Characterization of Fibronectin Assembly by Adherent Platelets Under Flow Conditions: Effect of Shear Stress and Role of β3 Integrins

Abstract

Platelet thrombi at site of vascular injury require resistance to shear stress generated by the flowing blood. Fibronectin assembly can contribute to platelet adhesion and aggregation. Here, we examined the effect of shear stress and the role of αIIbβ3 and αvβ3 in fibronectin assembly by adherent platelets under flow dynamic conditions in vitro. Platelets were placed onto immobilized fibronectin (50 µg/ml) with fluorescently labeled fibronectin. Shear rates (500 s⁻¹ or 5000 s⁻¹) were generated by a viscometer (Diamed) for 2 or 10 min. Adherent platelets were lysed with 2% deoxycholate buffer. Insoluble fibronectin fibrils were isolated by centrifugation for quantification by measuring fluorescence intensity. In some experiments, abciximab (anti-β3) or LM609 (anti-αvβ3) antibody, were added before experiments. When shear rates increased from 500 to 5000 s⁻¹, the amount of insoluble fibronectin detectable on platelets increased significantly (p < 0.05). However, prolongation of the exposure time to shear did not cause a significant difference in fibronectin assembly. No fibronectin fibrils were detectable on adherent platelets under static conditions. After 2 min at 5000 s⁻¹, platelets blocked with abciximab showed a significant decrease in fibril formation in comparison to control experiments (no antibody). A similar inhibitory effect was achieved with LM609. When platelets were exposed to 5000 s⁻¹ for 10 min, abciximab showed a higher inhibition on fibronectin assembly than LM609. We document that fibronectin assembly by adherent platelets is strongly affected by flow conditions. αvβ3 provides a significant contribution in initial phase, while αIIbβ3 probably exerts its effect in the later phase of fibronectin assembly under high shear conditions.