Important Role In Platelet Activation Research Articles

Icaritin Sensitizes Thrombin- and TxA2-Induced Platelet Activation and Promotes Hemostasis via Enhancing PLCγ2-PKC Signaling Pathways.

Vascular injury results in uncontrollable hemorrhage in hemorrhagic diseases and excessive antithrombotic therapy. Safe and efficient hemostatic agents which can be orally administered are urgently needed. Platelets play indispensable roles in hemostasis, but there is no drug exerting hemostatic effects through enhancing platelet function. The regulatory effects of icaritin, a natural compound isolated from Herba Epimedii, on the dense granule release, thromboxane A2 (TxA2) synthesis, α-granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by multiple agonists were investigated. The effects of icaritin on tail vein bleeding times of warfarin-treated mice were also evaluated. Furthermore, we investigated the underlying mechanisms by which icaritin exerted its pharmacological effects. Icaritin alone did not activate platelets, but significantly potentiated the dense granule release, α-granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by thrombin and U46619. Icaritin also shortened tail vein bleeding times of mice treated with warfarin. In addition, phosphorylated proteome analysis, immunoblotting analysis, and pharmacological research revealed that icaritin sensitized the activation of phospholipase Cγ2 (PLCγ2)-protein kinase C (PKC) signaling pathways, which play important roles in platelet activation. Icaritin can sensitize platelet activation induced by thrombin and TxA2 through enhancing the activation of PLCγ2-PKC signaling pathways and promote hemostasis, and has potential to be developed into a novel orally deliverable therapeutic agent for hemorrhages.

Apoptosis Signal-Regulating Kinase 1 (Ask1) Inhibition Prevents In Vivo Thrombosis without Compromising Hemostasis

Cardiovascular diseases are currently among the leading causes of death in mankind. Although significant progress has been made in prevention and treatment, the currently available pharmacological interventions have a significant rate of severe bleeding side effects due to their effect on hemostasis. Recently, we have shown that apoptosis signal-regulating kinase 1 (ASK1) plays an important role in platelet activation and thrombus formation. Here, we show that two chemically distinct ASK1 inhibitors, dose-dependently inhibited thrombin-induced phosphorylation of ASK1 and its downstream effectors MKK3/4/6, p38 MAPK, and cPLA2 in human platelets, as detected by western blotting using phospho-specific antibodies. ASK1 inhibitors also dose-dependently inhibited platelet a granule release and α IIbβ 3 activation which are the readouts for inside-out signaling events as analyzed by flow cytometry. Moreover, pretreatment of human platelets with ASK1 inhibitors completely impaired clot retraction and platelet spreading on immobilized fibrinogen two events regulated by outside-in signaling. To investigate the in-vivo effect of Ask1 inhibitor on thrombus formation, we intraperitoneally injected C57BL/6 mice with Ask1 inhibitor (1mg/kg), and the blood was withdrawn at 1-, 4-, and 24-hour time points and analyzed for in vitro thrombus formation on collagen matrix under arterial flow. Ask1 inhibitor significantly decreased thrombus growth ( P<0.01) which was sustained for at least 24 hours. Platelet adhesion to collagen was not affected as determined by surface coverage.In a 10% FeCl 3 -induced carotid artery injury model of thrombosis, we found that Ask1 inhibitor (100μg/kg)showed a significantly extended time of vessel occlusion ( P<0.01) . Moreover, Ask1 inhibitor (100μg/kg) treated C57BL/6 mice were protected from collagen/epinephrine-induced pulmonary thromboembolism ( P<0.001). Furthermore, in a well-established ischemic stroke model of transient middle cerebral artery occlusion, C57BL/6 mice treated with Ask1 inhibitor (1mg/kg) showed increased survival and improved neurological deficits than vehicle treated mice. The extent of the brain damage was assessed by staining the brain sections with 2% 2,3,5-triphenyltetrazoliun chloride, which showed significant reduction in infarct size and edema ( P<0.0001). Interestingly, mice treated with Ask1 inhibitor (1mg/kg), a 10-fold more than the amount needed to inhibit thrombosis, had no effect on hemostasis as observed by both the tail bleeding assay and laser-induced cremaster injury model. Whereas clopidogrel (5-30mg/kg) showed significantly increased ( P<0.0001) tail bleeding time. Next, we performed cerebral bleeding assay in which brain injury was induced by inserting a 26-gauge needle through the skull in the cerebrum 2mm laterally to the bregma and 4mm deep into the cerebrum and rotated 360 degrees. After 20 minutes the extent of bleeding in the brain was assessed by imaging. Treatment of C57BL/6 mice with clopidogrel (30mg/kg) showed significantly enhanced bleeding ( P<0.05) compared to vehicle treated mice. However, Ask1 inhibitor treated mice had same extent of bleeding as that of vehicle treated mice. Additionally, a liver bleeding assay was performed by excising a calibrated piece (~4-5mg) of inferior edge of the right lobe of the liver of the mouse under anesthesia. After 20 minutes the bleeding was assessed by counting red blood cells in the peritoneal lavage. As observed in brain bleeding assay, treatment of mice with clopidogrel (30mg/kg), showed significantly increased liver bleeding ( P<0.05) compared to vehicle treated mice whereas Ask1 inhibitor treated mice had same extent of liver bleeding as that of vehicle treated mice. Taken together, our results provide evidence that ASK1 inhibitors are novel therapeutic agents to combat thrombotic disorders without any side effects of bleeding, which is inherent to current anti-thrombotic drugs.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Blocking of PI3-kinase beta protects against cerebral ischemia/reperfusion injury by reducing platelet activation and downstream microvascular thrombosis in rats

Phosphoinositide 3-kinase beta (PI3Kβ) plays an important role in platelet activation and thrombosis, but its role in stroke pathology remains unknown. In this study, we investigated whether inhibition of PI3Kβ protects against cerebral ischemia/reperfusion (I/R) injury by preventing circulating platelet activation and downstream microvascular thrombosis. We used a rat intraluminal filament model of transient middle cerebral artery occlusion (tMCAO) because the rapid restoration of cerebral blood flow to the ischemic area in both tMCAO and endovascular thrombectomy provides clinical relevance for this model. The results showed that TGX221, a selective PI3Kβ inhibitor, treatment immediately before the onset of reperfusion dose-dependently reduced infarct volume and improved neurological function. The protective effects were associated with blocking platelet activation and thrombotic response, thereby reducing downstream microvascular thrombosis, and maintaining reperfusion efficiency. These results suggest that PI3Kβ might be a promising target for treating downstream microvascular thrombosis induced by cerebral I/R injury and offer a novel adjunctive treatment to improve reperfusion therapy for acute ischemic stroke.

The relative risk of ischemic cerebrovascular accident in patients with Von Willebrand disease.

Cerebrovascular accident (CVA) is one of the leading causes of death in the United States. Von Willebrand factor plays an important role in platelet activation and adhesion. It remains unclear whether Von Willebrand disease (vWD) is associated with a decreased risk of developing CVA. The study aimed to compare the relative risk (RR) of CVA in patients with and without vWD. We queried the National Inpatient Sample from 2009 to 2014 for discharge data and records for vWD and CVA using International Classification of Diseases, Ninth-Revision codes. The unadjusted and adjusted RR of CVA in patients with and without vWD were estimated using log-binomial model. Descriptive measures including means, medians, standard deviations, and range were presented based on normality test of continuous data. The prevalence of CVA was lower in patients with vWD than in those without vWD (1.31% vs 2.04%), with a RR of 0.64 (95% confidence interval (CI): 0.60-0.68). After adjusting for common CVA risk factors, the RR remained lower in vWD patients: 0.81 (95% CI: 0.76-0.86). vWD is associated with a lower RR of developing CVA. This suggests that deficiency of Von Willebrand factor is potentially protective against the development of CVA. To the best of our knowledge, this is the first study in humans to compare the RR of CVA in patients with and without vWD. Future studies are needed to explore causal relationships and therapeutic benefits.

One night of sleep deprivation induces release of small extracellular vesicles into circulation and promotes platelet activation by small EVs.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. Few studies have focused on EV levels in subjects with sleep disorders. Here, we aimed to explore the role of acute sleep deprivation on the quantity and functionality of circulating EVs, and their tissue distribution. EVs were isolated by ultracentrifugation from the plasma of volunteers and animals undergoing one night of sleep deprivation. Arterio‐venous shunt, FeCl3 thrombus test and thrombin‐induced platelet aggregation assay were conducted to evaluate the in vivo and in vitro bioactivity of small EVs. Western blotting was performed to measure the expression of EV proteins. The fate and distribution of circulating small EVs were determined by intravital imaging. We found that one night of sleep deprivation triggers release of small EVs into the circulation in both healthy individuals and animals. Injection of sleep deprivation‐liberated small EVs into animals increased thrombus formation and weight in thrombosis models. Also, sleep deprivation‐liberated small EVs promoted platelet aggregation induced by thrombin. Mechanistically, sleep deprivation increased the levels of HMGB1 protein in small EVs, which play important roles in platelet activation. Furthermore, we found sleep deprivation‐liberated small EVs are more readily localize in the liver. These data suggested that one night of sleep deprivation is a stress for small EV release, and small EVs released here may increase the risk of thrombosis. Further, small EVs may be implicated in long distance signalling during sleep deprivation‐mediated adaptation processes.

XJ‐8, a natural compound isolated from Sanguis draxonis, inhibits platelet function and thrombosis by targeting MAP3K3

BackgroundVascular injury initiates rapid platelet activation, which is critical for haemostasis, while it also causes fatal thrombotic diseases, such as myocardial infarction or ischemic stroke. ObjectivesTo study the inhibitory effects and underlying mechanisms of XJ‐8, a natural compound isolated from Sanguis draxonis, on platelet activation and thrombosis. MethodsThe regulatory effects of XJ‐8 on the dense granule release, thromboxane A2 (TxA2) synthesis, α‐granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by multiple agonists were investigated in in vitro experiments. The effects of XJ‐8 on bleeding time and FeCl3‐induced carotid artery thrombosis were also evaluated in in vivo experiments. Furthermore, we investigated the underlying mechanisms by which XJ‐8 exerted its pharmacological effects. ResultsXJ‐8 not only significantly inhibited the dense granule release, TxA2 synthesis, and aggregation of platelets induced by multiple agonists, but also exerted extending effects on bleeding time and therapeutic effects on thrombotic disease. In addition, XJ‐8 selectively and moderately inhibited the activity of mitogen‐activated protein kinase kinase kinase 3 (MAP3K3) and the activation of signalling pathways downstream MAP3K3, which play important roles in platelet activation. ConclusionXJ‐8 can inhibit platelet function and thrombosis by targeting MAP3K3 and has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.

Docking and mutagenesis studies lead to improved inhibitor development of ML355 for human platelet 12-lipoxygenase

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the “U” shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties.

Thymidine Phosphorylase Is Increased in COVID-19 Patients in an Acuity-Dependent Manner.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), is a human respiratory disease. Hitherto, there is no effective treatment has been established. Patients with cardiovascular or diabetes comorbidities are a high-risk cohort. COVID-19 is accompanied by excessive systemic thrombotic events, but the mechanism is not yet known. Recent studies have indicated that thymidine phosphorylase (TYMP) plays an important role in platelet activation, thrombosis, and TYMP expression is increased in diabetic patients. By using data provided by the MGH (Massachusetts General Hospital) Emergency Department COVID-19 Cohort with Olink Proteomics, here we show that plasma TYMP level is correlated with the COVID-19 associated thrombotic event, inflammation, and organ damage, as evidenced by the positive correlations with plasma D-dimer, CRP (C reactive protein), and LDH (lactate dehydrogenase), as well as Interferons (IFN). Plasma TYMP is also positively correlated with COVID-19 patients who had respiratory symptoms. TYMP thus could be an acuity marker for COVID-19 diagnosis. Targeting TYMP with tipiracil, a selective TYMP inhibitor, which has been approved by the Food and Drug Administration for clinical use, could be a novel effective medicine for COVID-19.

Associations of ozone exposure with urinary metabolites of arachidonic acid

BackgroundOzone (O3) exposure has been associated with biomarkers of platelet activation and oxidative stress. The metabolism of arachidonic acid (AA) plays an important role in platelet activation and oxidative stress. However, AA metabolic pathways have not been examined in relation to O3 and other air pollutants. MethodsEarly morning urine and fasting blood were longitudinally collected up to four times from 89 healthy adults (22–52 years old, 25 women) in Changsha City, China. We measured three urinary AA metabolites, namely 11-dehydro-Thromboxane B2 (11-dhTXB2) produced from the arachidonic cyclooxygenase pathway, 20-hydroxyeicosatetraenoic acid (20-HETE) from the CYPs pathway, and 8-isoprostane from the non-enzymatic pathway. Urinary malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured as indicators of oxidative damage to lipids and DNA, respectively. We measured soluble P-selectin (sCD62p) concentrations in plasma as an indicator of platelet activation. Indoor and outdoor air pollutants were measured and combined with participants’ time-activity pattern to calculate personal exposure to O3, PM2.5, NO2, and SO2 averaged over 12-hour, 24-hour, 1-week, and 2-week periods prior to biospecimen collection, respectively. Linear mixed-effects models were used to examine the relationships of AA metabolites with air pollutant exposures, plasma sCD62p, and urinary MDA & 8-OHdG. ResultsWe found that a 10% increase in 12 h and 24 h O3 exposure were associated with increases in urinary11-dhTXB2 by 1.4% (95%, 0.1% to 2.6%) and 1.3% (0.05% to 2.5%), respectively. These associations remained robust after adjusting for co-pollutant exposures. No significant associations were observed between 11-dhTXB2 and other pollutants or between O3 exposures and other AA metabolites. All the three AA metabolites were significantly and positively associated with urinary MDA and 8-OHdG, whereas only urinary 11-dhTXB2 was significantly and positively associated with plasma sCD62p. ConclusionsA metabolite of AA from the cyclooxygenase pathway was positively associated with short-term O3 exposure, and with a plasma marker of platelet activation and two urinary markers of oxidative stress. The results suggest that O3 exposure may contribute to increased platelet activation and oxidative damages via altering the metabolism of AA.

Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Platelet activation via dynamic conformational changes of von Willebrand factor under shear.

Shear-induced conformational changes of von Willebrand factor (VWF) play an important role in platelet activation. A novel approach describing VWF unfolding on the platelet surface under dynamic shear stress is proposed. Cumulative effect of dynamic shear on platelet activation via conformational changes of VWF is analysed. The critical condition of shear-induced platelet activation is formulated. The explicit expression for the threshold value of cumulative shear stress as a function of VWF multimer size is derived. The results open novel prospects for pharmacological regulation of shear-induced platelet activation through control of VWF multimers size distribution.

Low temperature induces von-willebrand factor expression via increased early growth response 1 transcriptional activity in splenic sinusoidal endothelial cells

von Willebrand factor (vWF) is a large plasma glycoprotein that plays an important role in hemostasis by forming molecular bridges with platelets following vascular injury. Previously, we reported that hypothermia enhanced vWF production in the spleen, which resulted in the activation of the platelet pool in a hypothermia-induced murine model. However, the mechanisms that regulate vWF expression under hypothermic conditions remain unclear. In this study, we focused on vWF expression under hypothermic conditions in splenic endothelial cell culture.Human splenic endothelial cells (HSEC) were incubated at 20 °C for 1 h. Total RNA was extracted from the cells, and cDNA microarray gene expression analysis was performed. Genes that may be associated with vWF expression in low temperature culture conditions were then selected for further analysis.Gene expression analysis showed that low temperature conditions increased the expression of FOS and EGR1. We then hypothesized that these factors upregulate vWF mRNA expression in HSEC. The transcriptional inhibitors of EGR1 significantly inhibited vWF mRNA expression in HSEC cultured at a low temperature.Our analysis revealed that low temperatures enhance the gene expression of EGR1, which transcriptionally increases vWF expression. This acute-phase reaction may play an important role in platelet activation in the spleen during hypothermia.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

The role of PI3K/Akt signaling pathway in non-physiological shear stress-induced platelet activation.

The PI3K/Akt signaling pathway has been implicated in playing an important role in platelet activation during hemostasis and thrombosis involving platelet-matrix interaction and platelet aggregation. Its role in non-physiological shear stress (NPSS)-induced platelet activation relevant to high-shear blood contacting medical devices (BCMDs) is unclear. In the context of blood cells flowing in BCMDs, platelets are subjected to NPSS (>100Pa) with very short exposure time (<1s). In this study, we investigated whether NPSS with short exposure time induces platelet activation through the PI3K/Akt signaling pathway. Healthy donor blood treated with or without PI3K inhibitor was subjected to NPSS (150Pa) with short exposure time (0.5s). Platelet activation indicated by the surface P-selectin expression and activated glycoprotein (GP) IIb/IIIa was quantified using flow cytometry. The phosphorylation of Akt, activation of the PI3K signaling, was characterized by western blotting. Changes in adhesion behavior of NPSS-sheared platelets on fibrinogen, collagen, and von Willebrand factor (vWF) were quantified with fluorescent microscopy by perfusing the NPSS-sheared and PI3K inhibitor-treated blood through fibrinogen, collagen, and vWF-coated microcapillary tubes. The results showed that the PI3K/Akt signaling was involved with both NPSS-induced platelet activation and platelet-matrix interaction. NPSS-sheared platelets exhibited exacerbated platelet adhesion on fibrinogen, but had diminished platelet adhesion on collagen and vWF. The inhibition of PI3K signaling reduced P-selectin expression and GPIIb/IIIa activation with suppressed Akt phosphorylation and abolished NPSS-enhanced platelet adhesion on fibrinogen in NPSS-sheared blood. The inhibition of PI3K signaling can attenuate the adhesion of unsheared platelets (baseline) on collagen and vWF, while had no impact on adhesion of NPSS-sheared platelets on collagen and vWF. This study confirmed the important role of PI3K/Akt signaling pathway in NPSS-induced platelet activation. The finding of this study suggests that blocking PI3K/Akt signaling pathway could be a potential method to treat thrombosis in patients implanted with BCMDs.

Tyro3, Axl, and Mertk receptors differentially participate in platelet activation and thrombus formation

BackgroundPreviously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood.MethodsUsing single receptor-deficient platelets from TAM knockout mice in the C57BL/6 J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo.ResultsDeficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl−/− and Tyro3−/− mice, but not in Mertk−/− mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl−/− and Tyro3−/− platelets, but not in Mertk−/− platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation.ConclusionsThese data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice

AbstractMAPKs play important roles in platelet activation. However, the molecular mechanisms by which MAPKs are regulated in platelets remain largely unknown. Real-time polymerase chain reaction and western blot data showed that MEKK3, a key MAP3K family member, was expressed in human and mouse platelets. Then, megakaryocyte/platelet-specific MEKK3-deletion (MEKK3−/−) mice were developed to elucidate the platelet-related function(s) of MEKK3. We found that agonist-induced aggregation and degranulation were reduced in MEKK3−/− platelets in vitro. MEKK3 deficiency significantly impaired integrin αIIbβ3–mediated inside-out signaling but did not affect the outside-in signaling. At the molecular level, MEKK3 deficiency led to severely impaired activation of extracellular signal–regulated kinases 1/2 (ERK1/2) and c-Jun NH2-terminal kinase 2 but not p38 or ERK5. In vivo, MEKK3−/− mice showed delayed thrombus formation following FeCl3-induced carotid artery injury. Interestingly, the tail bleeding time was normal in MEKK3−/− mice. Moreover, MEKK3−/− mice had fewer microthrombi, reduced myocardial infarction (MI) size, and improved post-MI heart function in a mouse model of MI. These results suggest that MEKK3 plays important roles in platelet MAPK activation and may be used as a new effective target for antithrombosis and prevention of MI expansion.

Role of Protein Kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis.

Sepsis is characterized by an intense systemic inflammatory response activating a cascade of proinflammatory events resulting in leukocyte dysregulation and host tissue damage. The lung is particularly susceptible to systemic inflammation, leading to acute lung injury. Key to inflammation-induced lung damage is the excessive migration of neutrophils across the vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are not well defined but there is growing evidence that platelets are actively involved and play a key role in microvascular permeability and neutrophil-mediated organ damage. We previously identified PKC-delta (PKCδ) as a critical regulator of the inflammatory response in sepsis and demonstrated PKCδ inhibition was lung protective. However, the role of PKCδ in sepsis-induced platelet activation and platelet-leukocyte interactions is not known. In this study, rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Following surgeries, a PKCδ inhibitor (200μg/kg) or vehicle (PBS) was administered intra-tracheally. At 24 hours post-surgeries, lung tissue, BAL fluid, and blood samples were collected. While sepsis caused thrombocytopenia, the remaining circulating platelets were activated as demonstrated by increased p-selectin expression, elevated plasma PF4, and enhanced platelet-leukocyte aggregate formation compared to Sham animals. Platelet activation was associated with increased platelet PKCδ activity. Inhibition of PKCδ attenuated sepsis-induced platelet activation, secretion and aggregate formation. Sepsis-induced thrombocytopenia was also significantly reduced and circulating platelet numbers were similar to sham animals. In the lung, sepsis induced significant influx of platelets and neutrophils and the development of lung injury. Administration of the PKCδ inhibitor decreased platelet and neutrophil influx, and was lung protective. Thus, PKCδ inhibition modulated platelet activity both locally and systemically, decreased neutrophil influx into the lung, and was lung protective. We demonstrate for the first time that PKCδ plays an important role in platelet activation and platelet-neutrophil interaction during sepsis.

DREAM plays an important role in platelet activation and thrombogenesis

AbstractDownstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5′-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iβ (PI3K-Iβ). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iβ activation. These results suggest that platelet DREAM regulates PI3K-Iβ activity and plays an important role during thrombus formation.

A-Disintegrin-And-Metalloproteinase (ADAM) 10 Activity on Resting and Activated Platelets.

The primary platelet collagen receptor, glycoprotein VI (GPVI), plays an important role in platelet activation and thrombosis. The ectodomain of human GPVI (sGPVI) is proteolytically shed from human platelets by a-disintegrin-and-metalloproteinase 10 (ADAM10). In this study, we used a novel ADAM10-sensitive fluorescence resonance energy transfer sensor to analyze ADAM10-mediated shedding of GPVI from human platelets in response to the exposure of GPVI ligands collagen-related peptide (10 μg/mL), collagen (10 μg/mL), and convulxin (0.1 μg/mL) to shear stress (1000-10000 s(-1), 5 min), or a generic activator of metalloproteinases, N-ethylmaleimide (NEM, 5 mM). Elevated shear, NEM, or ligand engagement of GPVI all induced shedding of GPVI, as detected by release of sGPVI; however, only shear or NEM significantly increased ADAM10 enzyme activity. ADAM10 activity was also detectable on the surface of thrombi formed on a collagen-coated surface under flow conditions. Our findings indicate different mechanisms regulate shear- and ligand-induced shedding and shear forces found within the vasculature can regulate ADAM10 activity.