Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population

Summary

Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation to activated protein C (APC) by thrombin down-regulates the clotting cascade by limited proteolysis of the pro-coagulant cofactors Va and VIIIa. In addition to its anticoagulant activity, APC also exhibits potent cytoprotective and anti-inflammatory activities. While the anticoagulant activity of APC is enhanced by the cofactor function of protein S on membrane phospholipids, the cytoprotective intracellular signalling activity of APC requires complex formation with endothelial protein C receptor (EPCR) expressed on the vascular endothelium. Two natural variants of APC [Arg-147 to Trp substitution (R147W) and Lys-150 deletion (K150del)] have been identified in the Chinese population as hotspot mutants occurring with high frequencies of 27.8% and 13.9%, respectively, among 36 protein C-deficient subjects. The affected individuals exhibit variable thrombotic tendencies. To understand the underlying cause of the thrombotic phenotype in these patients, we expressed these two protein C variants in mammalian cells and characterised their anticoagulant and anti-inflammatory properties using established in vitro and cellular assays. Our results suggest that both R147W and K150del variants have normal amidolytic and proteolytic activities in the absence of cofactors. However, the R147W mutant exhibits ∼3 times lower affinity for binding to EPCR and the K150del variant has ∼2–3-fold impaired anticoagulant activity in the presence of protein S. These results provide some insight into the possible pathogenic mechanism of protein C deficiency in Chinese patients carrying these mutations.

Keywords

Coagulation factors - deep-vein thrombosis - endothelial cells - protein C/S pathway - signal transduction

• References

• 1 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 29-35.
• 2 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 06: 2561-2567.
• 3 Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood 1991; 78: 1637-1651.
• 4 Mather T, Oganessyan V, Hof P. et al. The 2.8 Å crystal structure of Gla-domain-less activated protein C. EMBO J 1996; 15: 6822-6831.
• 5 Bode W, Mayr I, Baumann U. et al. The refined 1.9 Å crystal structure of human α-thrombin: interaction with D-Phe-Pro-Arg chlorometheylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 08: 3467-3475.
• 6 Mosnier LO, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 7 Fukudome K, Esmon CT. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 269: 26486-26491.
• 8 Taylor FB, Stearns-Kurosawa DJ, Kurosawa S. et al. The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood 2000; 95: 1680-1686.
• 9 Stearns-Kurosawa DJ, Kurosawa S, Mollica JS. et al. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-10216.
• 10 Fukudome K, Kurosawa S, Stearns-Kurosawa DJ. et al. The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor. J Biol Chem 1996; 271: 17491-17498.
• 11 Regan LM, Mollica JS, Rezaie AR. et al. The interaction between the endothelial cell protein C receptor and protein C is dictated by the gamma-carboxyglutamic acid domain of protein C. J Biol Chem 1997; 272: 26279-26284.
• 12 Dahlbäck B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res 1995; 77: 1-43.
• 13 Kinoshita S, Iida H, Inoue S. et al. Protein S and protein C gene mutations in Japanese deep vein thrombosis patients. Clin Biochem 2005; 38: 908-915.
• 14 Shen MC, Lin JS, Tsay W. High prevalence of antithrombin III, protein C and protein S deficiency, but no factor V Leiden mutation in venous thrombophilic Chinese patients in Taiwan. Thromb Res 1997; 87: 377-385.
• 15 Shen MC, Lin JS, Tsay W. Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese venous thrombophilic patients in Taiwan. Thromb Res 2000; 99: 447-452.
• 16 Ding Q, Shen W, Ye X. et al. Clinical and genetic features of protein C deficiency in 23 unrelated Chinese patients. Blood Cells Mol Dis. 2012 Epub ahead of print.
• 17 Zhu T, Ding Q, Bai X. et al. Normal ranges and genetic variants of antithrombin, protein C and protein S in the general Chinese population. Results of the Chinese Hemostasis Investigation on Natural Anticoagulants Study I Group. Haematologica 2011; 96: 1033-1040.
• 18 Yu T, Dai J, Liu H. et al. Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation. Pathology 2012; 44: 348-353.
• 19 Zhou RF, Cai XH, Xie S. et al. Molecular mechanism for hereditary protein C deficiency in two Chinese families with thrombosis. J Thromb Haemost 2006; 04: 1154-1156.
• 20 Tang L, Guo T, Yang R. et al. Genetic background analysis of protein C deficiency demonstrates a recurrent mutation associated with venous thrombosis in Chinese population. PLos One 07: e35773.
• 21 Tsay W, Shen MC. R147W mutation of PROC gene is common in venous thrombotic patients in Taiwanese Chinese. Am J Hematol 2004; 76: 8-13.
• 22 Mather T, Oganessyan V, Hof P. et al. The 2.8 A crystal structure of Gla-domain-less activated protein C. EMBO J 1996; 15: 6822-6831.
• 23 Mesters RM, Heeb MJ, Griffin JH. A novel exosite in the light chain of human activated protein C essential for interaction with blood coagulation factor Va. Biochemistry 1993; 32: 12656-12663.
• 24 Mosnier LO, Zampolli A, Kerschen EJ. et al. Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant. Blood 2009; 113: 5970-5978.
• 25 Yang L, Manithody C, Rezaie AR. Contribution of basic residues of the 70-80-loop to heparin binding and anticoagulant function of activated protein C. Biochemistry 2002; 41: 6149-6157.
• 26 Yang L, Rezaie AR. Calcium-binding sites of the thrombin-thrombomodulin-protein C complex: possible implications for the effect of platelet factor 4 on the activation of vitamin K-dependent coagulation factors. Thromb Haemost 2007; 97: 899-906.
• 27 Smirnov MD, Esmon CT. Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C. J Biol Chem 1994; 269: 816-819.
• 28 Yang L, Manithody C, Rezaie AR. Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C. Proc Natl Acad Sci USA 2006; 103: 879-884.
• 29 Yang L, Manithody C, Walston TD. et al. Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin. J Biol Chem 2003; 278: 37465-37470.
• 30 Manithody C, Fay PJ, Rezaie AR. Exosite-dependent regulation of factor VIIIa by activated protein C. Blood 2003; 101: 4802-4807.
• 31 Bae J-S, Yang L, Manithody C. et al. The ligand occupancy of endothelial protein C receptor switches the PAR-1-dependent signalling specificity of thrombin from a permeabilityenhancing to a barrier-protective response in endothelial cells. Blood 2007; 110: 3909-3916.
• 32 Kalafatis M, Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem 1993; 268: 27246-27257.
• 33 Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 2005; 105: 3178-3184.
• 34 Reitsma PH, Poort SR, Allaart CF. et al. The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects. Blood 1991; 78: 890-894.
• 35 Gandrille S, Aiach M. Identification of mutations in 90 of 121 consecutive symptomatic French patients with a type I protein C deficiency. The French IN-SERM Network on Molecular Abnormalities Responsible for Protein C and Protein S deficiencies. Blood 1995; 86: 2598-2605.
• 36 Tsay W, Greengard JS, Montgomery RR. et al. Genetic mutations in ten unrelated American patients with symptomatic type 1 protein C deficiency. Blood Coagul Fibrinolysis 1993; 04: 791-796.
• 37 Laszik Z, Mitro A, Taylor Jr FB. et al. Human protein C receptor is present primarily on endothelium of large blood vessels: implications for the control of the protein C pathway. Circulation 1997; 96: 3633-3640.
• 38 Xu J, Qu D, Esmon NL. et al. Metalloproteolytic release of endothelial cell protein C receptor. J Biol Chem 2000; 275: 6038-6044.
• 39 Zheng X, Li W, Song Y. et al. Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia. J Thromb Haemost 2007; 05: 1394-1400.
• 40 Miyata T, Sakata T, Yasumuro Y. et al. Genetic analysis of protein C deficiency in nineteen Japanese families: five recurrent defects can explain half of the deficiencies. Thromb Res 1998; 92: 181-187.
• 41 Miyata T, Sato Y, Ishikawa J. et al. Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis. Thromb Res 2009; 124: 14-18.
• 42 Reitsma PH. Protein C deficiency: from gene defects to disease. Thromb Haemost 1997; 78: 344-350.
• 43 Steinkamp M, Geva A, Joffe S. et al. Chronic disseminated intravascular coagulation and childhood-onset skin necrosis resulting from homozygosity for a protein C Gla domain mutation, Arg15Trp. J Pediatr Hematol Oncol 2002; 24: 685-688.