Platelet adhesion to collagen under flow causes dissociation of a phosphoprotein complex of heat-shock proteins and protein phosphatase 1.

Phosphorylation/dephosphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions. Using 32P-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the alpha2beta1 integrin resulted in a strong dephosphorylation of several protein bands. Neither adhesion to polylysine nor thrombin-induced aggregation caused similar protein dephosphorylation. In addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosphatases. Recent studies indicate that phosphatases may be associated with the heat-shock proteins. Immunoprecipitations with antibodies against either the heat-shock cognate protein 70 (hsc70) or heat-shock protein 90 (hsp90) showed the presence of a phosphoprotein complex in 32P-labeled, resting human platelets. Antibody probing of this complex detected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1C alpha and PP1C delta, as well as the M regulatory subunit of PP1 (PP1M). OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platelets adhering to collagen, hsc70 was completely dephosphorylated and hsp90, PP1 alpha, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platelet adhesion.