Incorporation of map kinases into the platelet cytoskeleton.

Erk1 (p44) and erk2 (p42) mitogen-activated protein (MAP) kinases are activated in agonist-stimulated platelets, although their role(s) in the activation process is unknown. In the present study, erk1, erk2 and the phosphorylated forms of both enzymes became associated with the contractile cytoskeleton in thrombin-stimulated platelets. Enzyme incorporation was accompanied by an increase in MAP kinase activity in the cytoskeleton, which was inhibited by PD98059. Pretreatment of the platelets with the arginine-glycine-aspartic acid-serine (RGDS) polypeptide enhanced both the cytoskeletal association and the enzyme activity, but cytochalasin D had no significant effect. Platelets from a patient with Glanzmann's thrombasthenia lack the alpha(IIb)beta(3) integrin and form only a rudimentary cytoskeleton, however, this cytoskeleton is enriched with both erk1 and erk2. These data suggest either that MAP kinases play a role in cytoskeletal rearrangement or that the cytoskeleton act as a frame to align MAP kinases with substrates in a highly integrated signal transduction pathway.

Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets.

The release of arachidonic acid, and its subsequent conversion to thromboxane A2, is an important component of platelet activation. The precise mechanism of arachidonic acid release is unknown although cytosolic phospholipase A2 (cPLA2) has been implicated. In the present study the effects of three agonists, the serine protease thrombin, the protein kinase C stimulant PMA and the calcium ionophore A23187 have been examined on the translocation and phosphorylation of cPLA2 and these have been correlated with arachidonic acid release. Thrombin, but neither PMA nor A23187, caused the release of [14C]-arachidonic acid from unstirred, prelabeled platelets. Immunoblot analysis was carried out on cytosolic and membrane fractions from control and activated platelets using an anti-cPLA2 antibody. In platelets stimulated by thrombin or A23187, but not by PMA, there was a translocation of cPLA2 to the membrane fraction. Immunoprecipitation of cPLA2 from [32P]-ortho-phosphate-prelabeled platelets, indicated enhanced phosphorylation on serine residues of cPLA2 from thrombin- or PMA-stimulated platelets. These results are consistent with two synergistic pathways mediating cPLA2 activity. Increased cytosolic calcium causes the translocation of cPLA2 to the membrane, and protein kinase either directly, or indirectly, phosphorylates the enzyme. Activation of both pathways, as occurs in response to thrombin, is required for arachidonic acid liberation.

Effects of the mitogen-activated protein (MAP) kinase kinase inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) on human platelet activation.

The role of mitogen-activated protein (MAP) kinase cascades in platelet function remains to be determined. Several studies have suggested a role in the activation of phospholipase A2; however, other functions seem likely. The object of the present study was to determine the role of the MAP kinase cascade in platelet function. An inhibitor of the mitogen-activated protein kinase kinase MEK1, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059), was used, at concentrations consistent with those reported to inhibit MEK1, to examine the role that this enzyme plays in platelet function. PD98059 inhibited aggregation in response to low-dose collagen and arachidonic acid, but not that in response to high-dose collagen, thrombin, thrombin receptor-activating peptide (TRAP), 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha (U46619), or phorbol ester. Thrombin, thrombin receptor-activating peptide, U46619, collagen, and arachidonic acid each caused the release of [3H]serotonin from dense granules, but only that elicited by low-dose collagen and arachidonic acid was inhibited by PD98059. The release of [3H]arachidonic acid in response to thrombin or collagen was unaffected by PD98059 pretreatment. In contrast, collagen- and arachidonic acid-induced thromboxane formation was inhibited by PD98059. These data suggest that MEK1 is not involved in the platelet response to thrombin or U46619. Furthermore, the inhibitory effects of PD98059 on collagen- and arachidonic acid-induced responses suggest that PD98059 may inhibit the conversion of arachidonic acid to thromboxane, in addition to its reported effects on MEK1.