Dennis M S, Henzel W J, Pitti R M, Lipari M T, Napier M A, Deisher T A, Bunting S, Lazarus R A
Department of Biomolecular Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Proc Natl Acad Sci U S A. 1990 Apr;87(7):2471-5. doi: 10.1073/pnas.87.7.2471.
The purification, complete amino acid sequence, and biological activity are described for several homologous snake venom proteins that are platelet glycoprotein (GP) IIb-IIIa antagonists and potent inhibitors of platelet aggregation. The primary structures of kistrin (from Agkistrodon rhodostoma), bitan (from Bitis arietans), three isoforms of trigramin (from Trimeresusus gramineus), and an isoform of echistatin (from Echis carinatus) were determined by automated sequence analysis and fast atom bombardment mass spectrometry analysis. Each of the protein in this family, which range from 47 to 83 residues, contains an Arg-Gly-Asp amino acid sequence found in protein ligands that binds to GPIIb-IIIa, a high (17 +/- 1%) cysteine content conserved in the primary sequence, and a homologous N-terminal region absent only in the echistatin isoforms. Each protein directly inhibits the interaction of purified platelet GPIIb-IIIa to immobilized fibrinogen about 100 times more effectively than does the pentapeptide Gly-Arg-Gly-Asp-Ser; IC50 values range from 1.1 to 3.0 nM. The IC50 value for the inhibition of platelet aggregation, using human platelet-rich plasma stimulated with ADP, ranges from 110 to 550 nM for the various proteins, about 1000-fold more potent than Gly-Arg-Gly-Asp-Ser. Kistrin binds reversibly to both resting and ADP-activated human platelets with high affinity (Kd = 10.8 nM and 1.7 nM, respectively) and to purified GPIIb-IIIa with a lower affinity (Kd = approximately 100 nM). Finally, kistrin injected at 1.0 mg/kg into rabbits reversibly inhibits platelet aggregation ex vivo over 30 min without induction of thrombocytopenia. We propose that these proteins are members of a general class of proteins found in the venom of pit vipers that inhibit platelet aggregation by antagonism of the GPIIb-IIIa-fibrinogen interaction and as such serve as potential antithrombotic agents.
描述了几种同源蛇毒蛋白的纯化、完整氨基酸序列和生物活性，这些蛋白是血小板糖蛋白（GP）IIb-IIIa拮抗剂和血小板聚集的强效抑制剂。通过自动序列分析和快原子轰击质谱分析确定了锯鳞蝰素（来自红口蝮蛇）、比特安（来自鼓腹咝蝰）、三种竹叶青毒素异构体（来自竹叶青）和一种echistatin异构体（来自锯鳞蝰）的一级结构。该家族中的每种蛋白质含有47至83个残基，包含在与GPIIb-IIIa结合的蛋白质配体中发现的精氨酸-甘氨酸-天冬氨酸氨基酸序列，在一级序列中具有高（17±1%）的半胱氨酸含量保守性，以及仅在echistatin异构体中不存在的同源N端区域。每种蛋白质直接抑制纯化的血小板GPIIb-IIIa与固定化纤维蛋白原的相互作用，其效力比五肽甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸高约100倍；IC50值范围为1.1至3.0 nM。对于使用ADP刺激的富含人血小板的血浆，抑制血小板聚集的IC50值对于各种蛋白质范围为110至550 nM，比甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸强约1000倍。锯鳞蝰素以高亲和力（分别为Kd = 10.8 nM和1.7 nM）可逆地结合静止和ADP激活的人血小板，并以较低亲和力（Kd = 约100 nM）结合纯化的GPIIb-IIIa。最后，以1.0 mg/kg注射到兔子体内的锯鳞蝰素在30分钟内可逆地抑制体外血小板聚集，且不诱导血小板减少症。我们提出这些蛋白质是蝰蛇毒液中发现的一类一般蛋白质的成员，它们通过拮抗GPIIb-IIIa-纤维蛋白原相互作用来抑制血小板聚集，因此可作为潜在的抗血栓形成剂。
