The contribution of amino acid residues 1508-1515 of factor V to light chain generation.

BACKGROUND: Factor (F) V is activated by alpha-thrombin following cleavages at Arg(709), Arg(1,018) and Arg(1,545). Amino acid region 1,490-1,520 of FV is essential for procofactor activation. AIM: To ascertain which amino acid residues from this region are important for light chain formation and procofactor activation, site-directed mutagenesis was used to create recombinant FV molecules missing amino acid 1,508-1,510 (FV(Delta1,508-1,510)) and 1,508-1,515 (FV(Delta1508-1515)). We have also created recombinant FV molecules with mutations (1508)DDY(1510)-->AAF (FV(AAF)), (1514)DY(1515)-->AF (FV(AF)) and Y(1510)-->F (FV(Y1510F)). METHODS AND RESULTS: The recombinant mutant molecules were expressed and purified to homogeneity. The clotting activities of all clotting recombinant mutant FV molecules were tested in a two-stage assay following activation by alpha-thrombin and were found to be impaired compared with the clotting activity observed with wild-type recombinant FV or plasma-derived FV, with the exception of FV(Y1510F), which had normal clotting activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FV demonstrated that incubation of 100 nm recombinant wild-type or plasma-derived FV with 1 nmalpha-thrombin for 5 min was sufficient to generate heavy and light chains and completely activate the procofactor. In contrast, similar experimental conditions were ineffective in fully activating the two deletion mutant molecules as well as FVa(AAF) and FVa(AF), resulting in accumulation of a M(r) 220,000 fragment representing amino acids 1,019-2,195. CONCLUSION: Our data demonstrate that amino acid residues 1,508-1,515 of FV are required for efficient cleavage by alpha-thrombin and light chain formation.

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Megakaryocytes endocytose and subsequently modify human factor V in vivo to form the entire pool of a unique platelet-derived cofactor.

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential cofactor in thrombin production catalyzed by the prothrombinase complex. Plasma-derived factor V (FV) is synthesized in the liver. The source(s) of the platelet-derived cofactor remains in question. We identified a patient homozygous for the FV(Leiden) mutation, who received a liver transplant from a homozygous wild-type FV donor. Eighteen days post-transplant, phenotypic analysis of the patient's platelet-derived FV indicated that the platelets were acquiring wild-type FV, consistent with the temporal differentiation of megakaryocytes and subsequent platelet production. Nine months post-transplant, the platelet-derived FV pool consisted entirely of wild-type FV. Consequently, megakaryocyte endocytosis of plasma-derived FV must account for the entire platelet-derived pool, because blood-borne platelets cannot bind or endocytose FV. Subsequent to this endocytic process, the patient's platelet-derived FV was cleaved to a partially active cofactor, and rendered resistant to phosphorylation catalyzed by a platelet-associated kinase, and hence less susceptible to activated protein C-catalyzed inactivation. These data provide the first in vivo demonstration of an endocytosed plasma protein undergoing intracellular modifications that alter its function. This process results in the sequestration of active FVa within the platelet compartment, poised for immediate action subsequent to release from platelets at a site of injury.

A matrix-assisted laser desorption/ionization time-of-flight based method for screening the 1691G --> A mutation in the factor V gene.

A point mutation, 1691 G --> A in the coagulation factor V gene results in an Arg506 --> Gln amino acid substitution in the factor V molecule. This mutation, defined as factor VLEIDEN, results in activated protein C (APC) resistance and is the most common genetic risk factor for familial thrombophilia. A new mini-sequencing method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed for the screening of the 1691G --> A substitution in factor V. In this method, a fragment of genomic DNA containing the 1691st base is first amplified, followed by mini-sequencing in the presence of dGTP and ddATP, ddCTP, and ddTTP. In this manner, the primer is extended by one base from one allele and two bases from the other allele. The extended products are analyzed using MALDI-TOF mass spectrometry. The base at position 1691 is identified based on the number of nucleotides added. We have used this method to genotype 16 APC-resistant patients previously identified by conventional methods and 11 normal control samples. The genotypes of all samples were correctly identified. This method is accurate, fast, and potentially allows for simultaneous multiplex genotyping of a number of mutation sites associated with thrombophilia and clot formation.

Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (protein C(R-1L/propeptide)).

A heterozygous G-->T transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.

Factor V: Dr. Jeckyll and Mr. Hyde.

The regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. The procoagulant enzymatic complexes (i.e. prothrombinase, intrinsic tenase and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities respectively because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events, which involve alpha-thrombin. In general bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. a-Thrombin, participates in its own down-regulation by binding to the endothelial cell receptor thrombomodulin, and initiating the protein C pathway, which in turn leads to the formation of activated protein C (APC). APC is required for efficient neutralization of factor Va cofactor activity which results in the inactivation of the prothrombin-activating complex. This inactivation can only occur in the presence of the appropriate membrane surface. APC down-regulates the prothrombinase complex by cleaving specific peptide bonds on the heavy chain of factor Va which results in the dissociation of the A2 domain of factor Va from the rest of the molecule. Irregularities in the mechanism of inactivation of factor Va by APC, are associated with thrombotic risk, presumably due to sustained prothrombin activation.

A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: identification of a novel microdeletion (delC556) and missense mutation (Lys(408)-->Asn) in the factor X gene. A study of an Italian family.

Low levels of factor X (F.X) were detected in a 4-year-old boy who experienced acute lymphoblastic leukemia and bleeding manifestations. Laboratory data suggested the presence of a dysfunctional F.X molecule. Two novel F.X gene mutations were identified in the proband that was double heterozygous for both: a microdeletion (delC556) in exon VI resulting in a frameshift leading to a termination codon at position 226. This deletion was found in six family members with reduced F.X antigen and activity levels. A second mutation characterised by a G(1344)-->C transversion in exon VIII was detected in the proband resulting in a Lys(408)-->Asn substitution. This latter mutation was present in several asymptomatic family members from the paternal and the maternal side. The proband's sister was homozygous for the Lys(408)-->Asn substitution and exhibited low F.X activity with a normal antigen level. The naturally occurring F.X Lys(408)-->Asn (F.X(K408N)) variant was isolated from plasma of either homozygous or double heterozygous individuals. NH(2)-terminal sequencing of the heavy chain of F.X(K408N) failed to show any sequence abnormality in patients who were also carriers of the delC556, suggesting that this latter lesion accounted for the lack of F.X synthesis. Purified F.X Lys(408)-->Asn had an identical behaviour to normal F.X as judged by SDS-PAGE and immunoblotting. Clotting assay using purified F.X(K408N) and F.X-deficient plasma resulted in a laboratory phenotype similar to that observed in a homozygous subject for F.X Lys(408)-->Asn substitution. This is the first characterisation of a naturally occurring F.X variant with a mutation at the COOH-terminal end of the molecule.

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function.

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a fragment of M(r) 47,000 containing the NH(2)-terminal part of the cofactor (amino acid residues 1-348) and a M(r) 42,000 fragment (amino acid residues 349-713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 fragment: Lys(309), Lys(310), and Arg(313). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 fragment containing the NH(2)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 fragment at Arg(306) by APC and appearance of a M(r) 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately 1.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.

Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family.

The study of the molecular bases of thrombophilia in a large family with 4 symptomatic members is reported. Three thrombophilic genetic components (FV R506Q, FV H1299R, and PT 20210G/A), all affecting the activity of the prothrombinase complex, were detected alone and in combination in various family members. In addition, a newly identified missense mutation (factor V [FV] Y1702C), causing FV deficiency, was also present in the family and appeared to enhance activated protein C (APC) resistance in carriers of FV R506Q or FV H1299R by abolishing the expression of the counterpart FV allele. The relationships between complex genotypes, coagulation laboratory findings, and clinical phenotypes were analyzed in the family. All symptomatic family members were carriers of combined defects and showed APC resistance and elevated F1 + 2 values. Evidence for the causative role of the FV Y1702C mutation, which affects a residue absolutely conserved in all 3 A domains of FV, factor VIII, and ceruloplasmin, relies on (1) the absolute cosegregation between the mutation and FV deficiency, both in the family and in the general population; (2) FV antigen and immunoblot studies indicating the absence of Y1702C FV molecules in plasma of carriers of the mutation, despite normal levels of the FV Y1702C messenger RNA; and (3) molecular modeling data that support a crucial role of the mutated residue in the A domain structure. These findings help to interpret the variable penetrance of thrombosis in thrombophilic families and to define the molecular bases of FV deficiency. (Blood. 2000;96:1443-1448)

A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition.

The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.

Phenotype and genotype expression in pseudohomozygous factor VLEIDEN : the need for phenotype analysis.

The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G1691-->A, resulting in an Arg506-->Gln amino acid substitution in the factor V molecule [factor VLEIDEN], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor VLEIDEN phenotype would occur if a heterozygous individual for factor VLEIDEN also did not express the "normal" (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor VLEIDEN form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G1691-->A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients' plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor VLEIDEN. Overall, these data suggest the existence of heterogeneous genetic "lesions," which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor VLEIDEN molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.

Molecular bases of pseudo-homozygous APC resistance: the compound heterozygosity for FV R506Q and a FV null mutation results in the exclusive presence of FV Leiden molecules in plasma.

Pseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient's non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient's plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.

Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding.

The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.

Human protein S cleavage and inactivation by coagulation factor Xa.

Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg60, a site that is distinct from those utilized by alpha-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-Gly-Arg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca2+, and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (Kd approximately 4 nM +/- 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.

Identification and partial characterization of factor Va heavy chain kinase from human platelets.

Factor Va, the essential cofactor for prothrombinase, is phosphorylated on the acidic COOH terminus of the heavy chain of the cofactor, at Ser692, by a platelet membrane-associated casein kinase II (CKII). Consistent with this observation, phosphorylation of the factor Va heavy chain by the platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was monitored using a polyclonal antibody to the alpha-subunit, and kinase activity in the various fractions was confirmed using human factor Va as a substrate. Immunoblotting experiments using polyclonal antibodies raised against synthetic peptides mimicking a portion of the deduced amino acid sequence of the alpha-, alpha'-, and beta-subunits of human CKII demonstrated the coexistence of both alpha- and alpha'-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha'beta2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alphaalpha/betabeta and one that is alpha'alpha'/betabeta. In the presence of the purified platelet-derived CKII, human factor Va incorporates between 0.8 and 1.3 mol of phosphate/mol of factor Va depending on the concentration of the beta-subunit in the kinase preparation. A peptide mimicking the sequence 687-705 of the human factor V molecule incorporates radioactivity in the presence of purified CKII and inhibits factor Va heavy chain phosphorylation by the platelet CKII. In contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation by the platelet CKII. Human factor Va is inactivated by activated protein C following three cleavages of the heavy chain at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure of the inactivating cleavage site at Arg306. The phosphorylated cofactor has increased susceptibility to inactivation by activated protein C, since phosphorylated factor Va was found to be inactivated approximately 3-fold faster than its native counterpart. Acceleration of the inactivation process of the phosphorylated cofactor occurs because of acceleration of the rate of cleavage at Arg506. These data suggest a critical role for factor Va phosphorylation on the surface of platelets in regulating cofactor activity.

Platelet-derived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.

We investigated the role of the thrombin-activated platelet in modulating the rate and extent of activated protein C (APC)-catalyzed inactivation of platelet-derived factor Va and factor VaLeiden. Platelet-derived factor Va and factor VaLeiden were inactivated by APC at near identical rates; however, complete inactivation of the cofactors was never achieved. Greater residual cofactor activity remained when using thrombin-activated platelets compared with that observed with synthetic phospholipid vesicles and platelet-derived microparticles, suggesting that thrombin-activated platelets protect the cofactors from APC-catalyzed inactivation. This apparent protection was not due to (1) an insufficient number of membrane binding sites for APC or factor Va; (2) the destruction of these sites; or (3) the presence of a platelet-associated APC inhibitor. Results from a plasma-based clotting assay (with or without APC) with platelets or PCPS vesicles added to induce clot formation indicated that, even in the presence of high concentrations of APC, platelets offered protection of the cofactor by delaying cleavage at Arg506. This resulted in incomplete proteolysis of the heavy chain, suggesting that platelets can also protect plasma-derived factor Va from APC-catalyzed inactivation. However, additional experiments indicated that the plasma-derived cofactor, bound to thrombin-activated platelets, was completely inactivated by APC, suggesting that the plasma and platelet-derived cofactor pools represent different substrates for APC. Collectively, these results indicate that platelets sustain procoagulant events by providing a membrane surface that delays cofactor inactivation and by releasing a cofactor molecule that displays an APC resistant phenotype. Thus, at sites of arterial injury, the factor VLeiden mutation may not as readily predict arterial thrombosis, because the normal and variant platelet-derived cofactors are equally resistant to APC at the activated platelet surface.

Protein C activation and factor Va inactivation on human umbilical vein endothelial cells.

The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10(5) cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg506 and Arg306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg506 was accelerated in the presence of HUVECs, while cleavage at Arg306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg506, generating an M(r) 75,000 species. Further proteolysis at Arg306 generated an M(r) 30,000 product. When protein C (0.5 mumol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10(-14) mol/min per cell was observed. With APC generated in situ, cleavage at Arg506 on the HUVEC surface is followed by cleavage at Arg306, generating M(r) 75,000 and M(r) 30,000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M(r) 97,000 fragment, which is further processed by APC to generate an M(r) 43,000 fragment. NH2-terminal sequence analysis of the M(r) 97,000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M(r) 97,000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg506 followed by a second cleavage at Arg306. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.

An in vitro analysis of the combination of hemophilia A and factor V(LEIDEN).

The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor V(LEIDEN) mutation, which leads to the substitution of Arg506 --> Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII-deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor V(LEIDEN) mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa x tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (approximately 6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor V(LEIDEN) mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor V(LEIDEN) concentration. When the factor V(LEIDEN) concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor V(LEIDEN) level to understand the effect of factor V(LEIDEN) in hemophilia A patients. The presented data support the hypothesis that the factor V(LEIDEN) mutation can increase thrombin formation in severe hemophilia A.