mechanism of αIIbβ3 antagonism by savignygrin and its implications for the evolution of anti-hemostatic strategies in soft ticks

• Published in: Insect biochemistry and molecular biology Vol. 34; no. 6; pp. 573 - 584
• Main Authors: Mans, B.J, Neitz, A.W
• Format: Journal Article
• Language: English
• Published: 01.06.2004
• Subjects: animal proteins; antagonists; anticoagulant activity; binding properties; binding sites; blood coagulation factors; blood platelets; evolution; hematophagy; hemostasis; integrin antagonist; membrane proteins; molecular models; Ornithodoros savignyi; platelet aggregation inhibition; platelet integrin; protein subunits; protein-protein interactions; structure-activity relationships; ticks
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2004.03.005

Savignygrin, a αIIbβ3 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 αIIbβ3 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~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 αIIbβ3 based on the recent crystal structure of αvβ3 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 αIIbβ3 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.