BACKGROUND Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity. RESULTS Recombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin. CONCLUSIONS These findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.

中文翻译：

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RGD-hirudin与凝血酶结合的结构基础：Tyr3和5个C末端残基对于抑制凝血酶活性至关重要。

背景技术水rud素是由药用水ech水rud的唾液腺产生的抗凝蛋白。它是一种功能强大且特异的凝血酶抑制剂。含有RGD基序的新型重组水rud素RGD-hirudin可竞争性抑制血纤蛋白原与血小板上GPIIb / IIIa的结合，从而抑制血小板凝集，同时保持其抗凝活性。结果基于分子模拟设计的重组RGD-hirudin和六个突变体变体（Y3A，S50A，Q53A，D55A，E57A和I59A）在毕赤酵母中表达。将蛋白质重折叠并通过凝胶过滤和阴离子交换色谱纯化为同质单体。测试了六个突变体和RGD-hirudin的抗凝血酶活性。进一步，我们使用BIAcore表面等离振子共振分析（SPR）评估了突变体变体和RGD-hirudin与凝血酶的结合。动力学和亲和常数表明，所有六个突变蛋白的KD值均高于RGD-hirudin。结论这些发现有助于人们对RGD-水in素与凝血酶之间的相互作用有了新的认识。