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

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

Blood ◽

10.1182/blood.v104.11.1933.1933 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1933-1933

Author(s):

Catherine P.M. Hayward ◽

Nola Fuller ◽

Shilun Zheng ◽

Frederic Adam ◽

Samira Jeimy ◽

...

Keyword(s):

Factor V ◽

Single Chain ◽

Platelet Factor ◽

Platelet Surface ◽

Disulfide Linkage ◽

Thrombin Cleavage ◽

Factor Va ◽

Plasma Factor ◽

Large Factor ◽

V Antigen

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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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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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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Human Plasma And Platelet Factor V Levels As Measured By Radioimmunoassay

10.1055/s-0038-1652216 ◽

1981 ◽

Author(s):

P B Tracy ◽

J M Peterson ◽

M E Nesheim ◽

J A Katzmann ◽

K G Mann

Keyword(s):

Human Factor ◽

Specific Activity ◽

Human Platelets ◽

Prolonged Storage ◽

Factor V ◽

Platelet Factor ◽

Average Value ◽

Standard Curve ◽

Plasma Factor ◽

Bioassay Data

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.

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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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Thrombin-Induced Platelet Factor Va Formation in Patients with a Gray Platelet Syndrome

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645967 ◽

1987 ◽

Vol 58 (02) ◽

pp. 768-771 ◽

Cited By ~ 6

Author(s):

Dominique Baruch ◽

Theo Lindhout ◽

Evelyne Dupuy ◽

Jacques P Caen

Keyword(s):

Normal Values ◽

Factor V ◽

Platelet Factor ◽

Normal Platelet ◽

Factor Va ◽

Platelet Disorder ◽

Plasma Factor ◽

Gray Platelet Syndrome ◽

Kinetics Of ◽

Functional Factor

SummaryThe present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on tlnumbin concentration was altered. Increasing the thrombin concentration 4-lold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor VQ activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.

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Subcellular Localization and Secretion of Factor V by Human Platelets

10.1055/s-0039-1682667 ◽

1977 ◽

Author(s):

D.D. Pifer ◽

R.W. Colman ◽

C.Mcl. Chesney

Keyword(s):

Molecular Weight ◽

Subcellular Localization ◽

Gel Filtration ◽

Molecular Size ◽

Apparent Molecular Weight ◽

Human Platelets ◽

Factor V ◽

Platelet Factor ◽

Dense Granules ◽

Plasma Factor

Previous studies have suggested that factor V in platelets is derived from plasma. In order to test this hypothesis, we studied the subcellular localization, release by aggregating agents, molecular size, and stability of factor V in human platelets. When platelet homoge-nates were fractionated on sucrose density gradient the factor V activity distributed primarily into the granules (0.076 units factor V activity/mg protein) with less amounts in the membrane fraction (0.014 units/mg protein) and virtually none in the dense granules or cytosol. Collagen released 79-3% of total platelet factor V clotting activity while ADP and epinephrine failed to liberate factor V activity, further supporting significant localization in the lysozomal granules rather than the dense granules. Platelet factor V can be solubilized by 0.1% Triton and has an apparent molecular weight of 470,000 as determined by 4% agarose gel filtration. In contrast, plasma factor V in the presence or absence of Triton has a molecular weight of 300,000. Factor V activity from the platelet homogenate exhibited biphasic decay at 37°C, suggesting more than one form, whereas plasma factor V shows a first order decay curve. These data suggest that platelet factor V is predominantly localized in the granules and is an intrinsic constituent of the platelet with different properties from plasma V.

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