Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors.

Abstract

The purification, complete amino acid sequence, and biological activity are described for several homologous snake venom proteins that are platelet glycoprotein (GP) IIb-Ila antagonists and potent inhibitors of platelet aggregation. The primary structures of kistrin (from Agkistrodon rhodostoma), bitan (from BiWs 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 proteins 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-Ila, 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-Hla 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 = x100 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-Ila-fibrinogen interaction and as such serve as potential antithrombotic agents.

Tailoring echistatin to possess higher affinity for integrin αIIbβ3

“…The ICss value of each echistatin is defined as the concentration necessary to inhibit receptor binding by 50%. than the natural cell adhesive proteins including the disintegrins [2,5,9]. The purpose of this study was to address the question of whether the conformational constraint works also in a natural disintegrin, echistatin.…”

“…They are cysteinerich, 50-80 residue proteins, and all contain a functional ArgGly-Asp (RGD) sequence. It is known that they inhibit platelet aggregation, through the binding to integrin an&, 500-1000 times more strongly than the GRGDS peptide [2,4].…”

“…These proteins include echistatin [l] from Echis carinatus, kistrin [2] from Agkistrodon rhodostroma,…”

Integrin αvβ3‐targeted cancer therapy

“…The first reported disintegrin was the snake venom protein trigramin [Huang et al, 1987]. Since then, many more members of this protein family have been discovered and isolated, such as kistrin [Dennis et al, 1990], bitistatin [Shebuski et al, 1989], barbourin [Scarborough et al, 1991], echistatin [Gan et al, 1988], and contortrostatin [Swenson et al, 2004]. Because of their high integrin binding affinities and their inhibition efficacy, several disintegrins and other integrin inhibiting snake venom components have been evaluated in pre-clinical tests for their capacity to inhibit tumor growth and progression both in vitro and in vivo [Calvete et al, 2003;McLane et al, 2001;Zhou et al, 2004].…”

Differences in binding of 99mTc-disintegrins to integrin αvβ3 on tumor and vascular cells

“…Bitistatin was produced as a recombinant product [18]. Other disintegrins were produced by reversed-phase (RP) HPLC purification from freeze-dried natural snake venom (Miami Serpentarium, Punta Gorda, FL): kistrin (from Calloselasma rhodostoma venom) [19]; echistatin (from Echis carinatus) [20]; flavoridin (from Trimeresurus flavoviridis) [21]; and VLO4 (homodimer from Vipera lebetina obtusa) [22]. The active component was identified by its bioactivity (e.g., ability to inhibit platelet aggregation in human platelet-rich plasma, or ability to inhibit adhesion of cultured cells [22]).…”