Determination of PAR4 numbers on the surface of human platelets: no effect of the single nucleotide polymorphism rs773902.

The thrombin receptor, protease-activated receptor 4 (PAR4), is important for platelet activation and is the target of emerging anti-thrombotic drugs. A frequently occurring single nucleotide polymorphism (SNP; rs773902) causes a function-altering PAR4 sequence variant (NC_000019.10:p.Ala120Thr), whereby platelets from Thr120-expressing individuals are hyper-responsive to PAR4 agonists and hypo-responsive to some PAR4 antagonists than platelets from Ala120-expressing individuals. This altered pharmacology may impact PAR4 inhibitor development, yet the underlying mechanism(s) remain unknown. We tested whether PAR4 surface expression contributes to the altered receptor function. Quantitative flow cytometry was used to determine the absolute number of PAR4 on platelets from individuals subsequently genotyped at rs773902. We detected 539 ± 311 PAR4 per platelet (mean ± SD, n = 84). This number was not different across rs773902 genotypes. This first determination of cellular PAR4 numbers indicates variations in platelet surface expression do not explain the altered pharmacology of the rs773902 PAR4 sequence variant.

High-content, label-free analysis of proplatelet production from megakaryocytes.

BACKGROUND: The mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets, which are cellular extensions originating from the MK cell body. OBJECTIVES: Proplatelet formation is an asynchronous and dynamic process that poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. METHODS: Phase-contrast images of live, primary MKs are captured over a 24-hour period. Pixel-based machine-learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphology. A subsequent reinforcement classification, by CellProfiler Analyst, improves the detection of cellular structures. RESULTS: This workflow yields the percent of proplatelet production, area, count of proplatelets and MKs, and other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. CONCLUSION: This advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.

Platelet-derived extracellular vesicles infiltrate and modify the bone marrow during inflammation.

During inflammation, steady-state hematopoiesis switches to emergency hematopoiesis to repopulate myeloid cells, with a bias toward the megakaryocytic lineage. Soluble inflammatory cues are thought to be largely responsible for these alterations. However, how these plasma factors rapidly alter the bone marrow (BM) is not understood. Inflammation also drives platelet activation, causing the release of platelet-derived extracellular vesicles (PEVs), which package diverse cargo and reprogram target cells. We hypothesized that PEVs infiltrate the BM, providing a direct mode of communication between the plasma and BM environments. We transfused fluorescent, wild-type (MPL+) platelets into recipient cMpl-/-mice before triggering systemic inflammation. Twenty hours postinfusion, we observed significant infiltration of donor platelet-derived particles in the BM, which we tracked immunophenotypically (MPL+ immunohistochemistry staining) and quantified by flow cytometry. To determine if this phenomenon relates to humans, we extensively characterized both megakaryocyte-derived and PEVs generated in vitro and in vivo, and found enrichment of extracellular vesicles in bone marrow compared with autologous peripheral blood. Last, BM from cMpl-/- mice was cultured in the presence or absence of wild-type (MPL+) PEVs. After 72 hours, flow cytometry revealed increased megakaryocytes only in cultures with added PEVs. The majority of CD41+ cells were bound to PEVs, suggesting a PEV-mediated rescue of megakaryopoiesis. In conclusion, we report for the first time that plasma-residing PEVs infiltrate the BM. Further, PEVs interact with BM cells in vivo and in vitro, causing functional reprogramming that may represent a novel model of inflammation-induced hematopoiesis.

New Insights Into the Differentiation of Megakaryocytes From Hematopoietic Progenitors.

Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells, through a hierarchical series of progenitor cells, ultimately to a mature megakaryocyte. Once mature, the megakaryocyte then undergoes a terminal maturation process involving multiple rounds of endomitosis and cytoplasmic restructuring to allow platelet formation. However, recent studies have begun to redefine this hierarchy and shed new light on alternative routes by which hematopoietic stem cells are differentiated into megakaryocytes. In particular, the origin of megakaryocytes, including the existence and hierarchy of megakaryocyte progenitors, has been redefined, as new studies are suggesting that hematopoietic stem cells originate as megakaryocyte-primed and can bypass traditional lineage checkpoints. Overall, it is becoming evident that megakaryopoiesis does not only occur as a stepwise process, but is dynamic and adaptive to biological needs. In this review, we will reexamine the canonical dogmas of megakaryopoiesis and provide an updated framework for interpreting the roles of traditional pathways in the context of new megakaryocyte biology. Visual Overview- An online visual overview is available for this article.

A function-blocking PAR4 antibody is markedly antithrombotic in the face of a hyperreactive PAR4 variant.

Thrombin activates human platelets via 2 protease-activated receptors (PARs), PAR1 and PAR4, both of which are antithrombotic drug targets: a PAR1 inhibitor is approved for clinical use, and a PAR4 inhibitor is in trial. However, a common sequence variant in human PAR4 (rs773902, encoding Thr120 in place of Ala120) renders the receptor more sensitive to agonists and less sensitive to antagonists. Here, we develop the first human monoclonal function-blocking antibody to human PAR4 and show it provides equivalent efficacy against the Ala120 and Thr120 PAR4 variants. This candidate was generated from a panel of anti-PAR4 antibodies, was found to bind PAR4 with affinity (KD ≈ 0.4 nM) and selectivity (no detectable binding to any of PAR1, PAR2, or PAR3), and is capable of near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant. Platelets from individuals expressing the Thr120 PAR4 variant exhibit increased thrombin-induced aggregation and phosphatidylserine exposure vs those with the Ala120 PAR4 variant, yet the PAR4 antibody inhibited these responses equivalently (50% inhibitory concentration, 4.3 vs 3.2 µg/mL against Ala120 and Thr120, respectively). Further, the antibody significantly impairs platelet procoagulant activity in an ex vivo thrombosis assay, with equivalent inhibition of fibrin formation and overall thrombus size in blood from individuals expressing the Ala120 or Thr120 PAR4 variant. These findings reveal antibody-mediated inhibition of PAR4 cleavage and activation provides robust antithrombotic activity independent of the rs773902 PAR4 sequence variant and provides rationale for such an approach for antithrombotic therapy targeting this receptor.

Perinatal lethality of Par4-/- mice delivered by primiparous dams reveals spontaneous bleeding in mice without platelet thrombin receptor function.

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4-/- mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4-/- mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2-/-), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4-/- pups. We observed significant postpartum loss of Par4-/- pups derived from Par4+/- intercrosses that was restricted to a dam's first litter; only 9% of surviving pups genotyped as Par4-/- in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2-/-; 10%) but not in those lacking fibrinogen (Fga-/-; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4-/- mice, suggest that a dam's first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets-and more specifically thrombin-induced platelet activation-in hemostasis during these more traumatic births.

Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets.

Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets-PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs.

Protease-activated receptor 4: from structure to function and back again.

Protease-activated receptors are a family of four GPCRs (PAR1-PAR4) with a number of unique attributes. Nearly two and a half decades after the discovery of the first PAR, an antagonist targeting this receptor has been approved for human use. The first-in-class PAR1 antagonist, vorapaxar, was approved for use in the USA in 2014 for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. These recent developments indicate the clinical potential of manipulating PAR function. While much work has been aimed at uncovering the function of PAR1 and, to a lesser extent, PAR2, comparatively little is known regarding the pharmacology and physiology of PAR3 and PAR4. Recent studies have begun to develop the pharmacological and genetic tools required to study PAR4 function in detail, and there is now emerging evidence for the function of PAR4 in disease settings. In this review, we detail the discovery, structure, pharmacology, physiological significance and therapeutic potential of PAR4. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Approval of the first protease-activated receptor antagonist: Rationale, development, significance, and considerations of a novel anti-platelet agent.

Twenty-three years after the discovery of the first thrombin receptor, now known as protease-activated receptor 1 (PAR1), the first drug targeting this receptor is available for human use. The PAR1 inhibitor, vorapaxar (Zontivity, MSD), was recently approved by the FDA for use in the USA for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral artery disease. In this review, we detail the rationale, development, as well as the clinical significance and considerations of vorapaxar, the original PAR antagonist and the latest anti-platelet agent in the pharmaco-armoury against arterial thrombosis.

Differential signaling by protease-activated receptors: implications for therapeutic targeting.

Protease-activated receptors (PARs) are a family of four G protein-coupled receptors that exhibit increasingly appreciated differences in signaling and regulation both within and between the receptor class. By nature of their proteolytic self-activation mechanism, PARs have unique processes of receptor activation, "ligand" binding, and desensitization/resensitization. These distinctive aspects have presented both challenges and opportunities in the targeting of PARs for therapeutic benefit-the most notable example of which is inhibition of PAR1 on platelets for the prevention of arterial thrombosis. However, more recent studies have uncovered further distinguishing features of PAR-mediated signaling, revealing mechanisms by which identical proteases elicit distinct effects in the same cell, as well as how distinct proteases produce different cellular consequences via the same receptor. Here we review this differential signaling by PARs, highlight how important distinctions between PAR1 and PAR4 are impacting on the progress of a new class of anti-thrombotic drugs, and discuss how these more recent insights into PAR signaling may present further opportunities for manipulating PAR activation and signaling in the development of novel therapies.