Project description:The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbβ3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.
Project description:We genetically manipulated the major platelet vesicle-associated membrane proteins (VAMP2, VAMP3, and VAMP8) to create mice with varying degrees of disrupted platelet secretion. As previously shown, loss of VAMP8 reduced granule secretion, and this defect was exacerbated by further deletion of VAMP2 and VAMP3. VAMP2?3?8-/- platelets also had reduced VAMP7. Loss of VAMP2 and VAMP3 (VAMP2?3?) had a minimal impact on secretion when VAMP7 and VAMP8 were present. Integrin ?IIb?3 activation and aggregation were not affected, although spreading was reduced in VAMP2?3?8-/- platelets. Using these mice as tools, we asked how much secretion is needed for proper thrombosis and hemostasis in vivo. VAMP2?3? mice showed no deficiency, whereas VAMP8-/- mice had attenuated formation of occlusive thrombi upon FeCl3-induced arterial injury but no excessive bleeding upon tail transection. VAMP2?3?8-/- mice bled profusely and failed to form occlusive thrombi. Plasma-coagulation factors were normal in all of the strains, but phosphatidylserine exposure was reduced in VAMP2?3? and VAMP2?3?8-/- platelets. From our data, an ?40% to 50% reduction in platelet secretion in vitro (dense and ? granule) correlated with reduced occlusive thrombosis but no compromise in hemostasis. At a >50% reduction, thrombosis and hemostasis were defective in vivo. Our studies are the first systematic manipulation of platelet exocytic machinery to demonstrate a quantitative linkage between in vitro platelet secretion and hemostasis and thrombosis in vivo. The animals described will be invaluable tools for future investigations into how platelet secretion affects other vascular processes.
Project description:CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin ?IIb?3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.
Project description:Platelet activation in blood flow under high, overcritical shear rates is initiated by Von Willebrand factor. Despite the large amount of experimental data that have been obtained, the value of the critical shear rate, above which von Willebrand factor starts to activate platelets, is still controversial. Here, we recommend a theoretical approach to elucidate how the critical blood shear rate is dependent on von Willebrand factor size. We derived a diagram of platelet activation according to the shear rate and von Willebrand factor multimer size. We succeeded in deriving an explicit formula for the dependence of the critical shear rate on von Willebrand factor molecule size. The platelet activation risk index was introduced. This index is dependent on the flow conditions, number of monomers in von Willebrand factor, and platelet sensitivity. Probable medical applications of the platelet activation risk index as a universal prognostic index are discussed.
Project description:Matrine is a naturally occurring alkaloid and possesses a wide range of pharmacological properties, such as anti-cancer, anti-oxidant, anti-inflammatory effects. However, whether it affects platelet function and thrombosis remains unclear. This study aims to evaluate the effect of matrine on platelet function and thrombus formation. Human platelets were treated with matrine (0-1 mg/ml) for 1 h at 37°C followed by measuring platelet aggregation, granule secretion, receptor expression by flow cytometry, spreading and clot retraction. In addition, matrine (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time, arterial and venous thrombus formation. Matrine dose-dependently inhibited platelet aggregation and ATP release in response to either collagen-related peptide (Collagen-related peptide, 0.1 μg/ml) or thrombin (0.04 U/mL) stimulation without altering the expression of P-selectin, glycoprotein Ibα, GPVI, or αIIbβ3. In addition, matrine-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction along with reduced phosphorylation of c-Src. Moreover, matrine administration significantly impaired the in vivo hemostatic function of platelets, arterial and venous thrombus formation. Furthermore, in platelets stimulated with CRP or thrombin, matrine significantly reduced Reactive oxygen species generation, inhibited the phosphorylation level of ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182) and AKT (Thr308/Ser473) as well as increased VASP phosphorylation (Ser239) and intracellular cGMP level. In conclusion, matrine inhibits platelet function, arterial and venous thrombosis, possibly involving inhibition of ROS generation, suggesting that matrine might be used as an antiplatelet agent for treating thrombotic or cardiovascular diseases.
Project description:Glutamate is a major signaling molecule that binds to glutamate receptors including the ionotropic glutamate receptors; kainate (KA) receptor (KAR), the N-methyl-d-aspartate receptor, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Each is well characterized in the central nervous system, but glutamate has important signaling roles in peripheral tissues as well, including a role in regulating platelet function.Our previous work has demonstrated that glutamate is released by platelets in high concentrations within a developing thrombus and increases platelet activation and thrombosis. We now show that platelets express a functional KAR that drives increased agonist induced platelet activation.KAR induced increase in platelet activation is in part the result of activation of platelet cyclooxygenase in a mitogen-activated protein kinase-dependent manner. Platelets derived from KAR subunit knockout mice (GluR6(-/-)) are resistant to KA effects and have a prolonged time to thrombosis in vivo. Importantly, we have also identified polymorphisms in KAR subunits that are associated with phenotypic changes in platelet function in a large group of whites and blacks.Our data demonstrate that glutamate regulation of platelet activation is in part cyclooxygenase-dependent and suggest that the KAR is a novel antithrombotic target.
Project description:The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3(-/-) mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A? (TXA?), but not collagen or VWF. In contrast, platelets from Akt1(-/-) or Akt2(-/-) mice are defective in platelet activation induced by thrombin, TXA?, and VWF, but only Akt1(-/-) platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3(-/-) platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3? (GSK-3?) at Ser9, which is known to inhibit GSK-3? function. Thus, Akt3 is important in inhibiting GSK-3?. Accordingly, treatment of Akt3(-/-) platelets with a GSK-3? inhibitor rescued the defect of Akt3(-/-) platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3? may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3(-/-) mice showed retardation in FeCl?-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.
Project description:Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.
Project description:Thrombosis and its complications are the leading cause of death in patients with diabetes. Metformin, a first-line therapy for type 2 diabetes, is the only drug demonstrated to reduce cardiovascular complications in diabetic patients. However, whether metformin can effectively prevent thrombosis and its potential mechanism of action is unknown. Here we show, metformin prevents both venous and arterial thrombosis with no significant prolonged bleeding time by inhibiting platelet activation and extracellular mitochondrial DNA (mtDNA) release. Specifically, metformin inhibits mitochondrial complex I and thereby protects mitochondrial function, reduces activated platelet-induced mitochondrial hyperpolarization, reactive oxygen species overload and associated membrane damage. In mitochondrial function assays designed to detect amounts of extracellular mtDNA, we found that metformin prevents mtDNA release. This study also demonstrated that mtDNA induces platelet activation through a DC-SIGN dependent pathway. Metformin exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing the release of free mtDNA, which induces platelet activation in a DC-SIGN-dependent manner. This study has established a novel therapeutic strategy and molecular target for thrombotic diseases, especially for thrombotic complications of diabetes mellitus.
Project description:Inherent to any stenting procedure is the prescription of dual antiplatelet therapy (DAPT) to reduce the platelet response. Clinical guidelines recommend 6-12 months of DAPT, depending on stent type, clinical picture and patient factors. Our hypothesis is that a nanostructured noble metal coating has the potential to reduce protein deposition and platelet activation. These effects would reduce subsequent thrombo-inflammatory reactions, potentially mitigating the need for an extensive DAPT in the acute phase. Here, a noble metal nanostructure coating on stents is investigated. Twelve pigs underwent endovascular implantation of coated and non-coated stents for paired comparisons in a blinded study design. The non-coated control stent was placed at the contralateral corresponding artery. Volumetric analysis of angiographic data, performed by a treatment blinded assessor, demonstrated a significant thrombus reduction for one of the coatings compared to control. This effect was already seen one hour after implantation. This finding was supported by in vitro data showing a significant reduction of coagulation activation in the coated group. This novel coating shows promise as an implant material addition and could potentially decrease the need for DAPT in the early phases of stent implementation.