Real-time analysis of the interaction of platelets with immobilized thrombospondin under flow conditions

The platelet granule protein (TS) is extracellularly secreted upon platelet activation and then binds to the platelet surface where it can interact with various adhesive proteins. Here, we have analyzed platelet interactions with a TS-coated surface under flow conditions, a model for platelet adhesion onto surface-bound TS under physiological conditions. Platelets exhibited temporary, very short-time adhesion on the TS surface, but no firm adhesion. This adhesion was inhibited by NNKY5-5 (anti-glycoprotein (GP) Ib antibody) and AJvW-2 (anti-von Willebrand factor (vWF)), indicating that both platelet GP Ib and plasma vWF contribute to this interaction. Antibodies against platelet collagen receptor integrin alpha(2)beta(1) had no significant effect. These results suggested that binding of vWF to TS is the first step in platelet interaction with the TS surface. By surface plasmon resonance spectroscopy, a dissociation constant (K(d)) of 3.97x10(-7) M was obtained for the binding reaction between immobilized TS and vWF. These results suggest the following model for platelet interaction with the TS surface under flow: plasma vWF first binds to the immobilized TS and then platelets interact with the TS-bound vWF. A low density of bound vWF would account for the observed weak interaction between TS and platelets under flow.