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Coordination of platelet agonist signaling during the hemostatic response in vivo.

ABSTRACT: The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation. We previously showed that thrombin activity at a site of vascular injury is spatially restricted, resulting in robust activation of a subpopulation of platelets in the hemostatic plug core. In contrast, adenosine 5'-diphosphate (ADP)/P2Y12 signaling contributes to the accumulation of partially activated, loosely packed platelets in a shell overlying the core. The contribution of the additional platelet agonists thromboxane A2 (TxA2) and epinephrine to this hierarchical organization was not previously shown. Using a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging, we show that TxA2 signaling is critical and nonredundant with ADP/P2Y12 for platelet accumulation in the shell region but not required for full platelet activation in the hemostatic plug core, where thrombin activity is highest. In contrast, epinephrine signaling is dispensable even in the presence of a P2Y12 antagonist. Finally, dual P2Y12 and thrombin inhibition does not substantially inhibit hemostatic plug core formation any more than thrombin inhibition alone, providing further evidence that thrombin is the primary driver of platelet activation in this region. Taken together, these studies show for the first time how thrombin, P2Y12, and TxA2 signaling are coordinated during development of a hierarchical organization of hemostatic plugs in vivo and provide novel insights into the impact of dual antiplatelet therapy on hemostasis and thrombosis.

SUBMITTER: Shen J

PROVIDER: S-EPMC5745130 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Coordination of platelet agonist signaling during the hemostatic response in vivo.

Shen Jian J Sampietro Sara S Wu Jie J Tang Juan J Gupta Shuchi S Matzko Chelsea N CN Tang Chaojun C Yu Ying Y Brass Lawrence F LF Zhu Li L Stalker Timothy J TJ

Blood advances 20171221 27

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation. We previously showed that thrombin activity at a site of vascular injury is spatially restricted, resulting in robust activation of a subpopulation of platelets in the hemostatic plug core. In contrast, adenosine 5'-diphosphate (ADP)/P2Y<sub>12</sub> signaling contributes to the accumulation of partially activated, loosely packed plat ...[more]

PMID: 29296928

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Project description:BackgroundBleeding anomalies have been reported in patients affected by Noonan syndrome. No study has been performed in patients with molecularly confirmed RASopathy. We aimed to characterize the frequency and types of bleeding disorders in patients with RASopathies and evaluate any significant association with laboratory findings.Patients and methodsForty-nine individuals (PTPN11, n = 27; SOS1, n = 7; RIT1, n = 3; SPRED1, n = 1; LZTR1, N = 3; RAF1, n = 2; BRAF, n = 4; MEK1, n = 1; MEK2, n = 1), and 49 age- and sex-matched controls were enrolled. The "Paediatric Bleeding Questionnaire Scoring Key" was administered to patients and families. Laboratory screening tests including clotting factors dosing, platelet count, Prothrombin Time and Partial Thromboplastin Time, were employed both in patients and controls to characterize the bleeding diathesis. A subgroup of 29/49 patients and 29/49 controls was also tested for platelet function.ResultsRegardless of the gene involved, pathological paediatric bleeding scores were recorded in 14/49 (28.5%) patients. Indeed, 7 were mutated in PTPN11, 3 in SOS1, 2 in RIT1, 1 in BRAF, and 1 in MEK1. Compared to patients with normal bleeding scores, those with pathologic bleeding score showed higher prevalence of splenomegaly (p = 0.006), prolonged aPTT (p = 0.04), lower levels of coagulation factor V (FV, p = 0.001), FVII (p = 0.003), FX (p = 0.0008) and FXIII (p = 0.002), higher vWAg (p = 0.04), and lower platelet sensitivity to Ristocetin (p = 0.001), arachidonic acid (AA) (p = 0.009) and collagen (p = 0.01). The presence of hematomas inversely correlated with factor V (p = 0.002), factor VII (p = 0.003), factor X (p = 0.002) and factor XIII (p = 0.004) levels, and directly correlated with platelet response to collagen (p = 0.02) and AA (p = 0.01). The presence of splenomegaly directly correlated with the presence of hematoma (p = 0.006), platelet response to Ristocetin (p = 0.04) and AA (p = 0.04), and inversely correlated with factor V levels (p = 0.03).ConclusionsPatients with RASopathies and a bleeding tendency exhibit multiple laboratory abnormalities, including platelet-related disorders. Splenomegaly is frequently detected and might be a suggestive sign for qualitative platelet dysfunction. A comprehensive clinical assessment should be carried out at diagnosis, during the follow-up and before any surgical procedures. Since there is currently no consensus on management of bleeding complications, it is important that physicians closely monitor these patients.

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Project description:In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

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Coordination of platelet agonist signaling during the hemostatic response in vivo.

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Coordination of platelet agonist signaling during the hemostatic response in vivo.

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