Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase

Abstract

p38 MAP kinase in human platelets is activated by platelet agonists including thrombin, thromboxane A 2 (TxA 2 ), ADP, and others. However, both upstream mechanisms of p38 MAP kinase activation, and their downstream sequelae, are presently controversial and essentially unclear. Certain studies report sequential activation of cGMP-dependent protein kinase (PKG) and p38/ERK pathways by platelet agonists, leading to integrin activation and secretion, whereas others establish an essential role of Src/ERKmediated TxA 2 generation for fibrinogen receptor activation in human platelets. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 release are important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation did not significantly contribute to calcium mobilization, P-selectin expression, ␣IIb␤3 integrin activation, and aggregation of human platelets in response to thrombin. Finally IntroductionIn vivo, circulating platelets are continually exposed to both adhesive and/or activating factors (fibrinogen, ADP, von Willebrand factor [VWF], thrombin, TxA 2 , etc), as well as inhibitory factors such as endothelium-derived nitric oxide (NO), prostacyclin (PG-I 2 ), and ADPase. 1 Most of these activating and inhibitory molecules bind to specific platelet receptors and stimulate signaling pathways that promote or inhibit platelet adhesion, aggregation, and secretion. A central role among platelet-activating factors is played by ADP, which induces multiple platelet responses and potentiates platelet aggregation caused by other agonists. 2,3 ADP is released from platelet-dense granules upon activation by agonists such as thrombin and collagen, and binds to purinergic receptors (P2Y 1 , P2Y 12 , P2X 1 ) to reinforce platelet aggregation and thrombus formation. 2,[4][5][6] Recently, new mechanisms for agonist-induced platelet activation and secretion were proposed to involve sequential activation of cGMP-dependent protein kinase (PKG)/p38/integrin or Akt/ eNOS/sGC/cGMP/PKG secretion, respectively, 7-9 whereas others indicate an essential role of Src/ERK-mediated TxA 2 generation for fibrinogen receptor activation in human platelets. 10 We 11,12 and others 10,13 were unable to reproduce the reported ERK activation by PKG. However, both the upstream mechanisms of p38 activation, and their downstream effects, are presently controversial and unclear. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 secretion are important mediators upstream of thrombin-evoked p38 activation. Furthermore, PKG activation does not stimulate, but rather inhibits, thrombin-evoked p38 activation, which alone has no significant effect on platelet stimulation/aggregation. Materials and methodsAnalysis of P-selectin expression, ␣IIb␤3 activation, aggregation, and intracellular calcium measurement Platelets were isolated from whole blood obtained from healthy volunteer...

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“…It was previously reported that p38 MAP kinase in human platelets could be activated by several platelet agonists, including thrombin, thromboxane A2, ADP, and von Willebrand factor, but the upstream and downstream pathways are not quite similar. 43,44 Therefore, the role and the underlying mechanism of p38 MAPK in IgG-mediated platelet activation will require further investigation.…”

p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

“…Human platelets possess 4 isoforms of p38 MAPK (␣, ␤, ␥, and ␦), but the most abundant forms are p38 MAPK-␣ and -␤. p38 MAPK-␣ (named p38 MAPK) was shown to be activated in response to several agonists, including thrombin, 10,11 TxA 2 , 12 collagen, 13 ADP, 14 and VWF, 15 but its role in platelet function remains controversial. Importantly, inhibition of p38 MAPK showed only minor effects on platelet aggregation induced by threshold concentrations of agonists, 12,16 and this effect, at least in part, may be the result of cross-reactivity of p38 inhibitors with cyclo-oxygenases and thus impairment of TxA 2 generation.…”

NO/cGMP/PKG pathway in platelets: inhibitory but not stimulatory

“…However, recently a new mechanism of platelet activation by vWF, mediated by PKG (that sequentially activates p38 and ERK MAP kinases), was proposed [1,2]. Here we present data that activation of PKG by cGMP analogs or NO donors does not stimulate, but rather inhibits, p38 and ERK MAP kinases [3]. However, some PKG stimulators and inhibitors do affect platelets independently of PKG activity [4].…”