Molecular mechanisms involved in human platelet aggregation by synergistic interaction of platelet-activating factor and 5-hydroxytryptamine.

Abstract

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated the effect of PAF on thromboxane A2 (TXA2) formation and phosphorylation of extracellularly regulated mitogen-activated protein kinases (ERK1/2). The synergism of 5-HT and PAF in platelet aggregation was inhibited by calcium (Ca2+) channel blockers, verapamil and diltiazem, phospholipase C (PLC) inhibitor, U73122, cyclooxygenase (COX) inhibitor, indomethacin, and MEK inhibitor, PD98059. These data suggest that synergistic effect of 5-HT and PAF on human platelet aggregation involves activation of PLC/Ca2+, COX and MAP kinase pathways.

Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation

“…PAF is also known to play an important role in various pathophysiological conditions that include modulation of blood pressure, hypotension, cardiac dysfunction, cardiac anaphylaxis, hemorrhagic, traumatic and septic shock syndromes (Anderson et al, 1991;Montrucchio et al, 2000). Because of its ability to stimulate endothelial migration and angiogenesis, a potential role of PAF is also known as a potent stimulator of thromboxane A2 (TXA2) production in human platelets (Shah et al, 2001). It is reported that PAF acts through the stimulation of pertussis toxin insensitive G-proteins (Gq/11) resulting in the stimulation of phospholipase C (PLC) and thus generation of second messenger diacylglycerol (DAG) and inositol-1, 4,5-triphosphate (IP3), which results in the activation of protein kinase C (PKC) and the mobilization of intracellular Ca 2+ , respectively (ObberghenSchilling and Pouyssegur, 1993).…”

Effect of Orthovanadate on Platelet Aggregation Induced by Platelet-Activating Factor

“…PAF, 5-hydroxytryptamine, or thrombin leads to platelet aggregation through activation of phospholipid-specific phospholipase C (PLC) and mitogen-activated protein kinase (MAPK) pathways. 24) The activated PLC stimulates a cleavage of inositol phospholipids to generate inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol, which results in the mobilization of intracellular Ca 2ϩ and the activation of protein kinase C, respectively. PKA activated by an increase in intracellular cAMP content inhibits activation of b 2 isoform of PLC through phosphorylation of serine residues of the enzyme molecule.…”

“…The PAF-induced platelet aggregation is inhibited by wortmannin, a potent inhibitor of phophatidylinositol 3-kinase (PI3K), 45) or PD98059, a specific inhibitor of MAPK kinase, 46) suggesting involvement of the MAPK activation via PI3K in the action of PAF. 24,47) MAPKs, a group of kinases responsive to a variety of environmental stimuli, are divided into three subfamilies of the p42/p44 MAPKs, c-Jun NH2-terminal kinase/stress-activated protein kinases, and p38 MAPKs. 48) These enzymes are activated by their upstream kinases and downregulated by phosphatases.…”

“…Although two forms of MAPKs, p42 and p44 proteins, have been identified in platelets, the increased phosphorylation and activation of p42 MAPK alone were observed with the aggregation induced by thrombin, involving a stimulated phosphorylation pathway similar to that by PAF. 24,49) Thus, the activation of p42 MAPK but not p44 MAPK appears to be involved in the induction of platelet aggregation. This may be the reason why the vanadate-induced MAPK activation is independent of the platelet aggregation.…”

“…24) To monitor the platelet aggregation, a decrease in absorbance at 650 nm was measured using a microtiter-plate reader at 30-s intervals after the addition of PAF. 25) Measurement of Clotting Time Assays were carried out essentially according to the method of Funakoshi et al 19,26) Briefly, the washed platelets (3ϫ10 8 cells/ml) were preincubated with or without test samples at 37°C for 15 min, washed to remove test samples, diluted with the platelet-poor plasma, then incubated with kaolin (50 mg/ml) for 3 min.…”