Quantitative Immunoblotting of Plasma and Platelet Protein S

Summary

Protein S, the activated protein C cofactor, was measured in plasma and in platelets by a quantitative immunoblotting assay using a double antibody technique. Either whole plasma or platelet lysates were electrophoresed on sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes by electroblotting. Based on the demonstration of proportional transfer of protein S from the gel to the nitrocellulose membrane, a reproducible and sensitive quantitative assay for protein S antigen (∼ 70,000 MW) was developed that correlated well with the Laurell rocket assay for plasma protein S. Pooled normal plasma contains 22 μg/ml protein S. Total platelet protein S antigen at ∼ 70,000 MW was determined in gel filtered platelets of ten healthy adults (mean, 163 ng per 10⁸8 platelets). In the supernatants of thrombin-stimulated platelets, 63% of the total platelet protein S antigen was measured. Thus, quantitative immunoblotting is a useful method to detect low levels of protein S in platelets or in plasma with the advantage of giving qualitative information, i.e. apparent MW, of protein S.

Key words

Protein S - Platelets - Immunoblotting - Protein C

• References

• 1 Walker FJ. The regulation of activated protein C by a new protein: A possible function for bovine protein S. J Biol Chem 1980; 255: 5521-5524
• 2 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
• 3 Walker FJ, Sexton PW, Esmon CT. The inhibition of blood coagulation by activated protein C through the selective inactivation of activated Factor V. Biochimica et Biophysica Acta 1979; 571: 333-342
• 4 Suzuki K, Stenflo J, Dahlback B, Teodorsson B. Inactivation of human coagulation Factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
• 5 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 6 Vehar GA, Davie EW. Preparation and properties of bovine Factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 2 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TA. Proteolytic inactivation of human Factor VIII procoagulant protein by activated human protein C and its analogy with Factor V. Blood 1984; 63: 486-489
• 8 Gardiner JE, McGann MA, Berridge CW, Fulcher C, Zimmerman T, Griffin JH. Protein S as a cofactor for activated protein C in plasma and in the inactivation of purified Factor VIII: C. Circulation 1984; 70: 820
• 9 Walker FJ. Regulation of activated protein C by protein S. The role of phospholipid in Factor Va inactivation. J Biol Chem 1981; 256: 11128-11131
• 10 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373
• 11 Broekmans AW, Veltkamp JJ, Bertina R. Congenital protein C deficiency and venous thromboembolism. N Engl J Med 1983; 309: 340-344
• 12 Schwarz HP, Fischer M, Hopmeier R, Batard MA, Griffin JH. Plasma protein S deficiency in familial thrombotic disease. Blood 1984; 64: 1297-1300
• 13 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 2082-2088
• 14 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528
• 15 Broekmans AW, Bertina RM, Reinalda-Poot J, Engesser L, Muller HP, Leeuw JA, Michiels JJ, Brommer EJ P, Briet E. Hereditary protein S deficiency and venous thromboembolism. A study in three Dutch familie. Throm Haemostas 1985; 53: 273-277
• 16 Schwarz HP, Heeb MJ, Wencel-Drake J, Griffin JH. Identification and characterization of protein S in human platelets. Blood 1985; 66: 1452-1455
• 17 Comp PC, Esmon CT. Activated protein C inhibits prothrombin converting activity. Blood 1979; 54: 1272-1281
• 18 Dahlback B, Stenflo J. Inhibitory effect of activated protein C on activation of prothrombin by platelet bound Factor Xa. Eur J Biochem 1980; 107: 331-335
• 19 Guinto ER, Esmon CT. Loss of prothrombin and of Factor Xa -Factor Va interactions upon inactivation of Factor Va by activated protein C. J Biol Chem 1984; 259: 13986-13992
• 20 Suzuki K, Nishioka J, Matsuda M, Maruayama H, Hashimoto S. Protein S is essential for the activated protein C-catalyzed inactivation of platelet associated Factor Va. J Biochem 1984; 96: 455-460
• 21 Harris KW, Esmon CT. Protein S is required for bovine platelets to support activated protein C binding and activity. J Biol Chem 1985; 260: 2007-2010
• 22 Towbin H, Gordon J. Immunoblotting and dot immunobinding. Current status and outlook. J Immunol Meth 1984; 72: 313-340
• 23 Lammle B, Berrettini M, Schwarz HP, Heeb MJ, Griffin JH. Quantitative immmunoblotting assay of blood coagulation Factor XII. Thromb Res 1986; 41: 747-749
• 24 Berrettini M, Lammle B, White T, Heeb MJ, Schwarz HP, Zuraw B, Curd J, Griffin JH. Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies. Blood 1986; 68: 455-462
• 25 Lammle B, Berrettini M, Griffin JH. Enhanced specificity of immunoblotting using radiolabeled antigen overlay. Studies of blood coagulation Factor XII and prekallikrein in plasma. Anal Biochem 1986; 156: 118-125
• 26 Heeb MJ, Schwarz HP, Nova CP, White T, Griffin JH. Quantitative immunoblotting of vitamin-K dependent coagulation factors in plasma. Thromb plasma Haemostas 1985; 54: 107
• 27 McConahey PJ, Dixon FJ. A method for trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol 1966; 29: 185-189
• 28 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 1970; 227: 680-685
• 29 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci USA 1979; 76: 4350-4354
• 30 Johnson DA, Gautsch JW, Sportsman JR, Elder JH. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Techn 1984; 1: 3-8
• 31 Tangen O, Berman HJ, Marfey P. Gel filtration. A new technique for separation of blood platelets from plasma. Thromb Diath Haemorrh 1971; 25: 268-278
• 32 Walsh PN, Griffin JH. Contributions of human platelets to the proteolytic activation of blood coagulation Factors XII and XI. Blood 1981; 57: 106-118
• 33 Bertina RM, van Wijngaarden A, Reinalda-Poot J, Poort SR, Bom VJ J. Determination of plasma protein S - The protein cofactor of activated protein C. Thromb Haemostas 1985; 53: 268-272
• 34 Fair DS, Revak DJ. Quantitation of human protein S in the plasma of normal and warfarin treated individuals by radioimmunoassay. Thromb Res 1984; 36: 527-535
• 35 Schwarz HP, Heeb MJ, Griffin JH. Human platelet protein S. Thromb Haemostas 1985; 54: 57
• 36 Nachman RL, Marcus AJ, Zucker-Frankliny D. Immunologic studies of proteins associated with subcellular fractions of normal human platelets. J Lab Clin Med 1967; 69: 651-658
• 37 Breederveld K, Giddings JC, Ten Cate JW, Bloom AL. The localization of Factor V within normal human platelets and the demonstration of a platelet Factor V antigen in congenital Factor V deficiency. Br J Haematol 1975; 29: 405-412
• 38 Koutts J, Walsh PN, Plow EF, Fenton JW, Bouma BN, Zimmerman TS. Active release of human platelet Factor VUI-related antigen by adenosine diphosphate, collagen, and thrombin. J Clin Invest 1978; 621: 1255-l263
• 39 Schmaier AH, Zuckerberg A, Silverman C, Kuchibhotla J, Tuszynski GP, Colman RW. High molecular weight kininogen. A secreted platelet protein. J Clin Invest 1983; 71: 1477-1489