凝血酶因子IIa

特异性

凝血酶是一种内切丝氨酸蛋白酶，可选择性地裂解纤维蛋白原的Arg -Gly键，形成纤维蛋白并释放纤维蛋白肽A和B。^1,2

凝血酶的最佳切割位点是：1）AB-Pro-Arg- || -XY，其中A和B是疏水性氨基酸，X和Y是非酸性氨基酸；2）Gly-Arg- || -Gly。²

任意哺乳动物的凝血酶可凝结其他哺乳动物的纤维蛋白原。³

凝血酶有两种仅作用于精氨酸位点的纤维蛋白原裂解方式。初级裂解产物纤维蛋白肽A是从纤维蛋白原第16位氨基酸后裂解，有时从第19位氨基酸后裂解，而次级裂解产物纤维蛋白肽B是在第14位氨基酸处裂解产生。⁴

凝血酶在pH 5-10.5的范围内有活性。⁵
催化最适pH是8.3。⁵
凝血酶在pH 5或更低时沉淀。⁵

凝血酶活化不需要二价金属离子或辅因子。但凝血酶的Na +依赖性变构激活与凝血酶在精氨酸残基位点切割的特异性密切相关。⁶血浆蛋白C活化时，血栓调节蛋白是凝血酶的辅因子。⁷

体内活化过程和物理性质

凝血酶在体内的主要形式是它的酶原，凝血酶原（因子II），由肝脏产生。正常人血浆中凝血酶原的浓度约为5-10mg / dL。⁸凝血酶原是一种糖蛋白，含糖量约为～12％。⁸

凝血酶原在体内被活化的因子X切割，释放活化肽并将凝血酶切割成轻链和重链，产生催化活性的α-凝血酶。 α-凝血酶由轻链（A链）（MW〜6,000）和重链（B链）（〜31,000）组成。这两条链通过一个二硫键连接。¹²人凝血酶的B链由肽部分（MW 29,485）和三个天冬酰胺位点上具有N连接糖基化的碳水化合物部分（2334）组成。^9,10牛凝血酶含有1.7 ％葡糖胺，1.8％唾液酸，0.61％半乳糖和0.95％甘露糖。¹¹凝血酶还含有γ-羧基谷氨酰胺残基。这些修饰的谷氨酰残基是微粒体酶，维生素K依赖性羧化酶羧化的结果。γ-羧基谷氨酰残基对于负电荷磷脂表面的钙依赖性相互作用是必需的，对于凝血酶原向凝血酶的转化也是必不可少的。¹²在体内，凝血酶原在磷脂膜的表面活化，所述磷脂膜结合氨基末端凝血酶原以及Va和Xa因子。活化起始缓慢，因为因子V本身是由最初的少量凝血酶激活。

在特定的储存条件下，α-凝血酶的自溶消化导致形成缺乏纤维蛋白溶解活性的β-纤维蛋白和γ-凝血酶，但保留一些除纤维蛋白原以外的抗肽底物和蛋白底物合成活性。¹³我们的凝血酶制剂主要是α凝血酶形式。 

人凝血酶由几个同工酶组成，等电点范围6.35-7.6。
牛凝血酶等电点范围是7.05 - 7.114

E^1%(280nm) = 18.3 (人)¹⁵
E^1%(280nm) = 19.5 (牛)¹⁶

凝血酶活性测定

我们的凝血酶实验方法是以与NIH凝血酶参考标准直接比较获得NIH单位来衡量。

根据改进的Biggs法，NIH测定方案使用0.2 mL稀释血浆（1：1添加生理盐水）和0.1 mL凝血酶样品（在pH 7.35的1％缓冲白蛋白溶液中固定）作为底物。¹⁷仅在15-25秒的凝血时间内测定凝血酶浓度。

文献中的凝血酶浓度通常有不同的活性单位。^18-20

凝血酶文献中的几个常识：
1 WHO 单位 = 1 NIH 单位
1 NIH 单位 = 1 USP 单位
1 NIH单位 = 0.324 +/- 0.073 µg
1 IOWA单位 = 0.83 NIH 单位

凝血酶（人和牛）将催化几种肽对硝基苯胺，甲苯磺酰基-硝基苄基酯和硫代苄基酯合成底物的水解。²¹

应用

血浆中纤维蛋白凝块的形成：
一到两个单位的凝血酶可凝结1毫升的血浆。

融合蛋白的切割：
在20微升50 mM pH 8.0碳酸氢铵溶液中，0.5 NIH unit /nM的凝血酶可在凝血酶识别位点处裂解多种肽。²²

凝血酶切割融合蛋白可以在1：500比率下进行。²³
融合蛋白可以在由50 mM Tris，pH 8.0，150 mM NaC，2.5 mM CaCl₂和0.1％2-巯基乙醇组成的凝血酶切割缓冲液中被切割。将2 mg融合蛋白与4 µg凝血酶在裂解缓冲液中室温孵育20分钟。²⁴

产品

 底物

天然底物

纤维蛋白原，凝血因子V，²⁵凝血因子VIII，²⁵凝血因子XIII，²⁵血小板反应蛋白，²⁶凝血酶原，¹²和蛋白C。²⁷

合成底物

N-苯甲酰基-Phe-Val-Arg 4-甲氧基-b-萘酰胺，N-苯甲酰基-Phe-Val-Arg-对硝基苯胺，Boc-β-苄基-Asp-Pro-Arg-7-酰胺基-4-甲基香豆素，Boc-Val-Pro-Arg-7-酰胺基-4-甲基香豆素，Sar-Pro-Arg对硝基苯胺，凝血酶形成显色底物，N-对甲苯磺酰基-Gly-Pro-Arg 7-酰胺基-4-甲基香豆素，N-(对甲苯磺酰基)-Gly-Pro-Arg对硝基苯胺。

抑制剂

氟磷酸二异丙酯，²⁸苯甲磺酰氟，²⁸AEBSF，水蛭素，²⁸四硝基甲烷，²⁸原黄素，²⁸抗凝血酶III，²⁸a1-抗胰蛋白酶²⁸，a1-抗纤溶酶，²⁸甲磺酸加贝酯，²⁹抗痛素³⁰和甲苯磺酰基-L-赖氨酸氯甲基酮。³¹

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