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.
Schools of Engineering and Medicine, State University of New York, Stony Brook, New York 11794, USA.
J
J
Jesty
D
D
Bluestein
D000107:Acetylation; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D005246:Feedback; D006801:Humans; D066298:In Vitro Techniques; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology; D011516:Prothrombin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology
D011516:Prothrombin; D013917:Thrombin