Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Vascular endothelium possesses multiple procoagulant properties, including synthesis and expression of Factor V. We studied the effects of homocysteine on the regulation of endothelial cell Factor V activity. Elevated levels of homocysteine are associated with the congenital thrombotic disorder homocystinuria. Treatment of cultured endothelial cells with 0.5-10 mM homocysteine had no effect on cell morphology, but did increase Factor V activity and prothrombin activation by Factor Xa. A radioimmunoassay for endothelial cell Factor V demonstrated that homocysteine treatment did not increase Factor V antigen levels. 125I-prothrombin was activated by treated endothelial cells and Factor Xa in the presence of thrombin inhibitors. Exogenous 125I-Factor V was cleaved by homocysteine-treated but not control endothelial cells. 125I-Factor V cleavage products distinct from those generated by thrombin and Factor Xa were identified. These data provide evidence for regulation of endothelial cell Factor V activity, and indicate that increased Factor V activity associated with homocysteine-treated vascular endothelium results primarily from induction of an activator of Factor V.

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor X.. A phage Agtll Hep G2 cell cDNA expression library was screened by using an affinity-purifiled antibody to human factor V, and 11 positive clones were isolated and plaque-purified. The clone containing the largest cDNA insert contained 2970 nucleotides and coded for 938 amino acids, a stop codon, and 155 nucleotides of 3' noncoding sequence including a poly(A) tail. The coding region includes 651 amino acids from the carboxyl terminus that constitute the light chain of human factor V. and 287 amino acids that are part of the connecting region of the protein. The predicted amino acid sequence agreed completely with 147 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the light chain. During the activation of factor V, several peptide bonds are cleaved by thrombin, giving rise to a heavy chain, a connecting fragment(s), and a light chain. The light chain is generated by the cleavage of an Arg-Ser peptide bond. The amino acid sequence of the light chain is homologous (40%) with the carboxyl-terminal fragment (Mr, 73,000) of human factor VIII. Both fragments have a similar domain structure that includes a single ceruloplasmin-related domain followed by two C domains. The carboxyl terminus of the connecting region, however, shows no significant amino acid sequence homology with factor VIII. It is very acidic and contains a number of potential N-linked glycosylation sites. It also contains about 20 tandem repeats of nine amino acids.Human coagulation factor V is a high molecular weight plasma glycoprotein that is required for rapid thrombin formation and normal hemostasis (1). It circulates in blood as a large single polypeptide chain (Mr, 330,000) with little or no coagulant activity (2-6). During the blood coagulation process, factor V is converted to factor Va by thrombin by limited proteolysis (2-6). This makes available binding sites for factor Xa (7) and prothrombin (8). Factor Va is composed of a heavy chain (Mr, 110,000) and a light chain (Mr, 76,000), and these two chains are held together by calcium ions (3, 6).The remainder of the original factor V molecule is released as a large connecting fragment(s) that is rich in carbohydrate (3,6,9). Factor Va binds to cell surfaces (9-12) and negatively charged phospholipid surfaces (13-16) through the light chain, and this increases the rate of prothrombin activation -z10,000-fold by factor Xa (17). Factor Va is readily inactivated by activated protein C (18), and this results in the cleavage of the heavy chain into two smaller fragments (19). In addition to its role in prothrombin activation, human factor Va stimulates the activation of protein C by thrombin on phospholipid and cell surfaces (12,20,21). Only the light chain is necessary for this reaction (22).Factor V has a number of physical and biological properties that are similar to factor VI...

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta- thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.