Protease-Activated Receptors Differentially Regulate Human Platelet Activation through a Phosphatidic Acid-Dependent Pathway

Abstract

Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through protease-activated receptor (PAR) 1 and PAR4, plays a central role in regulation of human platelet function in that it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intracellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study, we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion after PARinduced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Furthermore, these inhibitors significantly attenuated activation of Rap1 after stimulation by PAR1-AP but not thrombin or PAR4-AP. Because PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.Platelets are regulated through numerous agonist-induced signaling pathways, the most potent of which is thrombin (Davey and Luscher, 1967;Jamieson, 1997). Human platelets express two functional thrombin receptors, protease activated receptor-1 and -4 (PAR1 and PAR4) (Kahn et al., 1998;Coughlin, 2005), whereas mouse platelets express PAR3 and PAR4. Previous work from our lab has indicated that PAR1 and PAR4 mediate platelet activation through distinct signaling pathways (Holinstat et al., 2006). How differential signaling through the thrombin receptors is regulated and the potential physiological consequences of selective activation of PAR1 and PAR4 remain unclear. Several intermediates within this signaling scheme, such as PKC␣ and the small GTPase Rap1 have been identified to be important to regulating platelet function (Franke et al., 2000;Chrzanowska-Wodnicka et al., 2005). Genetic studies in the mouse have confirmed the role of Rap1b in platelet aggregation and subsequent clot formation (Chrzanowska-Wodnicka et al., 2005). How Rap1 is involved in the initial and irreversible phases of platelet aggregation (Franke et al., 2000) and whether Rap1 is differentially regulated in human platelets by PAR1 and PAR4 remain elusive.In mouse pla...

Lipid phosphate phosphatase inhibitors locally amplify lysophosphatidic acid LPA1 receptor signalling in rat brain cryosections without affecting global LPA degradation

“…The LPPs are likely to be involved in LPA dephosphorylation in brain cryosections, as brain sections efficiently generate P i from exogenous LPA largely in a NEM resistant and Mg 2+ -independent way. Propranolol and vanadate have been demonstrated to inhibit LPPs in various cell types [20,35,36,48], vanadate also in the rat brain [49]. Propranolol has been shown to act as a moderately effective inhibitor of LPPs [20] supporting our finding where the vanadate-induced response is relatively stronger when compared to the response observed with propranolol.…”

“…The LPP-mediated degradation of LPA is susceptible to the phosphatase inhibitor sodium orthovanadate (Na 3 VO 4 ) [35] and to propranolol [20,36], better known as a classical β-adrenoceptor blocking agent e.g. used in the treatment of hypertension.…”

“…used in the treatment of hypertension. Another β-blocker, nadolol, has no demonstrable LPP inhibiting capacity [36] and therefore can serve as a useful control compound. We tested the effects of these compounds on basal LPA 1 receptor signalling.…”

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

“…In the nineties Coorssen and Haslam proposed that PLD is important for human platelet secretion by using ethanol to block PLD mediated effects on degranulation [45]. In a recent study it was shown that 1-butanol abrogated platelet aggregation by PAR-1 but not PAR-4 activating peptide [46]. In the present study we observed similar effects induced by primary alcohols but obtained completely opposite results when FIPI was used to determine platelet secretion and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Elvers et al PLD as negative regulator of platelets 19 aggregation confirming the unspecific and PLD-unrelated effects of the (formerly) used alcohols.…”

Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

“…A selective PAR1 AP (TFLLR-NH2) on the other hand induces platelet aggregation and VEGF release but suppresses endostatin release. In addition, it is known that distinct signaling pathways are activated in platelets by PAR1 and PAR4 selective peptides (Holinstat et al, 2006(Holinstat et al, , 2007(Holinstat et al, , 2009) and these differences may form the basis of differential platelet responses to PAR activation.…”