Anti-thrombotic agents derived from snake venom proteins

Disintegrins as anti-thrombotic agents

Trigramin, a non-enzymatic small molecular weight polypeptide, was first discovered in 1987 [7]. Our earlier studies showed that disintegrins derived from T. gramineus, Agkistrodon halys, and Agkistrodon rhodostoma [8–10] inhibited platelet aggregation elicited by various aggregation agonists including ADP, epinephrine, sodium arachidonate, collagen and Ca²⁺ ionophone A23187 with similar IC50 concentrations. They neither affect shape change nor the cAMP level. Further studies showed that trigramin and echistatin purified from T. gramineus and Echis carinatus, inhibited fibrinogen binding to ADP-stimulated platelets [7, 11]. ¹²⁵I-trigramin showed less than 5 % binding to ADP-stimulated platelets in Glanzmann’s thrombasthenia patients (a genetic disease with ?IIb?3-defect) compared to normal platelets, suggesting that ?IIb?3 is the target of trigramin. In addition, 7E3 mAb raised against ?IIb?3 and RGDS inhibited ¹²⁵I-trigramin binding. Subsequent sequencing of trigramin showed that it is a RGD-containing single polypeptide with 72 amino acid residues and six disulfide bonds. Reduced trigramin lost its inhibitory activity for platelet aggregation and binding capacity to platelets, indicating that the biological activity of trigramin depends upon the presence of the RGD sequence and its steric structure maintained by disulfide bridges [7, 12]. Upon intravenous administration, trigramin prolonged the bleeding time of severed mesentery arteries, further supporting its in vivo antithrombotic activity [13]. Later, other disintegrins, such as kistrin and applagin were also shown to prevent experimental thrombosis in dogs [14, 15]. Since then, many pharmaceutical companies have developed potential antithrombotic agents based on the structure of these disintegrins. Among these disintegrins, barbourin, a KGD-containing polypeptide, showed a higher specificity toward platelet ?II?3 than to endothelial ?v?3 [16]. Thus, eptifibatide (Integrillin), a cyclic KGD-peptide, has been successfully developed as an ?IIb?3 antagonist, used clinically for prevention of restenosis during percutaneous transluminal coronary angioplasty (PTCA) [17]. Tirofiban is also designed to mimic the RGD-loop structure of the disintegrin, echistatin [18]. In contrast to the first anti- aggregation agent, the chimeric monoclonal c7E3Fab (Abciximab, Reopro) a mAb raised against ?IIb?3 [19], tirofiban and eptifibatide are small-mass ?IIb?3 antagonists derived from snake venom disintegrins. Vipegitide, a folded KTS-containing peptidomimetic molecule, inhibited the adhesion of ?1/?2 integrin toward collagen, and ADP and collagen-I induced platelet aggregation in PRP and whole human blood [20], providing strategy for developing ?2?1 antagonists as antiplatelet agents.