Phenotypic Comparison of Platelet- and Plasma-Derived Factor Va: Tyrosine Phosphorylation Differentiates the Two Cofactor Pools.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1696-1696
Author(s):  
Jeremy P. Wood ◽  
Paula B. Tracy
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Whole Blood ◽  
Membrane Surface ◽  
Tyrosine Phosphatase ◽  
Light Chains ◽  
Factor V ◽  
Phosphatase Inhibitors ◽  
Factor Va

Abstract Factor Va, the essential cofactor for thrombin formation via the Prothrombinase complex, exists in two pools in whole blood: 75–80% is found in the plasma, while platelet alpha-granules contain the remaining 20–25%. Platelet-derived factor Va possesses several modifications not present in its plasma counterpart, such as an O-glycosylation on Thr402, and is the more procoagulant molecule, exhibiting resistance to inactivation by activated protein C (APC) and plasmin when bound to the activated platelet membrane surface. As the platelet-derived cofactor pool originates from endocytosis of plasma-derived factor V by megakaryocytes, it can be hypothesized that, subsequent to its endocytosis, factor V is post-translationally modified, proteolytically processed, and packaged into alpha-granules to form the unique platelet-derived molecule. Platelet- and plasma-derived factor Va were purified from three individual donors. Platelets were isolated from whole blood and lysed with Triton X-100 in the presence of the tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid to prevent dephosphorylation by any released tyrosine phosphatases. Factor V in both the platelet lysates and the platelet-poor plasma was activated with thrombin, adsorbed onto a resin-coupled monoclonal antibody against human factor V, washed extensively, and eluted using 10% SDS. Western blotting analyses, using a monoclonal antibody against phosphotyrosine residues, revealed the presence of tyrosine phosphorylation on the light chain of plasma-derived, but not platelet-derived, factor Va. No such modification was observed on the heavy chain of either cofactor. Monoclonal antibodies against the heavy and light chains of factor V were used to verify that similar levels of factor V were present in all samples. This differential modification was observed in all three donors. Subsequent MALDI-TOF analyses of trypsin-digested plasma- and platelet-derived factor Va light chains were performed to identify the site(s) of tyrosine phosphorylation. These data identified phosphorylation of the C-terminal tyrosine residue (Tyr2196) in plasma-derived, but not platelet-derived, factor Va. The phosphorylated fragment (residues 2188–2196, m/z = 1152.5) is consistently present in spectra of plasma-derived factor Va, while only the non-phosphorylated fragment (m/z = 1072.5) has been observed in the platelet-derived cofactor. To demonstrate that the phosphatase inhibitors used were effective, the platelets were lysed in the presence of added plasma-derived factor Va. Tyrosine phosphorylation, as determined by immunoblotting, was still present on the light chain, indicating that the dephosphorylation was occurring prior to platelet lysis. Tyrosine phosphorylation of other proteins has been implicated in regulation of endocytosis. Further experiments will need to be performed to determine if the modification has a similar role in megakaryocyte endocytosis of factor V.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
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.

scholarly journals Mechanism of vanadate-induced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites

1991 ◽  
Vol 276 (3) ◽  
pp. 611-619 ◽  
Author(s):  
S Trudel ◽  
M R Pâquet ◽  
S Grinstein
Keyword(s):  
Tyrosine Phosphorylation ◽  
Respiratory Burst ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
O2 Consumption ◽  
Phosphatase Inhibitors ◽  
Hl60 Cells ◽  
Diphenylene Iodonium ◽  
Undifferentiated Cells

Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

1994 ◽  
Vol 72 (06) ◽  
pp. 947-956 ◽  
Author(s):  
Edward G Wyshock ◽  
Gwendolyn J Stewart ◽  
Robert W Colman
Keyword(s):  
Human Platelets ◽  
Molecular Forms ◽  
Light Chains ◽  
Factor V ◽  
Platelet Surface ◽  
Specific Antibodies ◽  
Factor Va ◽  
Activation Peptide ◽  
Platelet Pellet ◽  
Incomplete Cleavage

SummaryBinding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of a-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of α-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of a-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of α-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

Coagulation factor Va is an actin filament binding and cross-linking protein

1995 ◽  
Vol 73 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Emilia Furmaniak-Kazmierczak ◽  
Michael E. Nesheim ◽  
Graham P. Côté
Keyword(s):  
Actin Filament ◽  
Light Chain ◽  
Actin Filaments ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Actin Binding ◽  
Cross Linking ◽  
Factor V ◽  
Proteolytic Activation ◽  
Factor Va

Bovine coagulation cofactor factor Va is shown to bind to filaments of skeletal muscle actin with a dissociation constant of 40–50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-links actin filaments. Factor Va also bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va – factor Xa – phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.Key words: blood coagulation, factor V, actin.

scholarly journals The regulation of human factor V by a neutrophil protease

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 846-851 ◽  
Author(s):  
AM Oates ◽  
HH Salem
Keyword(s):  
Light Chain ◽  
Neutrophil Elastase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophil Elastase ◽  
Ionophore A23187 ◽  
Factor V ◽  
Single Chain ◽  
Prolonged Incubation ◽  
Factor Va

Abstract Neutrophils activated with serum opsonized zymosan, soluble heat- aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin- activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.

Evolutionary Adaptation of Factor V from the Venom of the Common Brown Snake into a Potent Procoagulant

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 586-586
Author(s):  
Mettine H.A. Bos ◽  
Michael Boltz ◽  
Liam St. Pierre ◽  
John de Jersey ◽  
Paul P Masci ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Light Chains ◽  
Factor V ◽  
Single Chain ◽  
Prothrombinase Complex ◽  
High Affinity ◽  
The Common ◽  
Cofactor Activity ◽  
Mechanistic Basis ◽  
Normal Requirement

Abstract Some of the most toxic snakes in the world are those from the Australian Elapid family, including the three most venomous land snakes Inland Taipan, Coastal Taipan, and Common Brown snake. Their venom is strongly procoagulant and they are the only species known to have acquired a powerful prothrombin activator in their venom, which consists of a factor Xa (FXa)-like and factor V (FV)-like component. Venom-derived FV (pt-FV) from the Common Brown snake P. textilis shares 44% sequence homology with mammalian FV and has a similar domain organization. Remarkably, the B domain length of pt-FV is dramatically shortened compared to human FV (46 vs. 836 aa). This adaptation provides a unique opportunity to gain new insight into the function of the B domain and to examine the mechanistic basis for the strong procoagulant nature of the venom-derived prothrombinase complex. Pt-FV was expressed in BHK cells, purified, and characterized in functional assays employing FXa purified from P. textilis venom (pt-FXa). SDS-PAGE analysis revealed that pt-FV migrated as a single chain protein (~180 kDa). Thrombin completely processed pt-FV to pt-FVa, yielding the characteristic heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are connected by a unique disulfide bond. Functional analysis of prothrombin and prethrombin-1 conversion using a purified component assay in the presence of pt-FXa and negatively charged phospholipids revealed that pt-FV exhibits kinetic parameters comparable to human prothrombinase. Proteolytic processing of single chain pt-FV to the heterodimer did not significantly increase cofactor activity, indicating that pt-FV is expressed as a constitutively active cofactor that has bypassed the normal requirement for proteolytic activation. These results were confirmed using an uncleavable variant, pt-FV-QQ. We speculate that the mechanistic basis for this constitutive cofactor activity is related to the absence of a key cluster of conserved B domain residues, which we have recently shown to play an important role in maintaining FV as an inactive procofactor (JBC2007;282:15033). Additional experiments revealed that the pt-FV–pt-FXa complex does not require a membrane surface to optimally function, as the kinetics of prethrombin-1 activation were equivalent in the presence or absence of membranes. Binding measurements indicated that this was due to the high affinity interaction (Kd ~8 nM) of pt-FV with pt-FXa in solution. Interestingly, human FVa did not bind soluble pt-FXa with high affinity, suggesting that pt-FXa binding involves unique molecular features on pt-FV. Additional studies revealed that pt-FV does not lose activity following incubation with high concentrations of activated protein C (APC), even though the pt-FV heavy chain was fully proteolyzed. Collectively, our findings provide new insights into FV structure/function as well as a biochemical rationale for the powerful procoagulant nature of the prothrombinase complex from P. textilis venom. Remarkably, pt-FV has acquired at least three gain of function elements: first, it is constitutively active and as such the first example of a naturally occurring active FV variant. Second, pt-FV has a unique conformation as it bypasses the normal requirement for a membrane surface to achieve high affinity FXa binding. Finally, pt-FV is functionally resistant to APC which could be due to its unique disulfide bond. Taken together, venom-derived P. textilis FV represents an exceptional example of a protein that has adapted into a potent biological weapon for host defense and to incapacitate prey. Uncovering the mechanistic details of these gain of function elements will provide a new level of understanding of FV/FVa function.

The regulation of human factor V by a neutrophil protease

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 846-851
Author(s):  
AM Oates ◽  
HH Salem
Keyword(s):  
Light Chain ◽  
Neutrophil Elastase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophil Elastase ◽  
Ionophore A23187 ◽  
Factor V ◽  
Single Chain ◽  
Prolonged Incubation ◽  
Factor Va

Neutrophils activated with serum opsonized zymosan, soluble heat- aggregated IgG, and ionophore A23187 in the presence of calcium release a material capable of initially activating factor V. Subsequent inactivation of factor V was only observed with neutrophil releasate derived from IgG and ionophore. In this study we examine the nature of this neutrophil activity and investigate its role in the regulation of factor V/Va. From early in the fractionation it was apparent that the cells contained different enzymes capable of cleaving factor V. The most active of these was isolated and found to be an isomer of human neutrophil elastase. The purified protease caused a dose-dependent activation of isolated factor V to a maximum of threefold. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single-chain factor V was cleaved to form intermediates of 100 and 91 kilodaltons (kD). Coagulant activity correlated with the formation of a 97-kD heavy and 77-kD light chain. On prolonged incubation the formed factor Va(e) was inactivated in association with proteolysis of the 97-kD band to smaller peptides and cleavage of the 77-kD light chain to a molecular weight of 75 kD, which is similar to thrombin-activated factor Va light chain. Neutrophil elastase also caused rapid inactivation of thrombin- activated factor V, factor Va(t). These observations suggest that elastase cleaves factor V at sites distinct from that by thrombin and therefore represents a novel factor V activation pattern. It is proposed that upon neutrophil activation elastase is secreted into the plasma milieu to initiate factor V activation. This serves to generate small amounts of thrombin that, in turn, by positive feedback fully activates factor V and thus amplifies the coagulation reaction.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
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

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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