Platelet Dense Granule Secretion Research Articles

Investigation of the impact of thirdhand e-cigarette exposure on platelet function: A pre-clinical study.

The use of e-cigarettes (ECs) has reached unprecedented levels, due to a variety of reasons, including the misconception regarding their safety. Thus, there have been efforts to characterize the effects of EC exposure, including in the context of thirdhand EC (THEC) on a host of disorders, such as cardiovascular disease (CVD). To address this issue, we sought to characterize the effects of THEC on platelet function and thrombus formation, using a novel mouse exposure protocol that resembles real life scenarios. To assess these effects, a host of related in vivo (i.e. tail bleeding time, and ferric chloride injury induced thrombosis model) assays and in vitro platelet specific (e.g. aggregation, and dense granule secretion) investigative assays were conducted. Our in vivo characterization demonstrated that THEC exposed mice exhibited a prothrombotic phenotype reflected by their shortened tail bleeding (THEC: 37 ± 15 seconds, versus clean air: 183 ± 56 s) and occlusion times (THEC: 188 ± 39 s, versus clean air: 519 ± 70 s), relative to those exposed to clean air. Importantly, we found no difference in the platelet counts between the THEC and clean air mice. As for the underlying mechanism, separate experiments revealed significantly enhanced platelet aggregation, dense and alpha granule secretion, as well as integrin/GPIIb-IIIa activation and phosphatidylserine exposure in response to thrombin and ADP agonist stimulation. Taken together, these results provide evidence that THEC does have the capacity to increase the risk of thrombotic disease, which should increase awareness regarding its underappreciated negative health effects.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity exvivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Exvivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Characterization of molecular basis of anti‐platelet effect of daphnetin

Coumarin derivates have been known for their antithrombotic, anti‐inflammatory, and antioxidant activities and daphnetin is one of the natural coumarin derivatives isolated from Daphne Korean Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect is not studied till date.We investigated the functional role of daphnetin and its molecular basis on the regulation of platelet activation using murine platelets.In order to check the effect of daphnetin on GPVI‐mediated platelet response, we first measured the effect of daphnetin on collagen‐induced platelet aggregation and secretion. Collagen‐induced platelet aggregation and dense granule secretion were partially inhibited in the presence of daphnetin. Interestingly, 2‐MeSADP‐induced secondary wave of aggregation and secretion were completely inhibited in the presence of daphnetin. It has been known that 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation are mediated by positive‐feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2‐MeSADP‐induced platelet aggregation in aspirin‐treated platelets where the contribution of TxA2 generation is blocked. Additionally, AYPGKF‐induced platelet aggregation and secretion were inhibited in the presence of daphnetin by blocking the positive‐feedback effect of TxA2 generation. Moreover, 2‐MeSADP‐induced TxA2 generation was completely inhibited in the presence of daphnetin confirming the role of daphnetin on TxA2 generation. Finally, 2‐MeSADP‐induced ERK phosphorylation was inhibited in the presence of daphnetin.We conclude that daphnetin plays a role in platelet function by inhibiting the TxA2 generation through the regulation of ERK phosphorylation.

Abstract 159: Effect Of Antiplatelet Agents And Tyrosine Kinase Inhibitors On OxLDL-mediated Platelet Procoagulant Activity

Background & Objective: Oxidized low density lipoprotein (oxLDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, the mechanisms by which oxLDL affects platelet activation, and how to best therapeutically target and safely prevent such responses remains unclear. Here, we investigate how oxLDL upregulates glycoprotein VI (GPVI) mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet activity ex vivo . Methods: Human platelets were treated with oxLDL and GPVI specific agonist, crosslinked collage-related peptide (CRP-XL) and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2Y; ticagrelor, AR-C 66096, MRS2179), cyclooxygenase (COX; aspirin, indomethacin), Bruton’s tyrosine kinase (BTK; ibrutinib), and spleen tyrosine kinase (Syk; fostamatinib/R406). Results: Ex vivo , oxLDL enhanced GPVI-mediated platelet dense granule secretion, alpha granule secretion, integrin activation, thromboxane generation, and platelet aggregation, as well as phosphatidylserine exposure and fibrin generation. Our study further demonstrated that CD36 signaling from oxLDL activation overlaps with the GPVI signaling pathway, in such that the phosphorylation of proteins along the Syk-BTK-PI3K/Akt axis was increased. P2Y antagonists (e.g., ticagrelor), BTK inhibitors (e.g., ibrutinib), and Syk inhibitors (e.g. fostamatinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (e.g., aspirin) had no significant effect. Conclusion: Together, our results demonstrate that oxLDL enhances ex vivo platelet responses and procoagulant activity downstream of GPVI signaling in a manner that may be reduced by P2Y antagonists and tyrosine kinase inhibitors, but not significantly affected by COX inhibitors.

207 Omega-3 and omega-6 fatty acids attenuate platelet reactivity in postmenopausal women

OBJECTIVES/GOALS: This study aimed to investigate the mechanistic effects of fish oil (âμ-3 fatty acids) or evening primrose oil (âμ-6 fatty acids) supplementation on platelet reactivity in postmenopausal women. METHODS/STUDY POPULATION: Postmenopausal women were recruited from the Ann Arbor community and the University of Michigan Medicine Center. All subjects were recruited under study protocols approved by the University of Michigan IRB between November 2015 and March 2017. We conducted a randomized, double-blind, two-period crossover trial, consisting of a 60-day supplementation period followed by a 14-day washout period in between and at the end of the study. Subjects were treated daily in random order with 2g of fish oil supplement and 2g of evening primrose oil. Blood was drawn at baseline, post-supplementation, and after washout. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion and activation of basal integrin âºIIbÎ23 were assessed following supplementation and washout period. RESULTS/ANTICIPATED RESULTS: The study started with 90 postmenopausal women. A total of 78 subjects completed the study, with 12 subjects dropping out due to non-compliance and medical reasons. Supplementation with fish oil attenuated the thrombin receptor PAR4-induced platelet aggregation, whereas primrose oil supplementation attenuated aggregation mediated by PAR4 or collagen. Supplementation with âμ-3 or âμ-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin âºIIbÎ23 activation. Post-washout following supplementation with primrose oil, the thrombin receptor PAR1-induced platelet aggregation was similarly attenuated. For either treatment, the observed effects post supplementation on dense granule secretion and basal integrin activation were sustained after the washout. DISCUSSION/SIGNIFICANCE: Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. This study indicates that supplementation with âμ-3 and âμ-6 fatty acids may offer significant protection for postmenopausal women against cardiovascular diseases and occlusive thrombotic events by reducing platelet reactivity.

Analysis of microvascular thrombus mechanobiology with a novel particle-based model

Platelet accumulation at the site of a vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood.Here we describe a novel computational model of microvascular clot formation for the detailed analysis of thrombus mechanics. We adopt a previously developed two-dimensional particle-based model focused on the thrombus shell formation and revise it to introduce the platelet agonists. Blood flow is simulated via a computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process.The new model qualitatively reproduces the enhanced thrombus formation due to dense granule secretion, in line with in vivo findings, and provides a mechanism for the thrombin confinement at the early stages of clot formation. Our calculations also predict that the release of platelet dense granules results in the additional mechanical stabilization of the inner layers of thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of a thrombus, which are expected to result in the mechanical disruptions at the later stages of clot formation.

Critical role of peroxisome proliferator-activated receptor α in promoting platelet hyperreactivity and thrombosis under hyperlipidemia

Platelet hyperreactivity and increased atherothrombotic risk are specifically associated with dyslipidemia. Peroxisome proliferator-activated receptor alpha (PPARα) is an important regulator of lipid metabolism. It has been suggested to affect both thrombosis and hemostasis, yet the underlying mechanisms are not well understood. In this study, the role and mechanism of PPARα in platelet activation and thrombosis related to dyslipidemia were examined. Employing mice with deletion of PPARα (Pparα-/-), we demonstrated that PPARα is required for platelet activation and thrombus formation. The effect of PPARα is critically dependent on platelet dense granule secretion, and is contributed by p38MAPK/Akt, fatty acid b-oxidation, and NAD(P)H oxidase pathways. Importantly, PPARα and the associated pathways mediated a prothrombotic state induced by a high-fat diet and platelet hyperactivity provoked by oxidized low density lipoproteins. Platelet reactivity was positively correlated with the levels of expression of PPARα, as revealed by data from wild-type, chimeric (Pparα+/-), and Pparα-/- mice. This positive correlation was recapitulated in platelets from hyperlipidemic patients. In a lipid-treated megakaryocytic cell line, the lipid-induced reactive oxygen species-NF-kB pathway was revealed to upregulate platelet PPARα in hyperlipidemia. These data suggest that platelet PPARα critically mediates platelet activation and contributes to the prothrombotic status under hyperlipidemia.

A Critical Role for the IKKβ/Lipid Rafts Axis in Regulating Platelet Exocytosis, Via the Spatial Regulation of SNARE Proteins

Lipid rafts, which are membrane microdomains that are rich in cholesterol and glycosphingolipids, have been implicated in the regulation of intracellular protein trafficking. Furthermore, we have previously shown that SNAREs mediate granule-plasma membrane fusion during exocytosis in platelets, and that IKKβ regulates SNAREs and granule exocytosis. Based on these considerations, we sought to investigate the regulatory relationship between IKKβ and lipid rafts, by employing the PF4-Cre/IKKβflox/flox platelets. We demonstrate that lipid rafts play a critical role in mediating IKKβ-/--dependent regulation of SNARE proteins and SNARE complexes. Specifically, we found that methyl β-cyclodextrin (MβCD, which disrupts lipid rafts) is able to inhibit platelet aggregation, dense and alpha granule secretion, PS exposure and αIIbβ3 integrin activation. We also observed that MβCD is able to inhibit platelet spreading, clot retraction, as well as 7S complex formation. In terms of co-localization, the SNARE protein SNAP-23 was found to associate with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in control platelets, but not in the IKKβ-/- platelets. Furthermore, in resting platelets, plasma membrane SNARE proteins such as syntaxin-11 and SNAP-23 or the granule-associated SNARE vesicle-associated membrane protein VAMP-8 were neither present in the microdomains of control platelets, nor in those from IKKβ-/- mice. Interestingly however, agonist/thrombin stimulation led to the relocation of syntaxin-11 and VAMP-8 into the lipid rafts, and as part of raft-associated phospho-SNAP-23/syntaxin-11/VAMP-8 complexes in control platelets. This seems to be IKKβ dependent, as these associations did not take place in the IKKβ-/- platelets. Taken together, these data reveal a critical role for the IKKβ/lipid rafts axis in regulating platelet exocytosis, via the spatial regulation of SNARE proteins. DisclosuresNo relevant conflicts of interest to declare.

Comparative antiplatelet and antithrombotic effects of red ginseng and fermented red ginseng extracts

BackgroundFermentation may alter the bioavailability of certain compounds, which may affect their efficacy and pharmacological responses. This study investigated the antiplatelet effects of red ginseng extract (RGE) and fermented red ginseng extract (FRG). MethodsA rodent model was used to evaluate the antiplatelet and antithrombotic effects of the extracts. Rats were orally fed with human equivalent doses of the extracts for 1 week and examined for various signaling pathways using standard in vivo and ex vivo techniques. Light transmission aggregometry was performed, and calcium mobilization, dense granule secretion, integrin αIIbβ3-mediated signaling molecules, cyclic nucleotide signaling events, and various protein molecules were evaluated ex vivo in collagen-stimulated washed platelets. Furthermore, antithrombotic properties were evaluated using a standard acute pulmonary thromboembolism model, and the effects on hemostasis were investigated using rat and mice models. ResultsBoth RGE and FRG significantly inhibited platelet aggregation, calcium mobilization, and dense granule secretion along with integrin-mediated fibrinogen binding and fibrinogen adhesion. cAMP levels were found to be elevated in RGE-treated rat platelets. Ginseng extracts did not exert any effect on prothrombin time and activated partial thromboplastin time. RGE-treated mice showed significantly better survival under thrombosis than FRG-treated mice, with no effects on hemostasis, whereas FRG-treated mice exhibited a slight increment in bleeding time. ConclusionBoth extracts, especially RGE, are remarkable supplements to maintain cardiovascular health and are potential candidates for the treatment and prevention of platelet-related cardiovascular disorders.

Pyk2 downstream of G12/13 pathways regulates platelet shape change through RhoA/p160ROCK

Rho/Rho-kinase downstream of G12/13 plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G12/13 pathways. In this study, we evaluated the role of Pyk2 in G12/13–induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of Gαq inhibitor YM254890 to selectively stimulate G12/13 pathways. We found that G12/13–induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in Gq −/− platelets was completely inhibited in the presence of AG17 or RhoA/p160ROCK inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G12/13-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160ROCK activation in platelets. These results demonstrate that Pyk2 downstream of G12/13 pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160ROCK.

An Optimized Assay for Assessing Platelet Dense Granule Secretion Using Diluted Whole Blood

Abstract Assessment of dense granule secretion following agonist stimulation is an integral part of platelet function testing and can be performed in the clinical laboratory using lumi-aggregometry. A significant limitation of traditional lumi-aggregometry is the relatively large volume of blood required and the normal platelet counts needed to perform the assay, which often precludes usage of this test in pediatric and thrombocytopenic patients. By optimizing the luciferase-luciferin reaction with commercially available reagents and using a conventional lumi-aggregometer, we have developed a method that measures platelet dense granule secretion following agonist stimulation in diluted whole blood, at up to 10-fold dilutions. With the goal of developing a testing method independent of platelet count, the assay intentionally does not include specimen stirring. Direct comparison of the optimized reagents with the standard Chrono-lume reagent in 10-fold diluted whole blood showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. We reproducibly observed dose-dependent responses in platelet ATP release using this assay upon stimulation with platelet agonists such as the thrombin receptor-activating peptide (TRAP) and the collagen receptor agonist convulxin. ATP release in 10-fold diluted whole blood, for example, was 168.6 ± 24.7 pmoles/107 platelets in response to 40 μM TRAP and 17.1 ± 2.9 pmoles/107 platelets in response to 1 nM convulxin (mean ± SEM, N = 6). Method comparisons of this assay were performed using 10-fold diluted whole blood without stirring, compared to standard methodology using undiluted platelet-rich plasma stirred at 1000 rpm. On concurrently run, matched specimens, ATP release in response to a series of agonists of varying stimulus intensity showed overall moderate concordance, with an r2 = 0.722. Based on ATP standard curves and the observed agonist responses to date, we are hopeful that this assay may prove capable of assessing platelet dense granule release in patient blood with platelet counts as low as 20,000/μL. Additionally, the assay requires less than 60 μL of whole blood per agonist with testing performed at two different agonist concentrations. The extended analytical range and robustness of the assay, with no need for centrifugation, offer promise that it may be useful for the assessment of platelet dense granule secretion in pediatric or thrombocytopenic patients who require assays amenable to limited blood volumes and low platelet counts.

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume.

Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

Characterisation of the Ral GTPase inhibitor RBC8 in human and mouse platelets

The Ral GTPases, RalA and RalB, have been implicated in numerous cellular processes, but are most widely known for having regulatory roles in exocytosis. Recently, we demonstrated that deletion of both Ral genes in a platelet-specific mouse gene knockout caused a substantial defect in surface exposure of P-selectin, with only a relatively weak defect in platelet dense granule secretion that did not alter platelet functional responses such as aggregation or thrombus formation. We sought to investigate the function of Rals in human platelets using the recently described Ral inhibitor, RBC8. Initial studies in human platelets confirmed that RBC8 could effectively inhibit Ral GTPase activation, with an IC50 of 2.2 μM and 2.3 μM for RalA and RalB, respectively. Functional studies using RBC8 revealed significant, dose-dependent inhibition of platelet aggregation, secretion (α- and dense granule), integrin activation and thrombus formation, while α-granule release of platelet factor 4, Ca2+ signalling or phosphatidylserine exposure were unaltered. Subsequent studies in RalAB-null mouse platelets pretreated with RBC8 showed dose-dependent decreases in integrin activation and dense granule secretion, with significant inhibition of platelet aggregation and P-selectin exposure at 10 μM RBC8. This study strongly suggests therefore that although RBC8 is useful as a Ral inhibitor in platelets, it is likely also to have off-target effects in the same concentration range as for Ral inhibition. So, whilst clearly useful as a Ral inhibitor, interpretation of data needs to take this into account when assessing roles for Rals using RBC8.

Platelet Shp2 negatively regulates thrombus stability under high shear stress

Essentials Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor-mediated platelet dense granule secretion. Through αIIbβ3 outside-in signaling, Shp2 targets calmodulin-dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. SUMMARY: Background Perpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. Objective To investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and results We demonstrate that the platelet-expressed Src homology region2 domain-containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride-induced mesenteric arteriole thrombosis model, megakaryocyte/platelet-specific Shp2-deficient mice showed less thrombi shedding than wild-type mice, although their occlusion times were comparable. In accordance with this invivo phenotype, a microfluidic whole-blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2-deficient platelets were more stable under high shear rates than those produced by wild-type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2-deficient platelets showed increased dense granule secretion when stimulated by the thromboxaneA2 analog U46619. Shp2 appears to act downstream of integrin αIIb β3 outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown tobind both Shp2 and Akt, linking Shp2 to Akt activation. Conclusions Platelet Shp2 negatively regulates thrombus perpetuation under high shear stress.This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Individual variations in platelet reactivity towards ADP, epinephrine, collagen and nitric oxide, and the association to arterial function in young, healthy adults

IntroductionPlatelet aggregation and secretion can be induced by a large number of endogenous activators, such as collagen, adenosine diphosphate (ADP) and epinephrine. Conversely, the blood vessel endothelium constitutively release platelet inhibitors including nitric oxide (NO) and prostacyclin. NO and prostacyclin are also well-known vasodilators and contribute to alterations in local blood flow and systemic blood pressure. Materials and methodsIn this study we investigated individual variations in platelet reactivity and arterial functions including blood pressure and flow-mediated vasodilation (FMD) in 43 young, healthy individuals participating in the Lifestyle, Biomarkers and Atherosclerosis (LBA) study. Platelet aggregation and dense granule secretion were measured simultaneously by light transmission and luminescence. FMD was measured with ultrasound. ResultsThe platelet function assay showed inter-individual differences in platelet reactivity. Specifically, a sub-group of individuals had platelets with an increased response to low concentrations of ADP and epinephrine, but not collagen. When the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) was combined with high doses of these platelet activators, the results indicated for sub-groups of NO-sensitive and NO-insensitive platelets. The individuals with NO-sensitive platelets in response to SNAP in combination with collagen had a higher capacity of FMD of the arteria brachialis. ConclusionsPlatelet reactivity towards ADP, epinephrine and NO differs between young, healthy individuals. Some individuals have a more effective response towards NO, both in the aspect of platelet inhibition ex vivo, as well as vasodilation in vivo.

Loss of the mitochondrial kinase PINK1 does not alter platelet function

PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson’s disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice. The data largely show an absence of such a role. Haematological analysis of blood counts from KO mice was comparable to wild type. Quantification of mitochondrial mass by citrate synthase activity assay or expression of mitochondrial markers were comparable, suggesting normal mitophagy in KO platelets. Analysis of mitochondrial permeability transition pore opening, changes in mitochondrial membrane potential and calcium signalling to platelet activation were unaffected by loss of PINK1, whereas subtle enhancements of activation-induced reactive oxygen species were detected. Platelet aggregation, integrin activation, α- and dense granule secretion and phosphatidylserine exposure were unaltered in KO platelets while mouse tail bleeding responses were similar to wild type. Together these results demonstrate that PINK1 does not regulate basal platelet mitophagy and is dispensable for platelet function.

Pharmacological actions of miltirone in the modulation of platelet function.

Salvia miltiorrhiza Bunge contains various active constituents, some of which have been developed as commercially available medicine. Moreover, some other ingredients in Salvia miltiorrhiza play roles in anti-platelet activity. The aim of the present study was to investigate the effects and the underlying mechanism of miltirone, a lipophilic compound of Salvia miltiorrhiza Bunge. The ability of miltirone to modulate platelet function was investigated by a variety of in vitro and in vivo experiments. Platelet aggregation and dense granule secretion induced by various agonists were measured with platelet aggregometer. Clot retraction and spreading were imaged by digital camera and fluorescence microscope. Ferric chloride-induced carotid injury model and pulmonary thromboembolism model were used to check miltirone antithrombotic effect in vivo. To elucidate the mechanisms of anti-platelet activity of miltirone, flow cytometry and western blotting were performed. Miltirone (2, 4, 8 µM) was shown to suppress platelet aggregation, dense granule, and α granule secretion in a dose-dependent manner. Meanwhile, miltirone inhibited the clot retraction and spreading of washed platelets. It reduced the phosphorylation of PLCγ2, PKC, Akt, GSK3βand ERK1/2 in the downstream signal pathway of collagen receptor. It also reduced the phosphorylation of Src and FAK in the integrin αIIbβ3-mediated "outside-in" signaling, while it did not suppress the phosphorylation of β3. In addition, miltirone prolonged the occlusion time and reduced collagen/epinephrine-induced pulmonary thrombi. Miltirone suppresses platelet "inside-out" and "outside-in" signaling by affecting PLCγ2/PKC/ERK1/2, PI3K/Akt, and Src/FAK signaling. Therefore, miltirone might represent a potential anti-platelet candidate for the prevention of thrombotic disorders.

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

The release of ADP from platelet dense granules and its binding to platelet P2Y12 receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y12 has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y12 receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y12 inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y12 with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca2+ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y12 with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.