Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function.
In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.
Savage
B
B
Department of Molecular and Experimental Medicine, and Scripps Clinic and Research Foundation, La Jolla, California 92037.
Marzec
U M
UM
Chao
B H
BH
Harker
L A
LA
Maraganore
J M
JM
Ruggeri
Z M
ZM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D003435:Crotalid Venoms; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism* / Q000494:pharmacology; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D012701:Serotonin / Q000097:blood; D013928:Thromboxane A2 / Q000096:biosynthesis / Q000097:blood; D014757:Viper Venoms / Q000378:metabolism* / Q000494:pharmacology
D003435:Crotalid Venoms; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D014757:Viper Venoms; C057986:echistatin; D012701:Serotonin; D013928:Thromboxane A2; D000244:Adenosine Diphosphate; D010740:Phospholipases; C041603:Agkistrodon platelet aggregation inhibitor; D010741:Phospholipases A