Witting J I, Bourdon P, Brezniak D V, Maraganore J M, Fenton J W
New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany 12201.
Biochem J. 1992 May 1;283 ( Pt 3)(Pt 3):737-43. doi: 10.1042/bj2830737.
Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.
水蛭素类似物-1 [D-苯丙氨酸-脯氨酸-精氨酸-脯氨酸-[甘氨酸]4-去硫酸水蛭素-(53 - 64) (HV1)] 的设计目的是，其前四个残基和最后12个残基分别与α-凝血酶催化位点和阴离子结合外位点结合，以识别纤维蛋白（原），在裂解位置有一个[甘氨酸]4桥和一个精氨酸-脯氨酸键。人α-、γ-和ζ-凝血酶，以及牛胰蛋白酶，在pH 7.4和约23℃时能快速水解光谱酶-TH（D-六氢酪氨酸-丙氨酸-精氨酸对硝基苯胺）。α-和ζ-凝血酶都具有高纤维蛋白原凝血活性（大于3000库单位/克），水蛭素类似物-1对这种底物能抑制它们 [Ki分别为2.56±0.35纳摩尔（n = 3）和1.84±0.15纳摩尔（n = 3）]，并能缓慢裂解抑制剂 [k分别为0.326±0.082分钟-1（n = 12）和0.362±0.056分钟-1（n = 18）]，而基本上没有凝血活性（约4库单位/克）的γ-凝血酶和胰蛋白酶，即使使用摩尔过量1000倍以上的水蛭素类似物-1也不会被抑制。在约23℃下，将水蛭素类似物-1与α-凝血酶或ζ-凝血酶一起孵育，并在37℃的凝血试验中用纤维蛋白原测量凝血酶活性，也得到了类似的抑制参数。α-或ζ-凝血酶对水蛭素类似物-1中精氨酸-3-脯氨酸-4键的裂解，通过高效液相色谱法上两种凝血酶相同的裂解产物以及对α-凝血酶产物的序列分析得以显示。这些数据表明，水蛭素类似物-1是具有高纤维蛋白原促凝活性的凝血酶形式的特异性抑制剂，并且其精氨酸-3-脯氨酸-4键会被这些凝血酶形式缓慢裂解。
