Substitution of Gly-548 to Ala in the Substrate Binding Pocket of Prothrombin Perijá Leads to the Loss of Thrombin Proteolytic Activity

Summary

Prothrombin Perijá is a dysprothrombin derived from a homozygous patient that manifests low thrombin activity upon activation in a onestage assay. Purified prothrombin Perijá showed normal appearance on SDS-PAGE, and was cleaved normally to form α-thrombin by the prothrombinase complex. The activated form, thrombin Perijá, however, did not show any proteolytic activity towards native substrates, fibrinogen, protein C or various synthetic substrates for α-thrombin, but it was able to bind to antithrombin III, although the binding capacity was markedly reduced even in the presence of heparin. Thrombin Perijá showed full reactivity toward a small inhibitor, DFP, indicating that the molecular defect is in the substrate binding site in the thrombin molecule but not in the active site itself. By DNA sequence analysis of the patient prothrombin gene, we identified a G to C mutation at nucleotide 20016 in exon 14, which predicts a Gly-548 to Ala substitution in the prothrombin Perijá molecule. The structural modeling of thrombin Perijá suggests that Ala-548 is located close to the limb of the cavity wall of the substrate binding pocket, and that the methyl group blocks protrusion of the guanidino group of Arg into the cavity. This steric hindrance may well inhibit the access of Arg-containing substrates to the catalytic Ser-525 leading to the loss of proteolytic activity.

Keywords

Prothrombin - dysprothrombin - proteolytic activity - substrate binding pocket - steric hindrance

^* This work was supported in part by Scientific Research Grants-in-Aid for Scientific Research 11470250, and for International Scientific Research Program, Joint Research Grants 11694308 from the Ministry of Education, Science and Culture of the Government of Japan and from the Research Foundation of Community Medicine.

• References

• 1 Davie E, Fujikawa K, Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 1991; 30: 10363-70.
• 2 Degen SJF. Prothrombin in Molecular Basis of Thrombosis and Hemostasis. High KA, Roberts HR. ed. New York: Dekker; 1995: 75-99.
• 3 Coughlin SR, Vu TK, Hung DT, Wheaton VI. Characterization of a functional thrombin receptor. Issues and opportunities. J Clin Invest 1992; 89: 351-5.
• 4 Soifer SJ, Peters KG, O’Keefe J, Coughlin SR. Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development. Am J Pathol 1994; 144: 60-9.
• 5 Morris R, Winyard PG, Blakeb DR, Morris CJ. Thrombin in inflammation and healing: relevance to rheumatoid arthritis. Ann Rheum Dis 1994; 53: 72-9.
• 6 Stiernberg J, Redin WR, Warner WS, Carney DH. The role of thrombin and thrombin receptor activating peptide (TRAP-508) in initiation of tissue repair. Thromb Haemost 1993; 70: 158-62.
• 7 Walz DA, Fenton JW. The role of thrombin in tumor cell metastasis. Invasion Metastasis 1994; 95: 303-8.
• 8 Degen SJ, Davie EW. Nucleotide sequence of the gene for human prothrombin. Biochemistry 1987; 26: 6165-77.
• 9 Girolami A, Scarano L, Saggiorato G, Girolami B, Bertomoro A, Marchioro A. Congenital deficiencies and abnormalities of prothrombin. Blood Coag Fibrin 1998; 09: 557-69.
• 10 Ruiz-Sáez A, Luengo J, Rodriguez A, Ojeda A, Gómez O, Acurero Z. Prothrombin Perijá: a new congenital dysprothrombinemia in an Indian family. Thromb Res 1986; 44: 587-98.
• 11 Ruiz-Sáez A, Bosch N, Echenagucia M, Arguello A, Rodriguez A. High prevalence of an abnormal prothrombin in an indian population of Venezuela. Thromb Haemost 1995; 73: 1438.
• 12 Sugo T, Watanabe K, Naraki T, Matsuda M. Chemical modification of -γ-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des--γ-carboxy)prothrombin. J Biochem (Tokyo) 1990; 108: 382-7.
• 13 Sugo T, Persson U, Stenflo J. Protein C in bovine plasma after warfarin Treatment. J Biol Chem 1985; 260: 10453-7.
• 14 Sugo T, Nakamikawa C, Takano H, Mimuro J, Yamaguchi S, Mosesson MW, Meh DA, DiOrio JP, Takahashi N, Takahashi H, Nagai K, Matsuda M. Fibrinogen Niigata with impaired fibrin assembly: an inherited dysfibrinogen with a Bβ Asn-160 to Ser substitution associated with extra glycosylation at Bβ Asn-158. Blood 1999; 3806-13.
• 15 Maekawa H, Sugo T, Yamashita N, Kamiya K, Umeyama H, Miura N, Naka H, Nishimura T, Yoshioka A, Matsuda M. Molecular defect in factor IX Tokyo: Substitution of Val182 by Ala at position P2’ in the second cleavage site by factor XIa resulting in the impaired activation. Biochemistry 1993; 32: 6146-51.
• 16 Iwahana H, Yoshimoto K, Shigekiyo T, Shirakami A, Saito S, Itakura M. Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia. Am J Hum Genet 1992; 51: 1386-95.
• 17 Iwahana H, Mizusawa N, Yoshimoto K, Itakura M. Detection of a new polymorphism of the human prothrombin (F2) gene by combination of PASA and mutated primer-mediated PCR-RFLP. Hum Genet 1992; 325-6.
• 18 Banner DW, Hadváry P. Crystallographic analysis at 3.0-Å resolution of the binding to human thrombin of four active site-directed inhibitors. J Biol Chem 1991; 266: 20085-93.
• 19 Ogata K, Umeyama H. An automatic homology modeling method consisting of data base searches and simulated annealing. J Mol Graph Model 2000; 18: 258-72.
• 20 Ogata K, Umeyama H. The role of played by environmental residues on side chain torsional angles within homologous families of proteins: A new method of side chain modeling. Proteins 1998; 31: 355-69.
• 21 Miyata T, Zheng YZ, Kato A, Kato H. A point mutation (Arg271 → Cys) of a homozygote for dysfunctional prothrombin, prothrombin Obihiro, which has a region of high sequence variability. Brit J Haematol 1995; 90: 688-92.
• 22 Rabiet MJ, Furie BC, Furie B. Molecular defect of prothrombin Barcelona. J Biol Chem 1986; 261: 15045-8.
• 23 Akhavan S, Rocha E, Zeinal S, Mannucci PM. Gly319 → Arg substitution in the dysprothrombin Segovia. Brit J Haematol 1999; 105: 667-9.
• 24 Owen CA, Henriksen RA, McDuffie FC, Mann KG. Prothrombin Quick. A newly identified dysprothrombinemia. Mayo Clin Proc 1978; 53: 29-33.
• 25 Henriksen RA, Mann KG. Identification of primary structural defect in dysthrombin thrombin Quick I: Substitution of cysteine for arginine-382. Biochemistry 1988; 27: 9160-5.
• 26 Henriksen RA, Mann KG. Substitution of valine for glycine-558 in the congenital dysprothrombin thrombin Quick II alters primary substrate specificity. Biochemistry 1989; 28: 2078-82.
• 27 O’Marcaigh AS, Nicols WL, Hassinger NL, Mullins JD, Mallouh AA, Gilchrist GS, Owen WG. Genetic analysis of functional characterization of prothrombin Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and hypoprothrombinemia. Blood 1996; 88: 2611-8.
• 28 Craik CS, Largman C, Fletcher T, Roczniak S, Barr PJ, Fletterick R, Rutter WJ. Redesigning trypsin: alteration of substrate specificity. Science 1985; 228: 291-7.
• 29 Phillips JE, Shirk RA, Whinna HC, Henriksen RA, Church FC. Inhibition of dysprothrombin Quick I and II by heparin cofactor II and antithrombin. J Biol Chem 1993; 268: 3321-7.
• 30 Bode W, Turk D, Karshikov A. The refined 1.9-Å X-ray crystal structure of D-Phe-Pro-Arg-chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active site geometry, and structure-function relationship. Protein Sci 1992; 01: 426-71.
• 31 Whinna HC, Church FC. Interaction of thrombin with antithrombin, heparin cofactor II and protein C inhibitor. J Prot Chem 1993; 12: 677-88.