Protease-Activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

Duvernay, Matthew T.; Young, Summer E.; Gailani, David; Schoenecker, Jonathan G.; Hamm, Heidi E.

doi:10.1124/mol.112.083477

Cited by 61 publications

(68 citation statements)

References 59 publications

(57 reference statements)

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“…However, results presented in the Bristol-Myers Squibb patent (Lawrence et al, 2013) around the efficacy of VU0652925 in reducing thrombus volume in a cynomolgus electrolytic carotid artery injury model of thrombosis with a PAR4 antagonist, and their movement into phase II clinical trials, speak to the relevance of these higher concentrations of thrombin and the efficacy of inhibiting their action on platelets. Precedent for the strong and superseding PAR4 response comes from work conducted by our group and others (Vretenbrant et al, 2007;Fälker et al, 2011;Duvernay et al, 2013). In our primary assay, we observed noncompetitive modes of inhibition for both SCH602539 and VU0652925.…”

Section: Discussionsupporting

confidence: 67%

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

et al. 2016

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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 Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4.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.

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Section: Discussionsupporting

confidence: 67%

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

et al. 2016

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“…Studies by Bevers et al in 1982 [5] clearly identified that collagen is more potent, but that the combination of collagen and thrombin greatly potentiated the capability of platelets to support thrombin formation [5]. The small percentage of phosphatidylserine (PS)-positive platelets reported in this paper are well in accordance with other studies [14,27,28], where only a small percentage of platelets turn PS positive even in response to high concentrations of thrombin, although it should be noticed that PS exposure determined by annexin V binding could vary considerably as a result of variations in experimental conditions [29]. The important contribution by collagen is also evident in this paper, as the combination of thrombin and collagenrelated peptide in Fig.…”

supporting

confidence: 90%

“…To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to study dose-responses to increasing concentrations of the specific receptor activating peptides. Such data are rare in previous publications, but studies by, for example, Duvernay et al and Vretenbrant et al [3,14] report a higher platelet activation with the most efficient PAR4-activating peptide, AYPGKF [15], as compared with the most potent PAR1-activating peptide, SFLLRN [16]. However, it should be kept in mind that the PAR4-activating peptides reach their maximum effects at concentrations 10-fold higher than the PAR1-activating peptides, which means that potential unspecific effects caused by the presence of the peptides themselves have to be considered as potential confounding factors.…”

mentioning

confidence: 93%

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Lindahl

Macwan

Ramström

2016

Journal of Thrombosis and Haemostasis

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To cite this article: Lindahl TL, Macwan AS, Ramstr€ om S. Protease-activated receptor 4 is more important than protease-activated receptor 1 for the thrombin-induced procoagulant effect on platelets. J Thromb Haemost 2016; 14:1639-41.See also French SL, Arthur JF, Lee H, Nesbitt WS, Andrews RK, Gardiner EE, Hamilton JR. Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood. This issue, pp 1642-54.Thrombin is the most important enzyme in coagulation and also a potent activator of platelets. It activates platelets via N-terminal cleavage of protease-activated receptors (PARs) 1 and 4. Although structurally similar, both PARs have distinct and complementary roles; for example, PAR1 is more sensitive than PAR4 to low concentrations of thrombin [1], whereas PAR4 gives rise to a more prolonged calcium mobilization after activation compared with a transient 'spike' by PAR1 [2]. Moreover, PAR4 is more important for clot lysis resistance [3]. After activation, a subpopulation of the platelets become procoagulant by allowing the formation of coagulation factor complexes on their surface. They do not participate in aggregation but facilitate generation of even more thrombin and, consequently, amplification of the response [4]. Experimental data suggest that collagen exposed during vessel damage in combination with thrombin is the most potent trigger for formation of procoagulant platelets [5], but the signaling processes regulating the formation of the aggregatory and procoagulant platelet subpopulations are still not known in detail.The article by Hamilton and co-workers in this issue of JTH [6] shows for the first time that activation of PAR4 is crucial for the thrombin-induced activation of platelets in an in vitro thrombus model; that is, platelets adhered on a collagen surface in a flow chamber system. Two tools were essential for this study, a rabbit polyclonal antibody directed towards the cleavage site for thrombin at the N-terminal of PAR4 and a fluorescence resonance energy transfer-based thrombin activity sensor linked to an anti-CD41a antibody, to prevent washing out by the flowing blood, which was invented by Welsh et al. [7]. The anti-PAR4 antibody only attenuated the peak of the thrombin-induced cytosolic calcium but abolished the sustained elevation of cytosolic calcium; thus PAR4 was responsible for the sustained elevation, in accordance with previous studies [8][9][10][11][12]. Presumably this sustained elevation is essential for the creation of the negatively charged surface and the procoagulant activity, which is supported by the known very strong effect in this direction by the calcium ionophore A23187 (see Fig. 4G in [6] and [13]).The relative importance of PAR1 and PAR4 for different aspects of platelet activation is far from clear; published studies have shown various, even contradicting, results. To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to st...

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“…Under conditions similar to those used in this study, Factor V has been shown to be slowly released over approximately 10 minutes 21. The observation that AET platelets secrete significantly less Factor V than normal platelets, despite having normal levels of Factor V, is important for 2 reasons.…”

Section: Discussionmentioning

confidence: 60%

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia

Norris

Pombo

Shirley³

et al. 2015

Veterinary Internal Medicne

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BackgroundTwo congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation.Hypothesis/ObjectivesPlatelet dysfunction in horses with this second thrombasthenia results from a secretory defect.AnimalsTwo affected and 6 clinically normal horses.MethodsEx vivo study. Washed platelets were examined for (1) expression of the αIIb‐β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α‐granules; (4) activation of the mammalian target of rapamycin (mTOR)‐protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol‐4‐phosphate‐3‐kinase, class 2B (PIK3C2B) and SH2 containing inositol‐5′‐phosphatase 1 (SHIP1).ResultsPlatelets from affected horses expressed normal amounts of αIIb‐β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α‐granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide‐dependent kinase 1 (PDK1) signaling were normal. SH2‐containing inositol‐5'‐phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation.Conclusions and clinical significanceDefects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia.

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Protease-Activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

Cited by 61 publications

References 59 publications

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

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

Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia

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