Abstract

Thrombin binding to the platelet membrane is a critical initial step in the coagulation cascade, driving platelet activation and the subsequent formation of a stable clot. While the biological significance is well-established, the precise physicochemical parameters governing this interaction remain incompletely defined. This study aimed to elucidate the thermodynamic and kinetic features of thrombin-platelet membrane binding, focusing on the influence of environmental factors such as pH, ionic strength, and membrane lipid composition. Using a combination of surface plasmon resonance (SPR) and fluorescence spectroscopy on model and native platelet membranes, we characterized the binding affinity and stoichiometry. Our results reveal a high-affinity, saturable binding site, with the interaction being significantly modulated by the presence of anionic phospholipids, particularly phosphatidylserine, suggesting a strong electrostatic component. Furthermore, kinetic analysis indicates a complex, multi-step binding mechanism, with the association rate being highly sensitive to changes in ionic strength. These findings provide a quantitative physicochemical framework for understanding thrombin's localization and activity on the platelet surface. The detailed characterization of these features is essential for developing targeted anti-thrombotic strategies that specifically interfere with the initial membrane-binding events of coagulation factors.