Kaminski M, Siebenlist K R, Mosesson M W
University of Wisconsin Medical School, Milwaukee Clinical Campus.
J Lab Clin Med. 1991 Mar;117(3):218-25.
Inhibition of thrombin proteolysis of fibrinogen with D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) results in irreversible inactivation of the thrombin catalytic site, but the PPACK-inhibited thrombin, through its exosite, retains its ability to bind to fibrinogen or fibrin. Hirudin inactivates thrombin at the catalytic site and also inhibits thrombin exosite binding to fibrin or fibrinogen. PPACK or hirudin was added to a clotting mixture of fibrinogen and active thrombin (enzyme-to-substrate ratio = 1:400 at ionic strength of 0.14; 1:800 at ionic strength of 0.09) before the onset of gelation. Subsequent fibrin assembly was evaluated by turbidity measurements at 350 nm and by determining the fibrin and fibrinogen content of the clots that ultimately formed. Polymerization rates and the fibrin-fibrinogen content of the clots that formed were greater in the PPACK-inhibited system than in the hirudin-inhibited system, and the effect was amplified at the lower ionic strength. PPACK-thrombin also promoted the polymerization of native or prepared mixtures of fibrin and fibrinogen. The results suggest that in addition to its well-recognized role in the proteolytic conversion of fibrinogen to fibrin, thrombin functions through exosite binding to fibrin as a cofactor in fibrin polymerization by accelerating fibrin clot assembly.
用D-苯丙氨酰-L-丙氨酰-L-精氨酸氯甲基酮（PPACK）抑制凝血酶对纤维蛋白原的蛋白水解作用会导致凝血酶催化位点不可逆失活，但PPACK抑制的凝血酶通过其外位点仍保留与纤维蛋白原或纤维蛋白结合的能力。水蛭素在催化位点使凝血酶失活，并且还抑制凝血酶外位点与纤维蛋白或纤维蛋白原的结合。在凝胶化开始前，将PPACK或水蛭素添加到纤维蛋白原和活性凝血酶的凝血混合物中（在离子强度为0.14时酶与底物的比例为1:400；在离子强度为0.09时为1:800）。随后通过在350nm处测量浊度以及测定最终形成的凝块中的纤维蛋白和纤维蛋白原含量来评估纤维蛋白的组装情况。在PPACK抑制的系统中，形成的凝块的聚合速率和纤维蛋白-纤维蛋白原含量高于水蛭素抑制的系统，并且在较低离子强度下这种效应会增强。PPACK-凝血酶还促进天然或制备的纤维蛋白与纤维蛋白原混合物的聚合。结果表明，除了其在纤维蛋白原向纤维蛋白的蛋白水解转化中公认的作用外，凝血酶还通过外位点与纤维蛋白结合，作为纤维蛋白聚合中的辅因子加速纤维蛋白凝块的组装发挥作用。
