PLATELET-ADHESION TO COLLAGEN UNDER FLOW CAUSES DISSOCIATION OF A PHOSPHOPROTEIN COMPLEX OF HEAT-SHOCK-PROTEINS AND PROTEIN-PHOSPHATASE-1

Phosphorylation/deposphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions, Using P-32-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the alpha(2) beta(1) integrin resulted in a strong dephosphorylation of several pro tein bands, Neither adhesion to polylysine nor thrombin-induced aggreg ation caused similar protein dephosphorylation. in addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phos phatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosph atases. Recent studies indicate that phosphatases may be associated wi th the heat-shock proteins, Immunoprecipitations with antibodies again st either the heat-shock cognate protein 70 (hsc70) or heat-shock prot ein 90 (hsp90) showed the presence of a phosphoprotein complex In P-32 -labeled, resting human platelets, antibody probing of this complex de tected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1 C alpha and PP1C delta, as well as the M regulatory subunit of PPI (PP 1M), OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platele ts adhering to collagen, hsc70 was completely dephosphorylated and hsp 90, PP1 alpha, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platele t adhesion. (C) 1997 by The American Society of Hematology.