Barnes C S, Krafft B, Frech M, Hofmann U R, Papendieck A, Dahlems U, Gellissen G, Hoylaerts M F
Department of Cardiovascular Research, Merck KGaA, Frankfurterstrasse 250, 64271 Darmstadt, Germany.
Semin Thromb Hemost. 2001 Aug;27(4):337-48. doi: 10.1055/s-2001-16887.
Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.
血小板以依赖于血管性血友病因子（vWF）和不依赖于vWF的方式与胶原蛋白结合。我们最近鉴定了一种从医用水蛭唾液中分离出的重组蛋白——沙瑞汀，在多形汉逊酵母中表达了该蛋白，并研究了其对血小板与胶原蛋白直接和间接相互作用的影响。沙瑞汀剂量依赖性地抑制纯化的人vWF与人I型和III型胶原蛋白（IC50分别为0.23±0.004和0.81±0.04μg mL-1）以及小牛皮肤胶原蛋白（IC50为0.44±0.008μg mL-1）的结合。此外，沙瑞汀对人、啮齿动物和猪血浆vWF与这些胶原蛋白的结合显示出相似的抑制效力。在高剪切力（2700 s-1）条件下的流动腔中，沙瑞汀剂量依赖性且有效地抑制了胶原蛋白包被表面上血小板聚集体的形成（IC50为0.96±0.25μg mL-1），但在低剪切力（1300 s-1）下，剂量反应曲线向右移动（IC50为5.2±1.4μg mL-1）。表面等离子体共振分析揭示了沙瑞汀在人III型胶原蛋白上具有高亲和力和低亲和力结合位点（Kd分别为5×10-8 M和2×10-6 M）。虽然低浓度的沙瑞汀在增加剪切力时抑制血小板黏附（即高亲和力结合位点饱和），对不依赖vWF的胶原蛋白诱导的血小板聚集没有影响，但高浓度（即低亲和力结合位点饱和）被发现可抑制血小板聚集。这些数据表明，沙瑞汀是vWF依赖性血小板与胶原蛋白黏附的有效抑制剂，因此作为抗血栓药物可能具有治疗潜力。
