De Cristofaro R, De Filippis V
Department of Internal Medicine, Hemostasis Research Center, Catholic University School of Medicine, 00168 Rome, Italy.
Biochem J. 2003 Jul 15;373(Pt 2):593-601. doi: 10.1042/BJ20030167.
Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII.
凝血酶对凝血因子VIII（FVIII）的激活在凝血级联反应的放大过程中起着关键作用，该过程通过在精氨酸（372）、精氨酸（740）和精氨酸（1689）处的特异性蛋白水解切割来实现。FVIII的完全激活需要在精氨酸（372）处进行切割，这一过程涉及α-凝血酶的外位点II，即肝素结合位点（HBS）。本研究旨在探究糖蛋白Ibalpha（GpIbalpha；1 - 282片段）与凝血酶HBS结合对FVIII激活的影响。还使用了基于GpIbalpha 268 - 282序列构建的合成肽进行了类似实验，该合成肽在其序列中所有酪氨酸残基（位于276、278和279位）上均成功进行了硫酸化修饰。通过凝血和FXa生成测定评估，GpIbalpha 1 - 282和硫酸化的GpIb 268 - 282肽均导致活化FVIII生成逐渐减少（高达70%）。此外，SDS/PAGE和蛋白质印迹实验表明，在存在GpIbalpha 1 - 282或GpIb 268 - 282肽的情况下，FVIII精氨酸（372） - 丝氨酸（373）肽键切割时44 kDa A2结构域的特异性出现显著延迟。此外，使用反相高效液相色谱研究了后者对凝血酶介导的具有FVIII 341 - 376序列的肽水解的影响。根据竞争性抑制作用，凝血酶对FVIII 341 - 376肽水解的k（cat）/K（m）值随GpIbalpha 268 - 282肽浓度呈线性下降。综上所述，这些实验表明GpIbalpha硫酸化的268 - 282区域与凝血酶HBS结合，并负责抑制精氨酸（372） - 丝氨酸（373）键的切割以及FVIII的激活。
