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The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist.

Keywords: Calcium; GPIIbIIIa; platelet signaling EN Calcium GPIIbIIIa platelet signaling 437 439 3 08/03/21 20210601 NES 210601 Glycoprotein (GP) IIbIIIa is the most abundant integrin on the platelet surface and plays a crucial role in hemostasis. Platelet inside-out signaling is triggered by platelet agonists including adenosine diphosphate (ADP), thrombin, or extracellular matrix proteins ranging from major to minor constituents including collagen, laminin, and nidogen, respectively [[3]]. [Extracted from the article]

Collagen, a strong platelet activator, is recognized by integrin α2β1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2β1 integrin binding site is a tool to specifically study how α2β1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2β1 integrin binding sites trigger signaling through dot-like clusters of α2β1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2β1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt. [ABSTRACT FROM AUTHOR]

Neuromedin U (NmU) is a pleiotropic hypothalamic neuropeptide involved in the gut–brain axis. It acts via both a Gαq/11-coupled receptor (NMUR1) and a Gαi-coupled receptor (NMUR2) in different cell types. Expression of both receptors was reported in platelets, but their significance for NmU signaling remains elusive. We studied the potential effects of NmU on human platelet activation. In platelet-rich plasma (PRP), NmU alone (up to 10 μM) did not induce any measurable aggregation, but at nanomolar concentrations, it potentiated platelet aggregation by low (mean 0.47 μM) ADP concentrations (from 25.9 ± 3.6% to 74.8 ± 2.7% maximal aggregation for ADP vs. ADP + NmU, 100 nM, mean ± SEM, n = 13), accompanied by platelet P-selectin expression and intracellular calcium mobilization. Accordingly, platelet preincubation with NmU for 2 min sensitized platelets for subsequent activation by ADP. When P2Y 1 was inactivated by 50 μM MRS2179, NmU comparably potentiated ADP-induced PRP aggregation, suggestive of cooperative activation with Gαi-coupled P2Y 12 . Likewise, NmU potentiated platelet aggregation by Gαi-operated epinephrine at subthreshold concentrations (99 ng/ml, mean), but not that by Gαq-dependent serotonin (20 μM). Platelet aggregation by NmU/epinephrine combination was fully inhibited by the Gαq inhibitor YM-254890 (1 μM). qPCR detection and western blot analysis substantiated platelet expression of NMUR1 in different donors, a finding collectively complying with functionally relevant Gαq/11-mediated activation of platelet NMUR1 by NmU. Our findings advocate further studies on platelet sensitization by NmU, released during vascular activation and injury, to define its role as a modifier of platelet responsiveness to the physiological activation signals, operational in cardiovascular health and disease. [ABSTRACT FROM AUTHOR]

Background: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count., Objectives: To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen)., Methods: By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n  = 23) or type-1 von Willebrand disease ( n  = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI-related disease with granule secretion defect)., Results: The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap., Conclusion: In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice., (© 2023 The Authors.)

Thrombin-dependent platelet activation has been shown to be important in the setting of angioplasty and stenting, which may cause ischemic complications including acute myocardial infarction and death. Inhibitors of the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), are now being evaluated in clinical trials for safety and efficacy in patients with atherothrombotic disease. However, it is unknown whether chronic inhibition of PAR1 in these large patient populations will have beneficial or possibly adverse effects on other biologic processes involved in blood vessel homeostasis and the response to vascular injury. Most recently, PAR1 was found to be cleaved at a distinct site by matrix metalloprotease-1 (MMP-1) to create a longer tethered ligand, which activates a distinct spectrum of G protein pathways in platelets. The differential activation by serine proteases such as thrombin and the metalloprotease MMP-1, places the protease receptor PAR1 at the junction of two major protease classes critically involved in thrombosis, matrix remodeling, and the response to vascular injury. © 2011 IUBMB IUBMB Life, 63(6): 412-418, 2011 [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α2A-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by Gaq but not Gα12. The compound inhibited thrombus formation in vivo following vascular injury with an IC50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation. [ABSTRACT FROM AUTHOR]

Abstract: Collagen, one of the major proteins of sub-endothelial vasculature get exposed following endothelium denudement, is a potent stimulator of platelet adhesion and aggregation. Adhesion of platelets following endothelial injury is the primary event usually associated with uncontrolled platelet activation culminating into intravascular thrombosis, thus needs to be intervened to prevent the pathology related to various peripheral, myocardial and cerebral ischemic episodes. Recent advances in the understanding of collagen mediated platelet adhesion and aggregation have led to the identification of two prominent receptors, glycoprotein Ia/IIa (GPIa/IIa or integrin α2β1) and glycoprotein VI (GPVI) and associated intracellular signaling, which are undoubtedly the new emerging targets for the development of more effective antithrombotic drugs. The optimism for collagen antagonism is based on results obtained so far by the use of monoclonal and polyclonal antibodies, peptide inhibitors, knockouts models and collagen-mimetics in various in vitro test systems and animal models. These findings have revealed that collagen receptor inhibition is an attractive and secure strategy for the new drug development to prevent intravascular thrombosis. [Copyright &y& Elsevier]

Platelets are small enucleated cell fragments specialized in the control of hemostasis, but also playing a role in angiogenesis, inflammation and immunity. This plasticity demands a broad range of physiological processes. Platelet functions are mediated through a variety of receptors, the concerted action of which must be tightly regulated, in order to allow specific and timely responses to different stimuli. Protein phosphorylation is one of the main key regulatory mechanisms by which extracellular signals are conveyed. Despite the importance of platelets in health and disease, the molecular pathways underlying the activation of these cells are still under investigation. Here, we review current literature on signaling platelet biology and in particular emphasize the newly emerging role of phosphatases in these processes. • Signal transduction in platelet function in health and disease still needs elucidation. • Stimulation of platelets results in large scale phosphorylation changes mediated by counteracting kinases and phosphatases. • Phosphatases can have both activating and stimulatory roles in platelet biology. [ABSTRACT FROM AUTHOR]

A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE 1 -induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

Cancer-associated mortality is frequently caused by metastasis, however, our understanding of this process remains incomplete and therapeutic options are limited. Metastasis is a dynamic multi-step process involving intravasation of tumor cells into the host's blood and lymphatic vessels, their dissemination within the circulation, and finally arrest and extravasation in a distant organ where they establish secondary tumors. It is generally conceived that platelets contribute to all steps of hematogenous tumor dissemination. In this review, we provide an overview of the current knowledge of the platelet receptors involved in tumor cell-induced platelet aggregation, an essential immune surveillance escape mechanism of circulating tumor cells. We discuss how platelets prevent immunological attack, contribute to tumor cell extravasation and thereby facilitate colonization of distant organs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)