Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.

[1]  G. Nelsestuen,et al.  Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change. , 1988, Biochemistry.

[2]  R. Henriksen,et al.  Characterization of the catalytic defect in the dysthrombin, Thrombin Quick. , 1987, The Journal of biological chemistry.

[3]  G. White,et al.  Structure-function relations in platelet-thrombin reactions. Inhibition of platelet-thrombin interactions by lysine modification. , 1981, The Journal of biological chemistry.

[4]  J. Fenton,et al.  Human thrombins. Production, evaluation, and properties of alpha-thrombin. , 1977, The Journal of biological chemistry.

[5]  J. Jesty,et al.  The activation of bovine coagulation factor X. , 1976, Methods in enzymology.

[6]  K. Mann,et al.  Identification of the primary structural defect in the dysthrombin thrombin Quick I: substitution of cysteine for arginine-382. , 1988, Biochemistry.

[7]  J. Jesty,et al.  Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma. , 1984, Blood.

[8]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[9]  H. Hemker,et al.  Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis". , 1996, The Journal of clinical investigation.

[10]  J. Dawes,et al.  Platelet alpha granule depletion: findings in patients with prosthetic heart valves and following cardiopulmonary bypass surgery. , 1983, Thrombosis research.

[11]  J. Rosing,et al.  The role of activated human platelets in prothrombin and factor X activation. , 1985 .

[12]  S. Morrison.  Origin of a fluorescence increase accompanying the limited proteolysis of fluorescein-labeled human prothrombin by Factor Xa. , 2009, International journal of peptide and protein research.

[13]  L V McIntire,et al.  Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow. , 1989, Biophysical journal.

[14]  J. Jesty,et al.  The use of acetylated factor X to prevent feedback activation of factor VIII during factor X activation: a tool for kinetic studies. , 1990, Analytical biochemistry.

[15]  G. Nelsestuen,et al.  Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1. , 1988, Biochemistry.

[16]  P. Sperryn,et al.  Blood. , 1989, British journal of sports medicine.

[17]  M. Jandrot-Perrus,et al.  Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin. , 1984, Blood.

[18]  J. Jesty,et al.  Calcium Stimulation of Procoagulant Activity in Human Erythrocytes , 1995, The Journal of Biological Chemistry.

[19]  S. Slack,et al.  The Effects of Flow on Blood Coagulation and Thrombosis , 1993, Thrombosis and Haemostasis.

[20]  W. Seegers,et al.  The acetylation of prothrombin. , 1960, Canadian journal of biochemistry and physiology.

[21]  J. Jesty,et al.  A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X , 1988 .

[22]  T. Byzova,et al.  Networking in the Hemostatic System , 1997, The Journal of Biological Chemistry.

[23]  J. Jesty,et al.  Effect of limited modification of amino groups on the reactivity of human factor Xa. , 1986, Biochimica et biophysica acta.

[24]  A. Schroit,et al.  Transbilayer movement of phospholipids in red cell and platelet membranes. , 1991, Biochimica et biophysica acta.

[25]  P. Comfurius,et al.  Platelet procoagulant activity: physiological significance and mechanisms of exposure. , 1991, Blood reviews.