Jesty J, Bluestein D
Schools of Engineering and Medicine, State University of New York, Stony Brook, New York 11794, USA.
Anal Biochem. 1999 Jul 15;272(1):64-70. doi: 10.1006/abio.1999.4148.
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.
人凝血酶原经乙酰化处理以产生一种修饰的凝血酶原，该修饰的凝血酶原在被血小板结合的凝血酶原酶激活后会生成一种凝血酶形式，这种凝血酶不会激活血小板，但在生色肽底物上保留其酰胺水解活性。如果在这样的测定中使用正常凝血酶原，生成的凝血酶会以反馈方式激活血小板，加速凝血酶生成速率，从而妨碍对初始血小板促凝活性的准确测量。凝血酶原的乙酰化在一系列磺基 -N -琥珀酰亚胺基乙酸酯（SNSA）浓度下进行。在室温下，于pH 8.2且不存在金属离子的条件下，用3 mM SNSA乙酰化30分钟，产生了一种修饰的凝血酶原，其凝血活性<0.1%（通过特异性凝血酶原凝血测定），但它可被因子Xa激活（在存在活化血小板或因子Va + 阴离子磷脂的情况下）以产生可用生色底物测量的凝血酶活性。由于对血小板的反馈作用被阻断，凝血酶生成呈线性，从而能够对初始血小板活化状态进行定量测量。
