Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.

Abstract Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

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Human platelets contain forms of factor V in disulfide-linkage with multimerin

Thrombosis and Haemostasis ◽

10.1160/th03-02-0123 ◽

2004 ◽

Vol 92 (12) ◽

pp. 1349-1357 ◽

Cited By ~ 12

Author(s):

Nola Fuller ◽

Shilun Zheng ◽

Frédéric Adam ◽

Samira Jeimy ◽

Ian Horsewood ◽

...

Keyword(s):

Disulfide Bonds ◽

Human Platelets ◽

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Disulfide Linkage ◽

Factor Va ◽

Plasma Factor ◽

Large Platelet ◽

Large Factor

SummaryFactor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factorV is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage.The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

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Cleavage of Factor Va Heavy Chain at Arg643 by Thrombin Partially Inactivates the Cofactor.

Blood ◽

10.1182/blood.v108.11.1703.1703 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1703-1703 ◽

Cited By ~ 1

Author(s):

Evrim Erdogan ◽

Michael A. Bukys ◽

Thomas Orfeo ◽

Kenneth G. Mann ◽

Michael Kalafatis

Keyword(s):

Endothelial Cells ◽

Heavy Chain ◽

Consensus Sequence ◽

Factor V ◽

Single Chain ◽

Chain Fragment ◽

Thrombin Cleavage ◽

Factor Va ◽

Plasma Factor ◽

Cofactor Activity

Abstract Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr 105,000) and light (Mr 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643 for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643→Q (factor VR643Q) and R643→A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105 and appearance of the Mr 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Q and factor VR643A with thrombin no cleavage of HC105 at Arg643 was observed and no presence of the Mr 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105 over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105 at Arg643 by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

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The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Blood ◽

10.1182/blood.v69.4.1188.1188 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1188-1195 ◽

Cited By ~ 5

Author(s):

RW Viskup ◽

PB Tracy ◽

KG Mann

Keyword(s):

Human Plasma ◽

Human Platelet ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Factor Va ◽

Plasma Factor ◽

End Products

Abstract Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

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The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Blood ◽

10.1182/blood.v69.4.1188.bloodjournal6941188 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1188-1195 ◽

Cited By ~ 21

Author(s):

RW Viskup ◽

PB Tracy ◽

KG Mann

Keyword(s):

Human Plasma ◽

Human Platelet ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Factor Va ◽

Plasma Factor ◽

End Products

Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

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Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽

10.1182/blood.v83.8.2180.bloodjournal8382180 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2180-2190

Author(s):

MD Rand ◽

M Kalafatis ◽

KG Mann

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Activated Protein C ◽

Coagulation Factor ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Factor V ◽

Platelet Factor ◽

Factor Va ◽

Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

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Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽

10.1182/blood.v54.5.1015.bloodjournal5451015 ◽

1979 ◽

Vol 54 (5) ◽

pp. 1015-1022 ◽

Cited By ~ 2

Author(s):

JP Miletich ◽

WH Kane ◽

SL Hofmann ◽

N Stanford ◽

PW Majerus

Keyword(s):

Binding Sites ◽

Factor Xa ◽

Coagulation Factors ◽

Severe Bleeding ◽

Factor V ◽

Platelet Factor ◽

Platelet Surface ◽

Factor Va ◽

Number Of Binding Sites ◽

Stimulation Of

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

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Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Blood ◽

10.1182/blood.v68.4.875.875 ◽

1986 ◽

Vol 68 (4) ◽

pp. 875-880 ◽

Cited By ~ 114

Author(s):

T Wiedmer ◽

CT Esmon ◽

PJ Sims

Keyword(s):

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Procoagulant Activity ◽

Ionophore A23187 ◽

Factor V ◽

Platelet Factor ◽

Platelet Surface ◽

Prothrombinase Complex ◽

Factor Va ◽

Exogenous Factors

Abstract The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.

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Interaction of Factor V and Factor Va with Platelets

10.1055/s-0039-1687300 ◽

1979 ◽

Author(s):

P.B. Tracy ◽

J. M. Peterson ◽

M.E. Nesheim ◽

F.C. McDuffie ◽

K. G. Mann

Keyword(s):

Binding Sites ◽

Intrinsic Factor ◽

Factor V ◽

Single Chain ◽

Apparent Dissociation Constant ◽

Platelet Factor ◽

High Affinity ◽

Factor Va ◽

Thrombin Activation ◽

Triton X

We have used homogeneous single chain bovine factor V to examine the binding of both factor V and factor Va to bovine platelets, as well as to develop a double-antibody radioimmunoassay (RIA) to measure intrinsic platelet factor V. Reaction of the protein with 125I Bolton-Hunter reagent produced a labelled product which retained 90% of its cofactor activity and gave products indistinguishable from native factor V following thrombin activation. When incubated separately with washed bovine platelets, both 125I-factor V and Va underwent saturable and exchangeable binding. There are high affinity binding sites to which 500-900 V(Va) molecules are bound per platelet with an apparent dissociation constant of 3 χ 10-10 M, as well as binding sites of slightly lower affinity (Kd = 3 χ 10-9M) to which as many as 3500 V (Va) molecules are bound per platelet. Thrombin pretreatment of the platelets was not required for the binding of either factor V or Va. The RIA data for Triton X-100 lysed, washed bovine platelets revealed that 400-1000 intrinsic factor V molecules were present per platelet. Factor V clotting assays produced results consistent with the RIA data. These studies suggest that the factor V molecules intrinsic to the platelet are equivalent to the number of high affinity factor V (Va) binding sites present on the platelet memhrane surface. (Supported by Grant HL-17430 and the hayo Foundation).

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Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽

10.1182/blood.v83.8.2180.2180 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2180-2190 ◽

Cited By ~ 52

Author(s):

MD Rand ◽

M Kalafatis ◽

KG Mann

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Activated Protein C ◽

Coagulation Factor ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Factor V ◽

Platelet Factor ◽

Factor Va ◽

Plasma Factor

Abstract Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

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Identification of Sulfo-Tyrosines of Factor V.

Blood ◽

10.1182/blood.v104.11.1731.1731 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1731-1731

Author(s):

Evrim Erdogan ◽

Michael Kalafatis

Keyword(s):

Amino Acid ◽

Factor Xa ◽

Factor V ◽

Wild Type ◽

Single Chain ◽

Amino Acid Region ◽

Thrombin Cleavage ◽

Factor Va ◽

Cofactor Activity ◽

Acid Region

Abstract The factor Va molecule is the essential cofactor of the prothrombinase complex. This complex composed of factor Xa and factor Va assembled on a platelet membrane-surface in the presence of Ca2+ ions converts membrane-bound prothrombin to thrombin. Single chain factor V does not bind factor Xa. Single-chain factor V is cleaved by thrombin first at Arg709 followed by cleavages at Arg1018 and Arg1545 to produce the heavy and light chains of the active cofactor (factor Va) and two activation peptides. Efficient thrombin cleavage and activation of factor V is essential for cofactor function and requires tyrosine sulfation. Tyrosine sulfation of factor V also appears to regulate its activity. Seven tyrosine residues in factor V, Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, and Tyr1565 have been identified as potential sites of sulfation. However, which residues are sulfated and their contribution to procofactor activation and cofactor function still remain to be elucidated. Two of the sulfation sites Tyr696 and Tyr698 are located in the acidic amino acid region near to the first required thrombin cleavage site at Arg709. Recent data demonstrated that these residues are essential for factor V activation and cofactor activity. Another acidic amino acid region, 1490–1520 is adjacent to the thrombin cleavage site at Arg1545 required for light chain formation. This region also contains three potential sulfation sites at residues 1494, 1510, and 1515 and was shown to be required for optimum procofactor activation. To ascertain which of these three residues is important for procofactor activation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations 1493DY1494→AF, 1508DDY1510→AAF and 1514DY1515→AF. The clotting and cofactor activity of the 1493DY1494→AF and 1514DY1515→AF mutants was similar to the clotting activity observed with the wild type recombinant factor Va molecule following activation by thrombin or RVV-V activator. In contrast, under similar experimental conditions recombinant factor V with the substitution 1508DDY1510→AAF was deficient in its clotting activity and had impaired cofactor activity. Moreover, following prolonged incubation with thrombin, no light chain formation was observed in the factor V molecule bearing the 1508DDY1510→AAF mutation. Thus, amino acid residues 1508–1510 of factor V are required for thrombin interaction with the procofactor which in turn appears necessary for cleavage at Arg1545. Studies of sulfated proteins have shown that the effect of sulfo-tyrosines on protein structure/function can be preserved by replacing them with glutamic acid. To explicitly identify the sulfated tyrosines on the factor V molecule, we mutated Tyr696, Tyr698 and Tyr1510 to glutamic acid and transfected them into COS-7L cells. Expression was performed in the presence of media containing or devoid of sulfate. In the presence of sulfate, the cofactor and clotting activities of the DY696DY698→DEDE and DDY1510→DDE mutants, separately were similar to the wild type recombinant factor Va molecule. However, in the absence of sulfate, the wild type and the mutant recombinant factor V molecules had both impaired cofactor activity and clotting activity following their activation with thrombin. However, their respective activity was higher than the activity of the factor V molecule bearing the 1508DDY1510→AAF mutation. Our data suggest that residues 696, 698, and 1510 of factor V appear to be sulfated and might be important for procofactor activation and cofactor function.

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