivation by APC (APC resistance).

A mathematical model for the spatio-temporal dynamics of intrinsic pathway of blood coagulation. I. The model description.

We developed and analyzed the mathematical model of the intrinsic pathway based on the current biochemical data on the kinetics of blood coagulation individual stages. The model includes eight differential equations describing the spatio-temporal dynamics of activation of factors XI, IX, X, II, I, VIII, V, and protein C. The assembly of tenase and prothrombinase complexes is considered as a function of calcium concentration. The spatial dynamics of coagulation was analyzed for the one-dimensional case. We examined the formation of active factors, their spreading, and growth of the clot from the site of injury in the direction perpendicular to the vessel wall, into the blood thickness. We assumed that the site of injury (in the model one boundary of the space segment under examination) becomes a source of the continuous influx of factor XIa. In the first part, we described the model, selected the parameters, etc. In the second part, we compared the model with experimental data obtained in the homogeneous system and analyzed the spatial dynamics of the clot growth.