Cloning of a cDNA Coding for Human Factor V, a Blood Coagulation Factor Homologous to Factor VIII and Ceruloplasmin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 83; no. 18; pp. 6800 - 6804
• Main Authors: Kane, William H., Davie, Earl W.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.09.1986; National Acad Sciences
• Subjects: Amino Acid Sequence; Amino acids; Biochemistry; Biological and medical sciences; Biotechnology; Blood coagulation factors; Ceruloplasmin - analysis; Cloning, Molecular; Complementary DNA; Connected regions; DNA - analysis; Factor V - analysis; Factor V - genetics; Factor VIII - analysis; Fundamental and applied biological sciences. Psychology; Generally accepted auditing standards; Genetic engineering; Genetic technics; Humans; Methods. Procedures. Technologies; Molecular cloning; Molecular Weight; Molecules; Nucleotides; Proteins; Human; Factor V; Complementary DNA; Molecular cloning; Coagulation factor
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.83.18.6800

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. A phage λ gt11 Hep G2 cell cDNA expression library was screened by using an affinity-purified 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 Va 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.