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.
Jesty
J
J
Schools of Engineering and Medicine, State University of New York, Stony Brook, New York 11794, USA.
Bluestein
D
D
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