Wild-type Platelets Research Articles

Distinct role of GRK3 in platelet activation by desensitization of G protein-coupled receptors.

Many platelet agonists mediate their cellular effects through G protein-coupled receptors (GPCRs) to induce platelet activation, and GPCR kinases (GRKs) have been demonstrated to have crucial roles in most GPCR functions in other cell types. Here, we investigated the functional role of GRK3 and the molecular basis for the regulation of GPCR desensitization by GRK3 in platelets. We used mice lacking GRK3 as well as β-arrestin2 which has been shown to be important in GPCR function in platelets. Platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in both GRK3 -/- and β-arrestin2 -/- platelets compared to wild-type (WT) platelets, while non-GPCR agonist collagen-induced platelet aggregation and secretion were not affected. We have previously shown that GRK6 is not involved in the regulation of Gq-coupled 5HT2A and Gz-coupled a2A adrenergic receptors. Interestingly, in contrast to GRK6, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate 5-HT2A and a2A adrenergic receptors, respectively, was significantly potentiated in GRK3 -/- platelets, suggesting that GRK3 was involved in general GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP was restored in GRK3 -/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, confirming that GRK3 contributes to general GPCR desensitization. Finally, 2-MeSADP- and AYPGKF-induced AKT and ERK phosphorylation were significantly potentiated in GRK3 -/- platelets. GRK3 plays a crucial role in the regulation of platelet activation through general GPCR desensitization in platelets.

LAIR-1 and PECAM-1 function via the same signaling pathway to inhibit GPVI-mediated platelet activation

BackgroundInhibition of platelet responsiveness is important for controlling thrombosis. It is well established that platelet endothelial cell adhesion molecule-1 (PECAM-1) serves as a physiological negative regulator of platelet-collagen interactions. We recently demonstrated that leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a negative regulator of platelet production and reactivity. It is however not known if LAIR-1 and PECAM-1 function in the same or different inhibitory pathways. ObjectivesIn this study, we investigated the role of LAIR-1 alongside PECAM-1 in megakaryocyte development and platelet production and determined the functional redundancy through characterization of a LAIR-1/PECAM-1 double knockout (DKO) mouse model. MethodsLAIR-1 and PECAM-1 expression in megakaryocytes were evaluated by western blotting. Megakaryocyte ploidy and proplatelet formation were evaluated by flow cytometry and fluorescent microscopy. Platelet function and signalling were compared in wild-type, LAIR-1−/−, PECAM-1−/− and DKO mice using aggregometry, flow cytometry and western blotting. Thrombosis was evaluated using the FeCl3 carotid artery model. ResultsWe show that LAIR-1/PECAM-1 DKO mice exhibit a 17% increase in platelet count. Bone marrow-derived megakaryocytes from all 3 mouse models had normal ploidy in vitro, suggesting that neither LAIR-1 nor PECAM-1 regulates megakaryocyte development. Furthermore, relative to wild-type platelets, platelets derived from LAIR-1, PECAM-1, and DKO mice were equally hyperresponsive to collagen in vitro, indicating that LAIR-1 and PECAM-1 participate in the same inhibitory pathway. Interestingly, DKO mice exhibited normal thrombus formation in vivo due to DKO mouse platelets lacking the enhanced Src family kinase activation previously shown in platelets from LAIR-1-deficient mice. ConclusionFindings from this study reveal that LAIR-1 and PECAM-1 act to inhibit GPVI-mediated platelet activation via the same signaling pathway. Mice lacking LAIR-1 and PECAM-1 do not however exhibit an increase in thrombus formation despite minor increase in platelet count and reactivity to collagen. This study adds to the growing evidence that immunoreceptor tyrosine-based inhibition motif–containing receptors are important regulators of platelet count and function.

Utility of thromboelastography with platelet mapping for monitoring platelet transfusion in qualitative platelet disorders

BackgroundPatients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) exhibit normal platelet counts but impaired platelet aggregation and αIIbβ3 activation. Moderate-to-severe bleeding episodes require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 (Rasgrp2−/− mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Thus, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. ObjectiveWe here studied the usefulness of thromboelastography with platelet mapping (TEG-PM) for ex vivo monitoring of the hemostatic potential in Rasgrp2−/− mice transfused with various amounts of wild-type (WT) platelets. MethodsWhole blood (WB) samples from WT and Rasgrp2−/− mice were tested in TEG-PM and parameters for clot formation and contraction (K time, α-angle, maximum amplitude [MA]) were measured. ResultsRasgrp2−/− WB samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbβ3 activation. Addition of WT platelets improved TEG parameters in a ratio-dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. ConclusionThis proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.

C-type lectin-like receptor-2 in platelets mediates ferric chloride–induced platelet activation and attenuates ferroptosis of endothelial cells

BackgroundAn iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. ObjectivesThis study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. MethodsPlatelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride–stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. ResultsFerric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride–induced platelet aggregation was almost lost in CLEC-2–depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2–deficient platelets, attenuated ferroptosis of endothelial cells in vitro. ConclusionFerric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.

#855 Cyclophilin D in cholesterol crystal-induced acute kidney injury

Abstract Background and Aims Cholesterol crystal embolism (CCE) occurs when cholesterol crystal (CC) clots move from an atherosclerotic plaque in a large artery to a smaller artery, leading to obstruction, ischemia, and organ failure. CCE directly impairs blood flow by provoking a local inflammatory response and triggering arterial thrombus formation, consisting of collagen and fibrin mesh, platelet and neutrophil activation, and extracellular DNA trap formation. Activated platelets form a procoagulant surface that supports adhesion to collagen and fibrin mesh, promoting thrombus growth and the recruitment of immune cells. CCE often targets kidney tissue, leading to thrombotic angiopathy, acute loss of glomerular filtration rate (GFR), and necrosis in tubular cells. Cyclophilin D (CypD) plays a central role in regulating platelet procoagulant activity by triggering calcium bursts and mitochondrial depolarization. While the increased number of procoagulant platelets contributes to thrombosis, CypD-mediated procoagulant pathomechanisms in the kidney during CCE remain unknown. In our study presented here, we hypothesized that CypD deficiency might attenuate procoagulant platelet activity, thereby inhibiting CCE-induced kidney infarction and organ injury. Method 6-week-old wild-type (WT) and CypD-/- mice were administered 10 mg/kg CC through injections into the left kidney arteries in vivo. Kidneys were collected 24 hours later, and kidney function, infarct size, necroinflammation and kidney injury were evaluated using GFR measurements, TTC colorimetric assay, immunohistochemistry, and immunofluorescence microscopy. For in vitro studies, whole blood samples were isolated from WT and CypD-/- mice and perfused on a collagen-coated surface in the presence or absence of CC under arterial shear conditions of 1000s-1. Results After CC injection in mice, CypD deficiency ameliorated immunothrombosis, acute kidney injury, and organ failure. CypD-/- mice were protected from the sudden drop in GFR, i.e., acute kidney injury, which resulted from the reduced number of arteries obstructed by crystal clots. Kidney infarct size consistently decreased in CypD-/- mice. CypD-/- mice displayed reduced neutrophil recruitment to thrombotic microangiopathic lesions, indicating diminished immunothrombosis. Preemptive injection of the CypD inhibitor cyclosporin A could prevent CC-mediated vessel obstruction and infarct size, thereby improving kidney function. Interestingly, WT platelets showed increased platelet adhesion and phosphatidylserine (PS) exposure on a collagen-coated surface in the presence of CC under shear conditions in whole blood, suggesting increased procoagulant activity, which was strongly reduced in CypD-/- platelets. Conclusion During CCE-mediated kidney injury, CypD-mediated platelet activation and consequent procoagulant activity accelerate ischemic tissue necrosis and the recruitment of neutrophils. Inhibition of CypD-mediated signaling could be a potential therapeutic approach to inhibit acute kidney injury in patients with CCE.

Abstract 2097: Jak2 V617f Clonal Hematopoiesis Promotes Arterial Thrombosis Via Platelet Activation And Cross-talk

JAK2 V617F ( JAK2 VF ) clonal hematopoiesis (CH) is associated with a marked increase in atherothrombotic cardiovascular disease (CVD). The role of JAK2 VF CH in arterial thrombosis has not been assessed. Using mice simulating JAK2 VF CH, we showed accelerated carotid artery thrombosis, with increased platelet counts and activation and increased leukocytes and red blood cells. There was increased platelet activation in both Jak2 VF and wild type (WT) platelets in Jak2 VF CH suggesting cross-talk between mutant and WT platelets. To assess platelet-specific effects, we used Gp1ba-Cre (VFGp1ba) to selectively express Jak2 VF in platelets. VFGp1ba increased platelet counts to a similar level as in 20% Jak2 VF CH mice but had no effect on leukocyte counts. Like Jak2 VF CH, VFGp1ba increased platelet activation and accelerated arterial thrombosis. Mechanistically, Jak2 VF platelets showed 2.8-fold up-regulation of COX-1, increased activation of cytosolic phospholipase A2, an enzyme important for arachidonic acid generation, and increased thromboxane A2 production. Conditioned media from activated Jak2 VF platelets induced WT platelet activation that was reversed by a thromboxane receptor antagonist. Low dose aspirin ameliorated carotid thrombosis in VFGp1ba and Jak2 VF CH but not in WT control mice. This study shows increased arterial thrombosis in Jak2 VF CH related to Jak2 VF platelet activation and cross-talk with WT platelets via thromboxane, suggesting a potential beneficial role of aspirin treatment in JAK2 VF CH.

Gelsolin regulates receptor-mediated and fluid-phase endocytosis in platelets

BackgroundEndocytosis is the process by which platelets incorporate extracellular molecules into their secretory granules. Endocytosis is mediated by the actin cytoskeleton in nucleated cells; however, the endocytic mechanisms in platelets are undefined. ObjectivesTo better understand platelet endocytosis, we studied gelsolin (Gsn), an actin-severing protein that promotes actin assembly. MethodsMouse platelets from Gsn-null (Gsn−/−) and wild-type (WT) controls were used. The uptake of fluorescent cargo molecules was compared as a measure of their endocytic efficiency. Receptor-mediated endocytosis was measured by the uptake of fibrinogen and transferrin; fluid-phase endocytosis was monitored by the uptake of fluorescent dextrans. ResultsAdenosine diphosphate (ADP)–stimulated WT platelets readily internalized both receptor-mediated and fluid-phase cargoes. In contrast, Gsn−/− platelets showed a severe defect in the endocytosis of both types of cargo. The treatment of WT platelets with the actin-disrupting drugs cytochalasin D and jasplakinolide also reduced endocytosis. Notably, the individual and combined effects of Gsn deletion and drug treatment were similar for both receptor-mediated and fluid-phase endocytosis, indicating that Gsn mediates endocytosis via its action on the actin cytoskeleton. ConclusionOur study demonstrates that Gsn plays a key role in the uptake of bioactive mediators by platelets.

Abstract 156: Pyruvate Kinase M2 Regulates SNAP23-mediated Platelet Granule Exocytosis, Neutrophil Extracellular Traps Formation And Experimental Venous Thrombosis

Introduction: Several factors released from activated platelets via exocytosis contribute to venous thrombosis (VT) by potentiating neutrophil extracellular traps formation (NETosis) at the sites of inflammation. The metabolic enzyme pyruvate kinase M2 (PKM2) contributes to platelet activation and arterial thrombosis. PKM2 possess protein kinase activity. The regulatory role of PKM2 in platelet granule exocytosis, NETosis, and VT has not been investigated yet. Aim: To test the hypothesis that dimeric PKM2 regulates SNAP23-mediated platelet granule exocytosis, and thereby contributes to NETosis, and VT. Methods: We utilized platelet-specific PKM2 –/– (PKM2 fl/fl PF4Cre + ), littermate (PKM2 fl/fl ) mice, and wild-type (WT) mice treated with small molecule ML265 (limits PKM2 dimerization) or vehicle to test the susceptibility to VT in inferior vena cava (IVC)-stenosis model. In vitro NETs formation was evaluated by co-incubating WT neutrophils with agonist (thrombin or convulxin)-stimulated platelets. The SNAP23 phosphorylation and PF4 release were measured using Western blot. Results: PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 exhibited reduced susceptibility to VT in the 48 hours IVC-stenosis model as evaluated by less thrombus burden (% incidence, length, and weight, n=11-12/group, P<0.05 vs. controls). Myography revealed that limiting PKM2 dimerization improves the venous wall function post-VT. Activated PKM2 –/– or ML265 pre-treated WT platelets exhibited decreased SNAP23 phosphorylation and PF4 secretion suggesting a regulatory role for PKM2 in SNAP23 exocytosis and PF4 secretion. Furthermore, neutrophils stimulated with activated platelets from either PKM2 fl/fl PF4Cre + or WT mice pre-treated with ML265 showed reduced NETosis. Finally, we demonstrate that the % of the area covered by the thrombus is profoundly reduced in the ML265-treated human whole blood perfused over a tissue factor-coated surface at the venous shear. Conclusions: Dimeric PKM2 is a novel regulator of SNAP23-mediated platelet granule exocytosis. Targeting dimeric PKM2 in platelets inhibits NETosis in neutrophils stimulated with activated platelets and reduces susceptibility to experimental VT.

Abstract 122: Deletion Of Pyruvate Dehydrogenase Kinases Reduces Susceptibility To Experimental Venous Thrombosis By Limiting Glycolysis In Neutrophils

Background: Neutrophils by releasing extracellular traps (NETs) provide a scaffold for platelet activation and coagulation, thereby facilitating venous thrombosis (VT) development. Emerging evidence suggests that neutrophil activation and NETs formation (NETosis) is an energy-intensive process, accompanied by an increased rate of aerobic glycolysis (lactate formation in the presence of oxygen). The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux (final product of glycolysis) from oxidative phosphorylation towards aerobic glycolysis. Recently, we reported the role of PDK2 and PDK4 (PDK2/4) in platelet activation and arterial thrombosis. The regulatory role of PDKs in neutrophils and VT has not been explored. Aim: To test the hypothesis that PDK2/4 promotes NETosis and VT by promoting glycolysis in neutrophils. Methods: Double-deficient PDK2/4 -/- and wild-type (WT) mice were subjected to 48 hours of inferior vena cava stenosis to test the susceptibility to VT. In vitro , neutrophils were stimulated with Phorbol 12-myristate 13-acetate (PMA) and the glycolytic proton efflux rate (glycoPER) was measured using the Seahorse XF Analyzer. Results: The PDK2/4 -/- mice were less susceptible to DVT as measured by the primary outcome of reduced thrombus burden (weight, length and % incidence) and marker of NETs such as citrullinated histones (P<0.05 vs WT). In the WT platelet aggregation assay mediated by PMA-stimulated WT or PDK2/4 -/- neutrophils, we observed reduced platelet aggregation evoked by PMA-stimulated PDK2/4 -/- neutrophils (P<0.05 vs WT). Impaired aggregation was associated with reduced release of cathepsin G and elastase from PMA-stimulated PDK2/4 -/- neutrophils (P<0.05 vs WT). Mechanistically, PMA-stimulated PDK2/4 -/- neutrophils exhibited reduced NETosis that was associated with decreased calcium mobilization and Erk1/2 phosphorylation (P<0.05 vs WT), known contributors of NETosis. Furthermore, PMA-stimulated PDK2/4 -/- neutrophils displayed reduced glycoPER (P<0.05 vs WT), suggesting reduced aerobic glycolysis. Conclusions: These findings for the first time demonstrate the regulatory role of PDK2/4 in NETosis and acute progression of VT.

Jak2V617F clonal hematopoiesis promotes arterial thrombosis via platelet activation and cross talk

AbstractJAK2V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre–mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.

Mitochondrial Fission Protein Drp1 Regulates Platelet Function

Background: Mitochondria in platelets contribute to platelet function and are altered by diseases that involve dysregulated hemostasis or thrombosis. Mitochondrial integrity, governed by its size, number and distribution is modulated by fusion and fission proteins. Pharmacological inhibition of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) has been shown to inhibit granule release in human platelets and impair thrombus formation in a murine model of thrombosis. However, the mitochondrial roles for Drp1 in platelet function are still largely unknown. Objective: To determine the mitochondrial roles of Drp1 in regulating platelet function. Methods: PF4-Cre mediated MK/platelet specific Drp1 null mice (Drp1 -/-) were used. Platelet morphology in Drp1 -/- and wild type (WT) platelets was analyzed by confocal microscopy. Procoagulant platelet formation (PS +ve platelets) after thrombin and the GPVI agonist convulxin (THR+CVX) stimulation was measured using Annexin V staining. Mitochondrial potential (ΔΨm) and mass at baseline and after stimulation was measured using TMRM and mitotracker green. Washed platelet activation in WT and Drp1 -/- platelets in response to convulxin was measured by flow cytometry. Platelet cytoplasmic calcium transients were measured using FURA-2AM after stimulation with THR+CVX. Phosphorylation of Drp1 (pDrp1 S616) in washed WT platelets in response to dual agonist THR+CVX was measured by western blotting. Results: Drp1 -/- platelets were bigger in size than WT platelets as measured by microscopy and had increased mitochondrial mass as determined by flow cytometry. Absence of Drp1 reduced activation of the αIIb/β3 fibrinogen binding receptor (P=0.035; n=5-6 per group) and reduced alpha granule release (P=0.025; n=5-6 per group) in response to CVX.Dual agonist stimulation resulted in reduced PS +ve platelets in Drp1 -/- compared to WT platelets (P=0.0035; n=7 per group). Since depolarization precedes PS exposure, we measured ΔΨm using TMRM in resting and dual agonist stimulated WT and Drp1 -/- platelets. No difference in ΔΨm was observed in resting Drp1 -/- vs WT platelets whereas, stimulated Drp1 -/- platelets exhibited significantly higher ΔΨm (P=0.038; n=4 per group) than stimulated WT platelets. Interestingly, in response to dual agonists, Drp1 -/- platelets also showed a significant increase (P<0.001) in cytoplasmic calcium transients than WT platelets. Western blot analyses of washed WT platelets stimulated with dual agonist showed phosphorylation of Drp1 at S616 (S616), indicating Drp1 activation. Conclusion: These findings highlight a role for Drp1 in murine platelet mitochondrial function, GPVI activation, and procoagulant platelet formation.

Interaction between JAK2-Mutated Neutrophils and Platelets Initiates Thrombosis Via Neutrophil Extracellular Traps

Thrombosis is one of the most common complications of myeloproliferative neoplasms with JAK2-V617F mutation. Recently, individuals with clonal hematopoiesis (CH) harboring JAK2-V617F mutation were also shown to have 12-fold higher risk of thrombosis compared to JAK2 non-mutated individuals (N Engl J Med. 2017;377:111-121). The finding that individuals with JAK2-V617F CH do not have altered blood counts suggests the mechanisms other than increases in the hematopoietic cells which induce thrombosis associated with JAK2-V617F hematopoiesis. JAK2-V617F neutrophils are primed to neutrophil extracellular traps (NETs), which can promote thrombosis (Sci Transl Med. 2018;10: eaan8292). NETs were initially reported as an immunological reaction against infection. However, NETs also play various roles in non-infectious (sterile) settings, and the mechanism how sterile NETs are initiated in vivo remains largely unknown. Here, we studied the mechanisms how NETs are initiated and promote thrombosis in the presence of JAK2-V617F hematopoiesis. First, we isolated neutrophils and platelets from Jak2-V617F transgenic ( Jak2VF) and wild-type (WT) mice, and co-cultured in each cell/genotype combination (see Figure1) at a 1:50 ratio. Of note, Jak2VF neutrophils formed NETs with Jak2VF platelets, but not with WT platelets. Also, Jak2VF platelets slightly increased formation of NETs by WT neutrophils. However, culture supernatant of Jak2VF platelets did not induce formation of NETs by Jak2VF neutrophils. These findings indicated that direct interaction, between Jak2VF neutrophils and Jak2VF platelets, initiates sterile NETs in the presence of JAK2-V617F hematopoiesis. Surprisingly, platelet activation of Jak2VF platelets measured by P-selectin release was inferior to WT platelets. Thus, it was unlikely that overactivation of Jak2VF platelets fully initiate NETs. Subsequently, we sought an initiator on Jak2VF platelets to form NETs in the presence of JAK2-V617F hematopoiesis. Unbiased proteomics analysis of WT and Jak2VF platelets identified several proteins significantly enriched in Jak2VF platelets, including a membrane protein PODXL, which is expressed on podocytes in kidney as well as platelets and other hematopoietic cells. Interestingly, PODXL on platelet is associated with aggregation with other hematopoietic cells depending on CD41, SLC44A2 and P-selectin. Therefore, we hypothesized that PODXL enhances aggregation of Jak2VF platelets with neutrophils and initiates formation of NETs (Figure 2). We confirmed overexpression of PODXL in Jak2VF platelets and in platelets of another murine model, hematopoietic cell-specific Jak2V617F/+ mice by FACS and immunofluorescence imaging. The formations of NETs measured by intensity of citrullinated histone H3 (citH3) correlated with aggregation of PODXL-expressing Jak2VF platelets on cell surface. Presently, we are conducting to confirm the findings utilizing patients' samples with JAK2-V617F. To test if a JAK inhibitor prevents PODXL-associated formation of NETs, we collected platelets from ruxolitinib-treated Jak2VF mice. Platelets from ruxolitinib-treated mice similarly expressed PODXL and induced formation of NETs with neutrophils from untreated Jak2VF mice, compared to platelets from untreated Jak2VF mice, suggesting that ruxolitinib does not alter platelets' ability to initiate formation of NETs. Next, we tested if these mechanisms induce venous and arterial thrombosis in vivo, utilizing models of half ligation of inferior vena cava (IVC) and photochemically induced thrombosis (PIT) in femoral artery. In both models, Jak2VF or Jak2V617F/+ mice formed thrombosis more quickly than WT controls. Jak2-mutated mice formed white thrombus presenting citH3-positive neutrophils aggregated with PODXL-expressing platelets at the core of the thrombus, while WT mice mainly formed red thrombus without any nucleated cells in these models. Therefore, we are generating platelet-specific knockout mice of PODXL to directly clarify if PODXL on Jak2VF-platelet is the initiator of NETs and thrombosis. In conclusion, PODXL on JAK2-V617F platelets may be a key player in the pathogenesis of thrombosis associated with formation of NETs with JAK2-V617F neutrophils in myeloproliferative neoplasms.

The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4.

The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study. Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy. The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbβ3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls. These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface.

Mitochondrial calcium uniporter b deletion inhibits platelet function and reduces susceptibility to arterial thrombosis

BackgroundMitochondrial calcium uniporter b (MCUb) is a negative regulator of the mitochondrial calcium uniporter (MCU) and is known to limit mitochondrial calcium ion (Ca2+) uptake. The role of MCUb in platelet function remains unclear. ObjectivesUtilizing MCUb−/− mice, we examined the role of MCUb in regulating platelet function and thrombosis. MethodsPlatelet activation was evaluated in agonist-induced standardized in vitro assays. Susceptibility to arterial thrombosis was evaluated in FeCl3 injury–induced carotid artery and laser injury–induced mesenteric artery thrombosis models. The glycolytic proton efflux rate and oxygen consumption rate were measured to evaluate aerobic glycolysis. ResultsUpon stimulation, MCUb−/− platelets exhibited reduced cytoplasmic Ca2+ responses concomitant with increased mitochondrial Ca2+ uptake. MCUb−/− platelets displayed reduced agonist-induced platelet aggregation and spreading on fibrinogen and decreased α and dense-granule secretion and clot retraction. MCUb−/− mice were less susceptible to arterial thrombosis in FeCl3 injury–induced carotid and laser injury–induced mesenteric thrombosis models with unaltered tail bleeding time. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with MCUb−/− platelets were less susceptible to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with wild type platelets, suggesting a platelet-specific role of MCUb in thrombosis. MCUb−/− stimulated platelets exhibited reduced glucose uptake, decreased glycolytic rate, and lowered pyruvate dehydrogenase phosphorylation, suggesting that mitochondrial Ca2+ mediates bioenergetic changes in platelets. ConclusionOur findings suggest that mitochondrial Ca2+ signaling and glucose oxidation are functionally linked in activated platelets and reveal a novel role of MCUb in platelet activation and arterial thrombosis.

Gelsolin Modulates Platelet Dense Granule Secretion and Hemostasis via the Actin Cytoskeleton

The mechanisms underlying platelet granule release are not fully understood. The actin cytoskeleton serves as the platelet's structural framework that is remodeled upon platelet activation. Gelsolin is a calcium-dependent protein that severs and caps existing actin filaments although its role in modulating platelet granule exocytosis is unknown. The hemostatic function of wild-type (WT) and gelsolin null (Gsn-/- ) mice was measured ex vivo by rotational thromboelastometry analysis of whole blood. Platelets were purified from WT and Gsn-/- mouse blood and activated with thrombin. Platelet aggregation was assessed by light-transmission aggregometry. Clot retraction was measured to assess outside-in integrin signaling. Adenosine triphosphate (ATP) release and surface P-selectin were measured as markers of dense- and α-granule secretion, respectively. The kinetics of agonist-induced aggregation, clot retraction, and ATP release were accelerated in Gsn-/- platelets relative to WT. However, levels of surface P-selectin were diminished in Gsn-/- platelets. ATP release was also accelerated in WT platelets pretreated with the actin-depolymerizing drug cytochalasin D, thus mimicking the kinetics observed in Gsn-/- platelets. Conversely, ATP release kinetics were normalized in Gsn-/- platelets treated with the actin polymerization agonist jasplakinolide. Rab27b and Munc13-4 are vesicle-priming proteins known to promote dense granule secretion. Co-immunoprecipitation indicates that the association between Rab27b and Munc13-4 is enhanced in Gsn-/- platelets. Gelsolin regulates the kinetics of hemostasis by modulating the platelet's actin cytoskeleton and the protein machinery of dense granule exocytosis.

Deletion of 12-Lipoxygenase Improves Platelet Function after Storage and Transfusion in Thrombocytopenic Mice

Introduction Human platelets are stored for up to 7 days for transfusion. During storage, platelets undergo numerous detrimental functional changes. Results from two recent randomized clinical trials challenge the hemostatic efficacy of stored platelets for transfusion. In a recent study in healthy humans, increased 12-HETE levels were associated with reduced platelet performance in vivo. In the current study, we sought to understand how genetic deletion of 12-LOX (the enzyme that generates 12-HETE) affects platelets during storage, before, and after transfusion in vitro and in mouse models. Methods and Results: We obtained platelets from wild-type (WT) and 12-LOX-/- mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) increased significantly in stored platelets from 12-LOX-/- mice, while oxylipins were significantly lower in 12-LOX-/- platelets than in WT platelets. The ω-6/ω-3 PUFA ratio decreased significantly in stored 12-LOX-/- compared to stored WT platelets, highlighting a pronounced increase in ω-3 PUFAs. The circulation time of stored 12-LOX-/- platelets did not differ from stored WT platelets. Baseline, pre-transfusion αIIbβ3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, once stored platelets were transfused, we observed significantly increased baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. To test whether this translates into better in vivo function, we exposed 24-hour stored platelets to an in vivo model of thrombosis and hemostasis. In brief, hIL4R-TG mice express the receptor for human interleukin 4 instead of GPIbα on the platelet surface. hIL4R-TG mice were rendered thrombocytopenic with an anti-human IL4R antibody. We transfused 24-hour stored WT, or 12-LOX-/- platelets, to thrombocytopenic hIL4R-TG mice, injured the carotid artery by FeCl3, and recorded the arterial blood flow. Indeed, stored 12-LOX-/- platelets had better in vivo function, as evidenced by more frequent and significantly faster vessel occlusions with stored and transfused 12-LOX-/- platelets than transfusion of stored WT platelets. Importantly, stored and transfused 12-LOX-/- occluded vessels in a similar time as normocytic WT mice. Untransfused, thrombocytopenic hIL4R-TG mice and untransfused, normocytic hIL4R-TG mice did not show vessel occlusion. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of ω-3 and ω-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.

Actin Bundling Protein L-Plastin Regulates Megakaryocyte Membrane Rigidity and Platelet Spreading

An adequate number of normally functioning platelets is required for normal hemostasis, while thrombocytopenia and platelet dysfunction are common causes of pathologic bleeding. Platelets are released into the blood from proplatelets (PPs) that extend from megakaryocyte (MK) intracellular invaginated membrane system (IMS) that is continuous with the plasma membrane. Tubulin provides the motor for PP extension, and actin reorganization is believed to be critical for MK migration and PP branching. However, the role for actin in PP formation (PPF) is less well understood. The initiation of PPF requires podosomes - actin-based structures with an F-actin core surrounded by a ring of adhesion molecules (e.g., integrins) and many actin-binding proteins. We have been intrigued by how podosomes form and how the IMS is able to penetrate the dense cortical actin cytoskeleton to form PPs. We recently showed that human MKs and platelets express a novel actin bundling protein, L-plastin. L-plastin levels were inversely correlated with platelet number, and its expression was down-regulated during MK maturation. L-plastin knockdown in cultured human CD34+ derived MKs caused increased PP, IMS, and podosome formation. These data indicate L-plastin is a negative regulator of MK platelet production. To assess the in vivo effects of L-plastin, we have investigated platelet production and function in vivo using L-plastin knockout (KO) mice (Lcp1-/-). Compared to wildtype (WT) littermate controls, Lcp1 KO mice showed both significantly increased platelet counts and increased PPF from cultured murine bone marrow MKs. Elegant work in both HUVECs and epithelial cells has shown high membrane proximal F-actin (MPA) prohibits membrane protrusions, and lowering MPA density is required to sensitize areas for membrane protrusion (Bisaria, Science 2020). We hypothesize that normal MK levels of L-plastin inhibits PPF in less mature MKs by F-actin bundling, which raises the MPA. Conversely, lower L-plastin levels result in lower MPA and enabling PP protrusion. Enhanced L-plastin-mediated F-actin bundling is expected to increase membrane stiffness, so we measured MK membrane stiffness at single cell level using a state-of-the-art Biomembrane Force Probe (BFP). MKs from KO mice showed 68% reduction in membrane stiffness as compared to WT controls (p<0.0001) (Fig 1). Ultrastructure of MKs also revealed increased IMS in Lcp1-/- MKs. Preliminary data suggests that L-plastin deficiency also associated with increased MK podosome formation in vitro, which is consistent with our findings in human CD34+ derived MKs. Although L-plastin levels decrease during terminal MK differentiation, they persist at moderate levels in platelets. Platelet integrin activation, granule release and clot retraction also depend on actin reorganization, but L-plastin function in platelets has not been studied. We observed no difference between WT and Lcp1 KO platelets for measures of inside-out integrin signaling (activation-induced JON/A binding or P-selectin expression). However, when plated on fibrinogen and stimulated with thrombin, platelets from KO mice showed significantly increased spreading compared to WT mice (Fig 2). A similar trend was observed for platelet clot retraction. Taken together these results show that L-plastin regulates MK membrane stiffness, which likely inhibits PPF. L-plastin also appears to participate in platelet outside-in, but not inside-out, integrin signaling. On-going studies are determining whether integrin beta3 is the molecular link between these two findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apoptosis in platelets is independent of the actin cytoskeleton.

Homeostasis between platelet production and clearance is essential for human health. A critical facet of the balance that facilitates platelet clearance from the circulation is apoptosis (programmed cell death). The precise cellular mechanisms that underpin platelet apoptosis are not defined. In nucleated cells, reorganization of the actin cytoskeleton is known to regulate platelet apoptosis. However, the role of the actin cytoskeleton in regulating apoptosis in platelets has not been extensively studied as they are anucleate and exhibit a distinctive physiology. Here, apoptosis was induced in washed human platelets using ABT-737, a BH3-mimetic drug. Mitochondrial depolarization was measured using the ratiometric dye JC-1; surface phosphatidylserine (PS) exposure was measured by annexin V binding; caspase-3 activation was measured by Western blotting. All three apoptotic markers were unaffected by the presence of either the actin depolymerizing drug cytochalasin D or the actin polymerizing drug jasplakinolide. Moreover, platelets were isolated from wild-type (WT) mice and mice deficient in gelsolin (Gsn), an actin-binding protein that is essential for normal cytoskeletal remodeling. In response to ABT-737, gelsolin-null (Gsn-/-) platelets initially showed accelerated PS exposure relative to WT platelets, however, both WT and Gsn-/- platelets exhibited similar levels of mitochondrial depolarization and caspase-3 activation in response to ABT-737. We conclude that ABT-737 induces established markers of platelet apoptosis in an actin-independent manner.

P-Selectin is a Critical Factor for Platelet-Mediated Protection on Restraint Stress-Induced Gastrointestinal Injury in Mice.

Psychological stress is associated with increased risk of gastrointestinal (GI) tract diseases. Evidence indicated that platelets facilitate GI tissue repair in intestinal anastomosis models. However, whether platelets are involved in native mechanism of the rescue of stress-induced GI injury for maintaining the GI homeostasis remains elusive. Because P-selectin-deficient (Selp−/−) mice displayed higher stress-induced GI injury compared to the wild-type (Selp+/+) mice, and P-selectin is specifically expressed in platelets, we hypothesize that P-selectin-expressing platelets play a protective role in the rescue of stress-induced GI injury. Our goal is to clarify the putative protective role of platelets in a GI system, thereby develop a feasible intervention strategy, such as platelet transfer, to overcome stress-induced GI injury. Through monitoring the plasma levels of GI-nonabsorbable Evans blue dye to reveal the progression course of GI injury in live mice, we found that intravenous treatments of purified platelets ameliorated stress-induced GI leakage. The transfer of platelets from wild-type mice was more potent than from Selp−/− mice in the rescue of stress-induced-GI leakage in the recipients. As such, platelet transfer-mediated rescue was conducted in a P-selectin dependent manner. Additionally, platelet-mediated protection is associated with corrections of stress-induced aberrant GI mRNA expressions, including tight junctions claudin 3 and occludin, as well as stress-induced genes activating transcription factor 3 and AMP-activated protein kinase, after the transfer of wild-type platelets into wild-type and Selp−/− mice. Furthermore, the stress-induced apoptosis of CD326+ GI epithelial cells was rescued by the transfer of wild type, but not P-selectin-deficient platelets. These results suggest that platelet plays a protective role for maintaining the GI homeostasis during stress in vivo, and that P-selectin is a molecular target for managing stress-induced GI tract injury.

Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation

AbstractProtein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22−/− mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide–treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.