The mechanism of alphaIIbbeta3 antagonism by savignygrin and its implications for the evolution of anti-hemostatic strategies in soft ticks.
Savignygrin, a alphaIIbbeta3 antagonist presents the RGD sequence on the substrate-binding loop of the (BPTI-fold). This study investigated whether this is the only integrin-targeting motif associated with its mechanism. It forms a tight-binding complex with alphaIIbbeta3 that is resistant to SDS dissociation under reducing and non-reducing conditions, but not to temperature or EDTA. The same complex is formed on resting and activated platelets, as well as aggregated platelets that have been disaggregated with savignygrin. Binding of FITC labeled savignygrin to platelets show that the binding kinetics and affinity of savignygrin is similar for resting and activated platelets (Kd approximately 50-70 nM). Binding to resting or activated platelets was significantly inhibited by two savignygrin peptide fragments, S2 (GSRGDEDATFG) and S3 (FDREDGGSRQG) that correspond with two specific loop-like areas in the structure of savignygrin that together form a continuous binding interface. The inability of S3 to inhibit platelet aggregation indicates that it targets a novel ligand-binding site. A model of alphaIIbbeta3 based on the recent crystal structure of alphavbeta3 into which the RGD sequence of savignygrin was docked shows that savignygrin lies along the interface formed by the two subunits. A novel mode of integrin antagonism is indicated that includes the targeting of distinct sites on the alphaIIbbeta3 subunits. The S2 and S3 loops are not involved in the mechanisms of the related soft tick blood coagulation inhibitors and suggest that this allowed their evolution as integrin targeting motifs.
Mans
Ben J
BJ
Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa. ben.mans@bioagric.up.ac.za
Neitz
Albert W
AW
D000595:Amino Acid Sequence; D000818:Animals; D026862:Argasidae / Q000235:genetics / Q000502:physiology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019143:Evolution, Molecular; D005434:Flow Cytometry / Q000379:methods; D006490:Hemostatics / Q000037:antagonists & inhibitors*; D019476:Insect Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*
D006490:Hemostatics; D019476:Insect Proteins; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011506:Proteins; C460366:savignygrin protein, Ornithodoros savignyi