Evolution and organization of the human protein C gene.

Abstract

We have isolated overlapping phage genomic clones covering an area of 21 kilobases that encodes the human protein C gene. The gene is at least 11.2 kilobases long and is made up of nine exons and eight introns. Two regions homologous to epidermal growth factor and transforming growth factor are encoded by amino acids 46-91 and 92-136 and are precisely delimited by introns, as is a similar sequence in the genes for coagulation factor IX and tissue plasminogen activator. When homologous amino acids of factor IX and protein C are aligned, the positions of all eight introns correspond precisely, suggesting that these genes are the product of a relatively recent gene duplication. Nevertheless, the two genes are sufficiently distantly related that no nucleic acid homology remains in the *ntronic regions and that the size of the introns varies dramatically between the two genes. The similarity of the genes for factor IX and protein C suggests that they may be the most closely related members of the serine protease gene family involved in coagulation and fibrinolysis.Protein C is a two-chain vitamin K-dependent serine protease that plays. a fundamental role in hemostasis by preventing coagulation and promoting fibrinolysis (1). The anticoagulant effects of protein C are achieved through cleavage of factors Va and Villa of the intrinsic pathway (2), while clot lysis appears to result from interaction of protein C with the inhibitor of plasminogen activator (3). First isolated from bovine plasma in 1976 by Stenflo (4), the human protein (5) has since been shown to be a 62-kDa dimer with a heavy chain that contains the active serine site and a light chain that contains, at its amino terminus, y--carboxylglutamic acid residues, which are highly conserved among the vitamin K-dependent factors (6). The protein circulates as a zymogen (7), is activated by thrombin coupled with an endothelial cofactor, thrombomodulin (8), and is inactivated by a specific plasma protease inhibitor (9). Heterozygous deficiency of protein C was first identified by Griffin et al. (10) and has been shown by several workers to be an autosomally dominant disorder manifested by a markedly increased tendency to clot (11). Homozygous protein C-deficient patients, with no detectable antigenic levels of protein C, suffer from massive venous thrombosis as neonates (12). A deficiency of protein S, the cofactor for protein C-mediated inactivation of factor Va, has been reported as also causing increased thrombosis (13 kilobase pairs (kbp), were mapped using the restriction enzymes BamHI, EcoRI, HindIII, Pst I, and Sma I. DNA Sequencing. Genomic subfragments (see Fig. 1) from isolated X phage were ligated into the appropriate restriction sites of plasmid pBR322, and the resulting chimeric plasmids were grown in Escherichia coli strain RR1 using standard procedures. Plasmid DNA was purified by CsCl banding as described elsewhere (36).The strategy utilized to locate the desired regions for sequencing (intron-exon junctions and all exonic segments) was...

Complex Association of Protein C Gene Promoter Polymorphism With Circulating Protein C Levels and Thrombotic Risk

“…5,6 The human PC gene maps to chromosome 2q13-q14, 7 spans over 11 kb, and comprises a coding region (exons II to IX) and a 5Ј untranslated region encompassing exon I. 8,9 Three polymorphic sites (-1654 C/T, -1641 A/G, and -1476 A/T) are located in the 5Ј nontranscribed region of the PC gene. In 44 unrelated individuals in whom familial studies could be performed, 3 of 8 possible haplotypes (CGT, TAA, and CAA) were observed with frequencies of 0.33, 0.30, and 0.25, respectively.…”

Interactions and inhibition of blood coagulation factor Va involving residues 311–325 of activated protein C

“…Residues 134-143 in trypsin or 148-157 in chymotrypsin, homologous to residues 3 11-320 in PC (Table l), form an exposed loop on the surface of the catalytic domain, whereas trypsin residues 144-148 or chymotrypsin 159-162, homologous to 321-325 in PC, are rather buried in the structure. "Amino acids in PC are numbered according to Plutzky et al (1986). Sequence alignment was performed as described (Mesters et al, 1991) with the additional alignment of the sequences of bovine PC (Fernlund & Stenflo, 1982;Stenflo & Fernlund, 1982) and bovine trypsinogen (Mikes et al, 1966).…”

Identification of two novel mutations associated with combined protein C and protein S deficiency

“…The human gene encoding PC (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons that code for 461 amino acid residues [3,4]. The human PS gene (PROS1) resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues [5][6][7].…”