Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets

Abstract

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, a-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe proteaseactivated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombinmediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.

Rac Regulates the TRAP-Induced Release of Phosphorylated-HSP27 from Human Platelets via p38 MAP Kinase but Not JNK

“…Therefore, it is most likely that Rac positively regulates the PAR-induced phosphorylation of HSP27 in human platelets, resulting in the release of phosphorylated-HSP27. Regarding PARs, it has been shown that human platelets express PAR1 and PAR4 [8]. In the present study, we clearly showed that NSC23766 suppressed Table 2.…”

“…This new amino terminus then serves as a tethered ligand and activates PARs, leading to platelet activation [7]. PARs belong to the GTP-binding proteincoupled receptor superfamily, and PAR1 and PAR4 among PARs are expressed on human platelets [8]. Thrombin receptor-activating peptide (TRAP), consisting of an identical amino acid sequence to the tethered ligand of PARs cleaved by thrombin, is a useful tool to evaluate the function of PARs [9].…”

Identification of the minimum PAR4 inhibitor pharmacophore and optimization of a series of 2-methoxy-6-arylimidazo[2,1- b ][1,3,4]thiadiazoles

“…This is in sharp contrast to 1 , which is a non-competitive inhibitor of PAR4 9 , and 2 which has a mixed competitive/non-competitive profile. 4 These results are exciting as until now small molecule tools with diverse modes of pharmacolgy and PAR4 antagonism to potentially assess in vivo effects of PAR4 inhibition have not existed.…”

“…This exercise led to the discovery that the most basic core of 2 , a 6-(benzofuran-2-yl)-2-methoxyimidazo[2,1- b ][1,3,4]thiadiazole 3 as a potent PAR4 inhibitor (PAR4 AP IC 50 = 1.69 nM, PAR4 γ-thrombin IC 50 = 58.8 nM), as the minimum pharmacophore (MW = 271) of 2 . 9 However, the potential liabitlities of an unsubstitiuted benzofuran, as in 3 , raised metabolic stabilty concerns. Thus, in this Letter, we describe efforts to survey alternative 6-position substituents on the 2-methoxyimidazo[2,1- b ][1,3,4]thiadiazole core to identify a minimum PAR4 pharmacophore that could then be further optimized with functional groups that would engender desirable DMPK properties.…”

Platelet-derived nanomedicine targets cancer

“…The platelet integrin a IIb was found broadly distributed at the plasma membrane of tumor cells and inside recycling endosomes, pointing to membrane fusion. No microparticles were detected in tumors of thrombocytopenic mice and mice with a deletion of the protease-activated receptor 4, a known driver of microparticle generation by platelets, 7 further confirming that microparticles of platelet origin reach tumors in the extravascular space and are associated with tumor cells. Platelet microparticles can transfer their cargo to endothelial cells, macrophages, and neutrophils.…”