Characterization of Cleaved Plasma Protein S with a Monoclonal Antibody-based Assay

Summary

A monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i. e. the carboxyterminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.

Key words

Cleaved plasma protein S - thrombin-sensitive region - monoclonal antibody - epitope mapping

^* Olivier Morbœuf was the recipient of a grant from Ministère de l’Education Nationale, de la Recherche et de la Technologie.

• References

• 1 Di Scipio RG, Davie EW. Characterization of protein S, a γ-carboxyglutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904.
• 2 Di Scipio RG, Hermodson MA, Yates SG, Davie EW. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor) and protein S. Biochemistry 1977; 16: 698-706.
• 3 Nicolaes GA, Tans G, Thomassen MC, Hemker HC, Pabinger I, Varadi K, Schwarz HP, Rosing J. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 21158-66.
• 4 Walker FJ, Chavin SI, Fay PJ. Inactivation of factor VIII by activated protein C and protein S. Arch Biochem Biophys 1987; 252: 322-8.
• 5 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 2082-8.
• 6 Dahlbäck B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci USA 1981; 78: 2512-6.
• 7 Dahlbäck B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4-binding protein. J Biol Chem 1986; 261: 12022-7.
• 8 Dahlbäck B. Purification of human vitamin-K dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-46.
• 9 Walker FJ. Regulation of vitamin K-dependent protein S, inactivation by thrombin. J Biol Chem 1984; 259: 10335-9.
• 10 Suzuki K, Nishioka J, Hashimoto S. Regulation of activated protein C by thrombin-modified protein S. J Biochem 1983; 94: 699-705.
• 11 Lu D, Xie RL, Rydzewski A, Long GL. The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S. Thromb Haemost 1997; 77: 1156-63.
• 12 Mitchell CA, Hau L, Salem HH. Control of thrombin mediated cleavageof protein S. Thromb Haemost 1986; 56: 151-4.
• 13 Long GL, Lu D, Xie RL, Kalafatis M. Human protein S cleavage and inactivation by coagulation factor Xa. J Biol Chem 1998; 273: 11521-6.
• 14 Borgel D, Gaussem P, Garbay C, Bachelot-Loza C, Morboeuf O, Liu W-Q, Brohard-Bohn B, Le Bonniec B, Aiach M, Gandrille S. Protein S interaction with membranes requires the presence of the thrombin-sensitive region (TSR). 1999 submitted.
• 15 Seitz ER, Egbring R, Kolb G, Havemann K. Protein S degradation in vitro by neutrophil elastase. Scand J Clin Lab Invest 1993; 53: 281-8.
• 16 Alhenc-Gelas M, Nicaud V, Gandrille S, van Dreden P, Amiral J, Aubry ML, Fiessinger J-N, Emmerich J, Aiach M. The factor V gene A4070G mutation and the risk of venous thrombosis. Thromb Haemost 1999; 81: 193-7.
• 17 Bauer KA, Kass BL, ten Cate H, Bednarek MA, Hawiger JJ, Rosenberg RD. Detection of factor X activation in humans. Blood 1989; 74: 2007-15.
• 18 Bauer KA, Mannucci PM, Gringeri A, Tradati F, Barzegar S, Kass BL, ten Cate H, Kestin AS, Brettler DB, Rosenberg RD. Factor IXa-factor VIIIa-cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo. Blood 1992; 79: 2039-47.
• 19 D’Angelo A, Vigano-d’Angelo S, Esmon CT, Comp PC. Acquired deficiencies of protein S Protein S activity during oral anticoagulation, in liver disease, and in disseminated intravascular coagulation. J Clin Invest 1988; 81: 1445-54.
• 20 Zöller B, Garcia de Frutos P, Dahlbäck B. Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease. Blood 1995; 85: 3524-31.
• 21 Heeb MJ, Mosher D, Griffin JH. Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation. Blood 1989; 73: 455-61.
• 22 Millenson MM, Bauer KA, Kistler JP, Barzegar S, Tulin L, Rosenberg RD. Monitoring “mini-intensity” anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels. Blood 1992; 79: 2034-8.