Determination of the putative binding sites for thrombin receptor activating peptide through a hydropathic complementary approach

Putative binding sites in a platelet thrombin receptor (PAR-1) for the acti vating peptide SFLLRNPNDKYEPF (AP) and for the bradykinin analogue MKRPPGFS PFRSSRIG were revealed using a computer program for identifying complementa ry peptide segments. The program is based on the assumption that interactio ns of agonist's peptides and protein's receptors can be elucidated by compl ementary average hydropathies as much as possible equal by size and opposit e by sign. Some of the computer-found putative binding sites were close to the supposed AP-PAR-1 contacts in the amino-terminal exodomain and in the s econd extracellulary loop of PAR-1. Peptides complementary to these binding sites were also computer-designed and were synthesized. They mostly inhibi ted the aggregation of gel filtered platelets by thrombin (0.025 U/mL) with IC50 in a high micromolar range of concentrations. The peptide complementa ry to site L258-Y269 of PAR-1 induced aggregation of gel filtered platelets with EC50 = 98 [mu mol/L] related to thrombin (0.025 U/mL) aggregation res ponse.