Demonstration of Exosite I-Dependent Interactions of Thrombin with Human Factor V and Factor Va Involving the Factor Va Heavy Chain:  Analysis by Affinity Chromatography Employing A Novel Method for Active-Site-Selective Immobilization of Serine Proteinases†

Biochemistry ◽  
1998 ◽  
Vol 37 (38) ◽  
pp. 13143-13152 ◽  
Author(s):  
Kumudini R. Dharmawardana ◽  
Paul E. Bock
Keyword(s):  
Heavy Chain ◽  
Active Site ◽  
Human Factor ◽  
Factor V ◽  
Serine Proteinases ◽  
Factor Va ◽  
Chain Analysis ◽  
Novel Method ◽  
Selective Immobilization ◽  
Site Selective

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 Human Neutrophil Elastase Activates Human Factor V but Inactivates Thrombin-Activated Human Factor V

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1065-1074 ◽  
Author(s):  
John A. Samis ◽  
Marilyn Garrett ◽  
Reginald P. Manuel ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Human Neutrophil ◽  
Human Factor ◽  
Time Dependent ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Terminal Sequence ◽  
Sds Page ◽  
Cofactor Activity

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or α-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2 -terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with α-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 μmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using α-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2 -terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2 -terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely α-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with α-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.

scholarly journals Monoclonal antibodies to human coagulation factor V and factor Va

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1060-1067 ◽  
Author(s):  
WB Foster ◽  
MM Tucker ◽  
JA Katzmann ◽  
RS Miller ◽  
ME Nesheim ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Factor ◽  
Solid Phase ◽  
Hybrid Cell ◽  
Coagulation Factor ◽  
Factor V ◽  
Binding Interaction ◽  
Apparent Dissociation Constant ◽  
Factor Va ◽  
Better Than

Abstract BALB/c mice were immunized with human factor V. The immunogen was a mixture of procofactor (factor V) and thrombin-activated cofactor (factor Va). Spleen cells were obtained from an immunized animal and fused with NS-1 murine myeloma cells. Hybrid cell cultures were assayed for the production of antibodies to human factor V and factor Va by a solid-phase radioimmunoassay. Factor V and/or factor-Va-specific antibodies were detected in 38 of the 96 cultures assayed. The cells from 10 of these positive cultures were subcloned by limiting dilution and grown as ascites tumors in BALB/c mice. Ascitic fluids were obtained and characterized with respect to their binding interaction with human factor V and factor Va. Three hybridoma cell lines produce monoclonal antibodies that react equally well with factor V and factor Va. Another antibody reacts with both antigens, but the reactivity with factor V is better than with factor Va. An additional two antibodies react with factor Va better than factor V in the radioimmunoassay (RIA). The remaining four antibodies react exclusively with factor V. A previously described murine monoclonal antibody to human factor V (alpha HFV-1) has been used to study the peptides produced during the thrombin-catalyzed activation of human factor V. This antibody binds both factor V and factor Va, releases them at high ionic strength, and has an apparent dissociation constant for factor Va of 3 x 10(-9)M. When human factor V (mol wt 330,000) is activated by thrombin and passed over an alpha HFV-1-Sepharose affinity resin, factor Va binds and subsequently can be eluted. The eluate in 1.2 M NaCl contains two fragments of apparent mol wt 93,000 and 70,000. EDTA, which inactivates factor Va, promotes release of the mol wt 93,000 fragment from factor Va bound to the antibody. Subsequent elution with 1.2 M NaCl releases the mol wt 70,000 fragment. These observations indicate that human factor Va is a two subunit protein and that the epitope for alpha HFV-1 is on the mol wt 70,000 fragment.

Platelet-Derived Factor Va Expressed on the Surface of the Activated Platelet Is GPI-Anchored

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 585-585 ◽  
Author(s):  
Jeremy P Wood ◽  
Ammon M. Fager ◽  
Jay R. Silveira ◽  
Paula B. Tracy
Keyword(s):  
Phase Separation ◽  
Heavy Chain ◽  
Consensus Sequence ◽  
Factor V ◽  
Gpi Anchor ◽  
Platelet Surface ◽  
Factor Va ◽  
Flow Cytometric ◽  
Activated Platelets ◽  
Triton X

Abstract Coagulation factor V exists in two pools in human blood. One is plasma-derived, originating from synthesis in the liver. The other is platelet-derived, originating from endocytosis of the plasma-derived cofactor by megakaryocytes and consisting of both unactivated and activated cofactor. Studies have demonstrated the presence of a non-dissociable, membrane-bound form of platelet-derived factor Va. When washed platelets were activated with thrombin and subjected to additional washing in the presence of EDTA to disrupt the calcium-dependent interaction between the factor Va heavy and light chains, western blotting and flow cytometric analyses revealed that ~35% of the heavy chain could not be removed from the platelet surface. Similarly, in a prothrombin time-based clotting assay, ~25% of the factor Va cofactor activity remained on the activated platelet surface after extensive washing, demonstrating that this platelet-bound cofactor pool functions in Prothrombinase. The mechanism by which this factor Va pool is bound to the membrane was investigated. Sequence analysis of factor Va has revealed a consensus sequence for glycosylphosphatidylinositol (GPI) anchor addition at Ser692 in the C-terminus of the heavy chain. Lipid raft domains in cell membranes are enriched in GPI-anchored proteins and are resistant to solubilization in Triton X-100 at 4°C but are soluble at 37°C. Compared to lysis at 4°C, ~50% more factor Va heavy chain was solubilized when platelets were lysed at 37°C. When cells are solubilized in Triton X-114, membrane-anchored and trans-membrane proteins segregate to the detergent phase, and when activated platelets were subjected to this procedure, a portion of the factor Va heavy chain segregated to the detergent phase. Flow cytometric analyses of activated platelets have demonstrated that phosphatidylinositol-specific phospholipase C (PI-PLC), which can cleave GPI-anchored proteins from cell surfaces, is able to remove ~45% of the non-dissociably bound factor Va heavy chain from the platelet surface in a dose-dependent manner. Subsequent analysis of these samples by Triton X-114 phase separation corroborated these results, indicating that ~50% of the heavy chain was removed from the detergent phase upon treatment with PI-PLC. After cleavage by PI-PLC, GPI-anchored proteins express an epitope known as the cross-reacting determinant (CRD), which encompasses the remainder of the GPI anchor on the modified protein. Western blotting analyses of platelet supernatants after PI-PLC treatment have demonstrated that the platelet-derived factor Va heavy chain contains the CRD epitope. To investigate the origin of the non-dissociably bound pool of factor Va on the platelet surface, platelets were isolated from a factor V-deficient individual with undetectable levels of the cofactor, who receives therapeutic transfusions of fresh frozen plasma. When the individual’s platelets were subjected to Triton X-114 phase separation, a population of factor Va heavy chain was detected in the detergent phase. These results are consistent with the non-dissociable portion of the platelet-derived factor Va pool being formed by post-translational modification of factor V from plasma subsequent to its endocytosis by megakaryocytes. The presence of a GPI anchor consensus sequence in the factor Va heavy chain, combined with the washing, solubilization, phase separation, PI-PLC treatment, and anti-CRD immunoblotting data strongly suggest that non-dissociably bound, platelet-derived factor Va is linked to the activated platelet membrane via a GPI anchor on its heavy chain. GPI-anchored, platelet-derived factor Va is functional in Prothrombinase, and, as it is retained on the platelet surface at the site of vascular injury, it is likely a physiologically significant source of cofactor activity.

Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

1996 ◽  
Vol 75 (01) ◽  
pp. 036-044 ◽  
Author(s):  
Thomas L Ortel ◽  
Karen D Moore ◽  
Mirella Ezban ◽  
William H Kane
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Secreted Protein ◽  
Factor V ◽  
Minimal Effect ◽  
Repetitive Domain ◽  
Human Factor Viii ◽  
A1 Domain ◽  
A2 Domain

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

scholarly journals The Carbohydrate Moiety of Factor V Modulates Inactivation by Activated Protein C

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4348-4354 ◽  
Author(s):  
José A. Fernández ◽  
Tilman M. Hackeng ◽  
Kazuhisa Kojima ◽  
John H. Griffin
Keyword(s):  
Risk Factor ◽  
Heavy Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Activated Partial Thromboplastin Time ◽  
Resistance Ratio ◽  
Factor V ◽  
Potential Influence ◽  
Apc Resistance ◽  
Factor Va

AbstractAn important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.

scholarly journals Phosphorylation of factor Va and factor VIIIa by activated platelets

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 704-719 ◽  
Author(s):  
M Kalafatis ◽  
MD Rand ◽  
RJ Jenny ◽  
YH Ehrlich ◽  
KG Mann
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Human Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor Va ◽  
Activated Platelets ◽  
Human Factor Viii ◽  
Phosphoamino Acid Analysis ◽  
Carboxyl Terminal

Abstract Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.

scholarly journals Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 139-146 ◽  
Author(s):  
AE Annamalai ◽  
AK Rao ◽  
HC Chiu ◽  
D Wang ◽  
AK Dutta-Roy ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Factor Xa ◽  
Direct Interaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor V ◽  
Factor Va ◽  
Direct Binding ◽  
First Time ◽  
Murine Monoclonal Antibodies ◽  
Murine Mabs

Abstract We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

scholarly journals Role of Regulatory Exosite I in Binding of Thrombin to Human Factor V, Factor Va, Factor Va Subunits, and Activation Fragments

1999 ◽  
Vol 274 (26) ◽  
pp. 18635-18643 ◽  
Author(s):  
Kumudini R. Dharmawardana ◽  
Steven T. Olson ◽  
Paul E. Bock
Keyword(s):  
Human Factor ◽  
Factor V ◽  
Factor Va
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