External calcium facilitates signalling, contractile and secretory mechanisms induced after activation of platelets by collagen

Abstract

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca²⁺. We have studied the involvement of exogenous Ca²⁺ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca²⁺ levels ([Ca²⁺]_i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca²⁺ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca²⁺, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca²⁺]_i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca²⁺. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca²⁺, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca²⁺ depends on a functional cytoskeleton.

• Tyrosine phosphorylation
• cytoskeleton
• secretion
• external Ca2+
• collagen