The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2ß1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response.

Platelet activation by Streptococcus sanguinis is accompanied by MAP kinase phosphorylation.

There is increasing interest in the role of infections in atherothrombotic conditions. In particular, bacteria, notably those of oral origin, have been shown to activate platelets using a variety of mechanisms. Previous studies have shown that S. sanguinis strain 2017-78 induces platelet aggregation which requires the presence of both vWF and IgG. This aggregation is accompanied by the consecutive phosphorylation/desphosphorylation/rephosphorylation of several signalling proteins. The first two phases are thromboxane-dependent whereas the rephosphorylation phase is mediated by engagement of the αIIbß3 integrin. Here signalling events, specifically the potential role of MAP kinases, associated with S. sanguinis strain 2017-78-induced platelet activation have been further examined using an immunoblotting approach. The addition of S. sanguinis strain 2017-78 caused a similar triphasic phosphorylation profile of the platelet MAP kinase Erk2 to that seen with other phosphoproteins. Pretreatment with aspirin or RGDS did not affect 2017-78-induced Erk2 phosphorylation or desphosphorylation but both inhibited the rephosphorylation phase. In contrast the level of 2017-78-induced platelet MAP kinase p38 phosphorylation remained at an elevated level, and this was unaffected by aspirin. Similarly, 2017-78-induced cPLA(2) phosphorylation remained above basal levels during the aggregation process. The p38 inhibitor SB203580 inhibited S. sanguinis-induced aggregation with no effect on the phosphorylation of either p38 or cPLA(2). Thus the current study demonstrates the activation of both the Erk2 and p38 forms of MAP kinases, and of cPLA(2), in platelets stimulated with S. sanguinis strain 2017-78, and is consistent with a role for Erk2, but not for p38, in the cPLA(2) phosphorylation in response to S. sanguinis.

Cyclic nucleotides inhibit MAP kinase activity in low-dose collagen-stimulated platelets.

Collagen-induced platelet activation is a complex process involving multiple signaling pathways. The role(s) of MAP kinases (ERKs and p38(MAPK)) are unclear, although at high, but not low, collagen concentrations p38(MAPK) is involved in cPLA(2)-mediated arachidonic acid release, prior to thromboxane generation. Cyclic nucleotides are conventionally regarded as mediators of platelet inhibition. However recent studies suggested a role for cGMP early in a MAP kinase pathway in platelet activation. In the current study the roles and relationships of MAP kinases, cyclic nucleotides and cPLA(2) in platelet activation by low-dose collagen and a thromboxane analogue (U46619) have been evaluated. Stimulants of neither adenylate cyclase (PGI(2)) nor guanylate cyclase (NaNP) alone had any effect on the basal phosphorylation of either MAP kinase. PGI(2) inhibited ERK/p38(MAPK) phosphorylation in response to both agonists which was unaffected by a cPLA(2) inhibitor (AACOCF(3)). NaNP inhibited collagen-induced ERK/p38(MAPK) phosphorylation, which was enhanced by AACOCF(3) and reversed by a guanylate cyclase inhibitor (ODQ). However NaNP had no effect on U46619-induced p38(MAPK) phosphorylation. Thus adenylate cyclase activation inhibits low-dose collagen-induced MAP kinase phosphorylation both prior, and distal, to thromboxane release. The study also supports an inhibitory, rather than stimulatory, role for guanylate cyclase in platelet signaling.

Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets.

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.