Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin.

Abstract

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...

Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insG in exon 16 of factor 5 gene

“…The following oligonucleotides used in polymerase chain reaction (PCR) amplification and sequencing were designed from factor V gene sequence 18 : e1F, 5Ј-cccacagcctctagagctc-3Ј; e1R, 5Ј-cccggactccacacct-3Ј; e2F, 5Ј-ttagtttttgtattttatttccag-3Ј; e2R, 5Ј-gtttctataaattttcagtaaatgg-3Ј; e5F, 5Ј-ctgcagtgctactgaaaacat-3Ј; e5R, 5Ј-tttccttcttgatagggagttg-3Ј; e7F, 5Ј-ttcttctcttgagttatttcattg-3Ј; e7R, 5Ј-tttgcccagtggtatgaa-3Ј; e8F, 5Ј-atttgagaaagtggtttaatttt-3Ј; e8R, 5Ј-catttgaatttaaaattatatgagc-3Ј; e17F 5Ј-ctgtgtcaacagattttaattgattt-3Ј; e17R, 5Ј-aagaaatgagaaggagttacagatt-3Ј; e21F, 5Ј-gaatttaggcagtgtgtgacttgtt-3Ј; e21R, 5Ј-tctagagattcagatagaaatatgcacaca-3Ј; e22F 5Ј-taaactttcctcttttcttctag-3Ј; e22R, 5Ј-tcccaaatcttgattcttt-3Ј; e24F, 5Ј-caaaggttttaacatcttccttatct-3Ј; e24R, 5Ј-gcacagtcttcagattgctttc-3Ј; e25F, 5Ј-tttctcttatttggctttcag-3Ј; e25R 5Ј-attctaaatggtttgaggtctt-3Ј. Exon 13 was amplified in 4 overlapping fragments by means of the following primers: e13aF, 5Ј-gattattgtgttttcatgtct-3Ј; e13aR, 5Ј-cttgggtcccttatgctta-3Ј; e13bF, 5Ј-atacgtctactttcacttg-3Ј; e13bR, 5Ј-tgggaagagatgtttcatt-3Ј; e13cdF, 5Ј-caacacattttcagaaagaag-3Ј, e13cdR, 5Ј-cattgagagtaggagatg-3Ј; e13eF, 5Ј-ttgatcagatattctacc-3Ј; e13eR, 5Ј-tcagcagtaatggaaaaatg-3Ј.…”

Structural Requirements for Expression of Factor Va Activity

“…Plasma factor V circulates as a large single chain protein of M r 330,000 (4 -6). The cDNA sequences for human, murine, porcine, and bovine factor V have been reported previously (7)(8)(9)(10)(11). The factor V molecule is composed of triplicated "A" domains, duplicated "C" domains, and a "B" region.…”

Defining the Factor Xa-binding Site on Factor Va by Site-directed Glycosylation

“…Factor V (FV) is a single-chain procofactor (330 kDa), having the domain organization A1-A2-B-A3-C1-C2 in common with the homologous Factor VIII (FVIII) (5)(6)(7). FV circulates in human plasma at a concentration of 7 g/ml but is also present in platelets from where it is released and activated during hemostasis (8).…”