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 upo n activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a c hromogenic peptide substrate. Lf normal prothrombin is used in such an assa y, the thrombin that is generated activates the platelets in a feedback man ner, accelerating the rate of thrombin generation and thereby preventing ac curate measurement of the initial platelet procoagulant activity. Acetylati on 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 proth rombin that has <0.1% clotting activity (by specific prothrombin clotting a ssay), but it is activated by factor Xa (in the presence of either activate d platelets or factor Va + anionic phospholipid) to produce thrombin activi ty that is measurable with a chromogenic substrate. Because the feedback, a ction on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state. (C) 1999 Academic Press.