Bryan Voss

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.

PAR1, but Not PAR4, Activates Human Platelets through a Gi/o/Phosphoinositide-3 Kinase Signaling Axis

Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to Gi/o in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin αIIbβ3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of Gi/o by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the Gi/o-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated Gi/o signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.

Protease-activated receptors differentially regulate human platelet activation through a phosphatidic acid-dependent pathway.

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 thrombins 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 PAR-induced 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.

Pharmacokinetics, safety, and tolerability of a rapid infusion of i.v. ibuprofen in healthy adults

PURPOSE The pharmacokinetics, safety, and tolerability of a rapid infusion of i.v. ibuprofen in healthy adults were evaluated. Methods In this randomized, double-blind, placebo-controlled, single-dose, crossover study, 12 healthy subjects age 18-65 years were randomized to receive a single dose of either 800 mg i.v. ibuprofen (infused over five to seven minutes) concomitantly with an oral placebo or 800 mg oral ibuprofen with concomitant i.v. placebo (0.9% sodium chloride injection). After a six-day washout period, subjects received the treatment not previously received. Blood samples were taken 1 hour before each dose of study medication was administered and throughout the 12 hours thereafter. Plasma ibuprofen concentrations were determined using validated liquid chromatography-tandem mass spectrometry methods. The frequency and severity of treatment-emergent adverse effects were monitored throughout the study. RESULTS The maximum plasma concentration (C(max)) of i.v. ibuprofen was approximately twice that of oral ibuprofen, and the (t(max)) of i.v. ibuprofen was 0.11 hour, compared with 1.5 hours for oral ibuprofen. However, the elimination half-life of i.v. and oral ibuprofen did not differ, both of which were approximately 2 hours. Oral ibuprofen was 100% bioavailable; therefore, the area under the concentration-time curve did not differ between i.v. and oral ibuprofen. In addition, i.v. ibuprofen infused over five to seven minutes did not differ in terms of safety or tolerability when compared with oral ibuprofen. CONCLUSION I.V. ibuprofen, when administered over five to seven minutes in healthy subjects, achieved a higher C(max) and a more-rapid t(max) than did oral ibuprofen and was found to be safe and well tolerated.

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites.

A prospective, multicenter, randomized, double-blind trial of IV ibuprofen for treatment of fever and pain in burn patients.

This prospective study evaluated the efficacy and safety of IV ibuprofen for the reduction of fever and treatment of pain in patients with thermal burn injury. A total of 61 patients with second- and/or third-degree thermal burns covering >10% TBSA were randomly assigned in a 2:1 ratio to receive either 800 mg IV ibuprofen or placebo every 6 hours for 120 hours (5 days). Antipyretic medications were restricted during the first 24 hours of the study, but analgesics were allowed throughout. The primary efficacy endpoint was area under the curve for temperature (AUC-T°) within the first 24 hours of treatment. After 24 hours of dosing, there was a significant reduction in temperature in patients who received IV ibuprofen compared with those who received placebo (P = .008). The temperature remained reduced over the entire 120-hour dosing period in the patients who received IV ibuprofen, although the difference beyond 24 hours did not reach statistical significance. Because of enrollment of patients unable to perform self-assessments of pain, an inadequate number of patients were enrolled to detect differences in pain scores. There was no significant difference in the incidence of serious adverse events. Fever was reduced significantly by IV ibuprofen in burn patients over the initial 24-hour dosing period and remained reduced throughout the dosing period. Exposure to the maximum daily recommended dose of 3200 mg (800 mg every 6 hours) for a total of 120 hours (5 days) was well tolerated.

Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling.