Irreversible Platelet Activation Requires Protease-Activated Receptor 1-Mediated Signaling to Phosphatidylinositol Phosphates

Abstract

Thrombin induces platelet activation through an early, reversible stage of platelet aggregation, which is followed by a later, irreversible stage of platelet aggregation. Without intervention, events leading to pathological platelet activation can result in vessel occlusion, acute coronary syndrome, and stroke. Therefore, a better understanding of events leading to platelet-mediated clot formation may provide insight into new therapeutic targets. Once activated, protease activated receptors (PARs) are essential in regulating events leading to platelet aggregation. We have determined a signaling cascade through PAR1, which involves phosphatidylinositol (PI) kinases, phosphatidylinositol bisphosphate (PIP 2 ), and Rap1 activation (independent of P2Y12) in the formation of a stable platelet aggregate. The putative phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 was found to reduce basal and PAR-stimulated PIP 2 levels by mass spectrometry and to inhibit PAR1-mediated stable platelet aggregation. Rap1 activation in platelets (during time points corresponding to the late, irreversible phase of aggregation) was found to require the PI signaling pathway. Perturbation of PI3K signaling by isoform-selective inhibitors had differential effects on Rap1 activation through PAR1 and PAR4. Hence, it is possible to disrupt lipid signaling pathways involved in stable clot formation without inhibiting early clot formation, offering a new potential target for antiplatelet therapy.Research spanning nearly three decades has firmly established the role of platelet activation in the pathophysiology of cardiovascular disease and acute coronary syndromes (Elwood et al., 1991;Ross, 1999;Gurbel and Bliden, 2003), because platelet activation plays a critical role in the formation of intravascular thrombus at the site of arterial injury or plaque rupture (Fuster et al., 1992). Under pathological conditions, a local increase in thrombin concentrations may result in a perpetual feed-forward activation of platelets, resulting in subsequent occlusion of coronary vessels and stroke. In platelets, thrombin signals through the activation of protease-activated receptors (PAR1 and PAR4). Inhibition of specific points in PAR signaling pathways leads to the attenuation of normal aggregation kinetics , suggesting that regulation of PAR1-and PAR4-mediated aggregation does not occur through identical signaling cascades.Phosphatidylinositol phosphates PIP, PIP 2 , and PIP 3 (PIP n ) are produced upon the activation of PI-kinases. In human platelets, PI(4,5)P 2 (PIP 2 ) is the major substrate for PI(3,4,5)P 3 , (PIP 3 ) and both of these have been implicated in thrombin-mediated signaling processes in platelets (Hartwig et al., 1995;Yang et al., 2004). A number of studies have investigated the role of PI3-kinase (PI3K) ␤ in outside-in signaling, and studies suggest a potential role for other PI3K isoforms in inside-out signaling (Jackson et al

Impaired Feedback Regulation of the Receptor Activity and the Myofilament Ca2+ Sensitivity Contributes to Increased Vascular Reactiveness after Subarachnoid Hemorrhage

“…Furthermore, the observations with trypsin and YM254890 support the persistent signaling activity of PAR 1 during the thrombin-induced contraction. The removal of the tethered ligand region of PAR 1 by proteinases converts PAR 1 to the inactive conformation, thereby terminating the irreversibly persisted signals (Holinstat et al, 2009). Trypsin is one such proteinase and it removes the tethered ligand region of PAR 1 (Nakayama et al, 2004;Nakayama et al, 2003).…”

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

“…Platelet activation is regulated in large part by the small GTPase Rap1 ( 39 ). To identify whether 12-LOX metabolites from the various fatty acids regulate the level of Rap1 activation, washed platelets were treated with or without 40 M of each metabolite [12(S)-HETE, 12(S)-HETrE, 12(S)-HEPE, or 13(S)-HOTrE] or 10 M fatty acid (AA, DGLA, EPA, LA, or EDA) followed by stimulation with PAR1-AP for 5 min ( Fig.…”

“…Rap1 activity was measured using GST-RalGDS-Rap1-binding domain that specifi cally interacts with activated Rap1 as described elsewhere ( 39,40 ). Activated Rap1 was detected by immunoblotting with the anti-Rap1 antibody.…”

Characterization of Phospholipid Molecular Species by Means of HPLC-Tandem Mass Spectrometry

“…These values were found to be higher in brain, heart, lymphocytes, spleen, macrophages and polymorphonuclear leukocytes. Phosphatidylinositol (PI) is the precursor of phosphatidylinositol phosphates upon the activation of PI-kinases, which are implicated in thrombin-mediated signaling processes in platelets (Holinstat et al, 2009). Phosphatidylinositol transfer proteins (PITPs) bind and facilitate the transport of phosphatidylinositol (PI) and phosphatidylcholine between membrane compartments; they are able to sequester PI in their hydrophobic pocket.…”