Platelet Granule Secretion Research Articles

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

Angiopoietin-like protein 2 inhibits thrombus formation.

Acute myocardial infarction is mainly caused by a lack of blood flood in the coronary artery. Angiopoietin-like protein 2 (ANGPTL2) induces platelet activation and thrombus formation in vitro through binding with immunoglobulin-like receptor B, an immunoglobulin superfamily receptor. However, the mechanism by which it regulates platelet function in vivo remains unclear. In this study, we investigated the role of ANGPTL2 during thrombosis in relationship with ST-segment elevation myocardial infarction (STEMI) with spontaneous recanalization (SR). In a cohort of 276 male and female patients, we measured plasma ANGPTL2 protein levels. Using male Angptl2-knockout and wild-type mice, we examined the inhibitory effect of Angptl2 on thrombosis and platelet activation both in vivo and ex vivo. We found that plasma and platelet ANGPTL2 levels were elevated in patients with STEMI with SR compared to those in non-SR (NSR) patients, and was an independent predictor of SR. Angptl2 deficiency accelerated mesenteric artery thrombosis induced by FeCl3 in Angptl2-/- compared to WT animals, promoted platelet granule secretion and aggregation induced by thrombin and collogen while purified ANGPTL2 protein supplementation reversed collagen-induced platelet aggregation. Angptl2 deficiency also increased platelet spreading on immobilized fibrinogen and clot contraction. In collagen-stimulated Angptl2-/- platelets, Src homology region 2 domain-containing phosphatase (Shp)1-Y564 and Shp2-Y580 phosphorylation were attenuated while Src, Syk, and Phospholipase Cγ2 (PLCγ2) phosphorylation increased. Our results demonstrate that ANGPTL2 negatively regulated thrombus formation by activating ITIM which can suppress ITAM signaling pathway. This new knowledge provides a new perspective for designing future antiplatelet aggregation therapies.

Zixue Powder attenuates septic thrombosis via reducing neutrophil extracellular trap through blocking platelet STING activation

Ethnopharmacological relevanceMicrothrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear. Aim of the studyInvestigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis. Materials and methodsZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments. ResultsUPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation. ConclusionOur study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.

Abstract 154: Hierarchical Redundancy And Contextual Roles Of Vesicle-associated Membrane Proteins (VAMPs) In Platelet Function

Background: Platelet granule secretion, driven by the SNARE complex involving Vesicle-Associated Membrane Proteins (VAMPs) and t-SNAREs, is essential for vascular integrity. We dissected individual and combinatorial contributions of all four platelet VAMPs to granule secretion, hemostasis, and intracellular cargo trafficking. Research Questions: 1. Are specific VAMPs critical for granule fusion, or does total VAMP quantity (mass action) dictate secretion? 2. How do differential VAMP deficiencies impact hemostasis in arterial and venous microenvironments? 3. Do VAMPs influence platelet cargo loading and gene expression? Goals: Investigate individual and combinatorial roles of VAMPs in platelet biology and their context dependent contributions to hemostasis. Approach: We generated V7 -/- 8 -/- and V2 Δ 3 Δ 7 -/- 8 -/- mice, assessing granule secretion (dense, alpha, lysosomal) via ATP, PF4/P-selectin, and β-hexosaminidase/LAMP1 release assays, respectively. Hemostasis in arterial and venous models was evaluated with tail bleeding, FeCl3, and venous puncture injury assays. Cargo levels were analyzed using proteomic arrays, and platelet RNA sequencing identified transcriptional changes. Results: Global V7/V8 deletion modestly reduced secretion compared to isolated V8 deficiency. Surprisingly, while platelet-specific V2 Δ 3 Δ 7 -/- 8 -/- exhibited the most dramatic decrease, complete abolition was absent (p<0.01). Alpha and lysosomal secretion were more affected than densegranule release. Both knockout models bled profusely and failed to form stable arterial thrombi (p<0.01). Notably, only V2 Δ 3 Δ 7 -/- 8 -/- mice displayed significantly prolonged venous bleeding (p<0.01). RNA sequencing revealed >800 upregulated and >500 downregulated transcripts in V7 - /- 8 -/- and V2 Δ 3 Δ 8 -/- platelets, suggesting shared transcriptional control by V7 and secondary VAMPs (V2 and V3). Conclusions: VAMP functions exhibit hierarchical redundancy, with V8 presence minimally impacting dense granule secretion and arterial hemostasis. Alpha granule release is more sensitive to VAMP loss. The requirements for platelet granule secretion for hemostasis are context dependent. The deletion of VAMPs disrupts platelet cargo trafficking and transcription.

Abstract 3094: Traditional Chinese Medicine Buyang Huanwu Decoction Regulates The Platelet Gpvi And Its Downstream Protein Signaling Pathways,inhibiting Thrombosis And Preventing Cardiovascular Disease.

Background: Coronary heart disease Qi-Deficiency and Blood Stasis Syndrome will lead to the body of platelets disorders, blood viscosity, conducive to the formation of thrombosis, Buyang Huanwu Decoction (BYHWD) is a classic Chinese medicine treatment of Qi-Deficiency and Blood Stasis Syndrome. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets. GPVI has become the focus of new approaches to antithrombotic therapy. Methods: After 6 weeks of irregular sleep deprivation in rats with syndrome of deficiency of qi and blood stasis formed, the coronary heart disease model was established by ligation of the left anterior descending coronary artery in rats. The dosage of 1 mL/100 g was given by intragastric administration for 14 days after operation. Results: The purpose of this study was to investigate the effects of BYHWD on platelet granule secretion and platelet function in vitro. We found that BYHWD can significantly reduce the platelet aggregation induced by ADP and collagen, significantly reduce the median expression of CD62p and the ratio of activated platelets, and reduce the concentration of fluorescent calcium ions in platelets. Under the electron microscope observation found that under the action of BYHWD, the ultrastructure of rat platelets pseudopodia reduced, aggregation reduced, rat coronary inner wall platelets, thrombus mass adhesion reduced, and the inner wall was smoother. In addition, BYHWD can regulate GPVI and its downstream proteins. Compared with the coronary heart disease group with syndrome of blood stasis, BYHWD can significantly reduce the expression of GPVI. Significantly reduced the expression of Syk, CALDAG-GEFI, significantly reduced the expression of IP 3 and significantly reduced the degree of PLCγ 2 of rats. Decreased the degranulation reaction of the rat blood, significantly decreased the level of TXA 2 , 5-HT in the rat blood, and significantly decreased the content of the rat blood COX-1 in the rat serum. Conclusion: Therefore, BYHWD can regulate GPVI and its downstream protein signaling pathways, reduce platelet over-activation, degranulation reaction, thereby inhibiting thrombosis, protecting heart function and preventing cardiovascular disease.

Abstract 139: Contribution Of Platelet Granule Cargo Uptake And Secretion To The Initiation And Progression Of Aortic Aneurysms

Aortic aneurysms present as aortic widening and often catastrophic rupture. While precipitated by a thrombo-inflammatory environment, underlying pathological mechanisms are unclear and adequate treatment is lacking. Antithrombotics have been implied as therapy, but few studies have defined how platelets contribute to aortic aneurysms. We studied the role of platelet granule cargo uptake and secretion in aortic aneurysm formation. Aneurysms were induced in mice defective in platelet α-granule biogenesis (Nbeal2 -/- , Serglycin -/- ), endocytosis (Arf6 -/- , VAMP2/3 Δ ), and exocytosis (Munc13-4 Jinx ) via: i.p. injection of an AAV vector with a mouse PCSK9 gain-of-function mutation; western diet; and continuous s.c. infusion of angiotensin II. Thoracic and abdominal aneurysm formation was assessed with ultrasound. At 4 and 12 weeks, or after rupture, aortas were harvested, analyzed, and prepared for histology. During the study, 30% (3/10) of Nbeal2 -/- and 21.4% (3/18) of Munc13-4 Jinx mice suffered aortic rupture, compared to 0% (0/26) of wildtype mice. In the survivors, the inner diameter of neither the thoracic nor abdominal aorta of Nbeal2 -/- mice changed over time. Ex vivo , Serglycin -/- mice showed a clear, more distributed, widening of the thoracic and descending aorta instead of local aneurysm formation with an increased maximum outer diameter of the ascending region. Munc13-4 Jinx mice had an all-or-nothing phenotype; either early-stage massive aneurysm formation or rupture, or a more diffused phenotype. Mice with platelet endocytosis defects survived and at early stages, the differential increase in the inner diameter of both the thoracic and abdominal aorta was higher in female Arf6 -/- mice. Similarly, VAMP2/3 Δ mice had the largest inner thoracic diameter increase after 28 days. Arf6 -/- and VAMP2/3 Δ mice showed a smaller maximum outer diameter of the descending aorta as wildtype. Preliminary histology shows reduced elastin breaks in all genetic strains, especially in Nbeal2 -/- and VAMP2/3 Δ mice. Our data show that platelet granule cargo exerts significant but perhaps contrasting effects on aortic aneurysm formation. By comparing phenotypes, more detailed mechanistic insights into the role of platelets in aortic aneurysms are possible.

Abstract 2098: A Novel Mechanism Of Focal Adhesion Kinase Activation By Integrins And Its Role In Outside-in Signaling In Platelets

Ligand binding to integrin αIIbβ3 mediates platelet adhesion/aggregation and transmits outside-in signaling, leading to platelet spreading, granule secretion, and significant expansion of platelet thrombi resulting in thrombosis. Focal Adhesion Kinase (FAK) has been recognized in many cells to play an important role during integrin outside-in signaling. Whereas FAK activation downstream of integrins is characterized by autophosphorylation of FAK at Y 397 followed by Y 576/7 phosphorylation, it is not clear how FAK phosphorylation and activation is regulated and how FAK play roles in outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) plays a key role in integrin-dependent stimulation of FAK phosphorylation and activation and ROS-stimulated FAK activation is downstream of the Gα13-integrin interaction, NOX2 activation, and the Src signaling pathway. Furthermore, FAK, following ROS-dependent activation, is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We further show that FAK is important in amplification of platelet activation and aggregation as well as in vivo thrombosis. Thus, we have identified a novel mechanism of FAK activation and a key role of FAK in integrin outside-in signaling and integrin-dependent ITAM pathway activation, leading to amplification of platelet activation, aggregation, and thrombosis.

Ayurvedic preparations of Raudra Rasa inhibit agonist-mediated platelet activation and restrict thrombogenicity without affecting cell viability.

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3β. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.

Clinical, laboratory and ultrastructural findings in patients with storage pool disease: A case series

IntroductionPlatelet storage pool diseases (SPD) are a heterogeneous group of bleeding disorders associated with defects in the storage, secretion, or release of platelet granules. Patients with SPD present with a life-long mucocutaneous bleeding diathesis. Our goal was to study the clinical, laboratory, and ultrastructural changes in platelets of patients diagnosed with SPD using a transmission electron microscope (TEM). MethodsIn this retrospective, cross-sectional cohort study, medical records of all patients referred for evaluation of a platelet function disorder were screened for an underlying diagnosis of SPD during the period 2010–2020. ResultsSixty-eight patients were identified, among whom 62 (91.2%) had a platelet function assay (PFA) study, of whom 21 (33.9%) were abnormal. Clinical, laboratory, and light transmission aggregometry (LTA) suggested that 10 (14.7%) patients had SPD; five had gray platelet syndrome (GPS), three had Hermansky Pudlak syndrome (HPS), and two had Chedia-Higashi syndrome (CHS). Most of these cases presented with mucocutaneous bleeding diathesis, but a few had oculocutaneous albinism. They were associated with variable abnormalities in the PFA and LTA studies. However, EM studies using TEM showed a reduction/absence of alpha or delta granules in GPS and HPS patients, respectively, but no abnormality in the granules of CHS patients. ConclusionsAlthough patients with SPD presented with bleeding diathesis, PFA and platelet aggregation studies were inconclusive. Abnormalities in platelet ultrastructure on EM studies demonstrated corroborative evidence for SPD with absent/reduced alpha or delta granules.

STING activation in platelets aggravates septic thrombosis by enhancing platelet activation and granule secretion

Sepsis is a dysregulated inflammatory consequence of systemic infection. As a result, excessive platelet activation leads to thrombosis and coagulopathy, but we currently lack sufficient understanding of these processes. Here, using the cecal ligation and puncture (CLP) model of sepsis, we observed septic thrombosis and neutrophil extracellular trap formation (NETosis) within the mouse vasculature by intravital microscopy. STING activation in platelets was a critical driver of sepsis-induced pathology. Platelet-specific STING deficiency suppressed platelet activation and granule secretion, which alleviated sepsis-induced intravascular thrombosis and NETosis in mice. Mechanistically, sepsis-derived cGAMP promoted the binding of STING to STXBP2, the assembly of SNARE complex, granule secretion, and subsequent septic thrombosis, which probably depended on the palmitoylation of STING. We generated a peptide, C-ST5, to block STING binding to STXBP2. Septic mice treated with C-ST5 showed reduced thrombosis. Overall, platelet activation via STING reveals a potential strategy for limiting life-threatening sepsis-mediated coagulopathy.

Sulforaphane attenuates platelet granule secretion through down-regulating glycoprotein VI-mediated p38 MAPK/cPLA2 signaling pathway

ABSTRACT Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and is important in mediating granule secretion and atherothrombosis. Sulforaphane (SFN), a dietary isothiocyanate enriched in cruciferous vegetables, exerts multiple biological activities. This study aimed to investigate the efficacy of SFN on GPVI-mediated signaling and platelet granule secretion in vitro. We herein demonstrated that SFN greatly attenuated granule secretion in human gel-filtered platelets in response to collagen, including inhibiting CD62P and CD63 expression and PF4, CCL5, and ATP release. Mechanistically, these inhibitory effects of SFN were mainly mediated by its attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation and thromboxane A2 (TxA2) generation. Moreover, SFN did not further decrease the inhibitory effects of p38 MAPK inhibitor SB203580 on cPLA2 phosphorylation, TxA2 generation and granule secretion. Thus, SFN attenuates platelet granule secretion via down-regulating GPVI-mediated p38 MAPK/cPLA2/TxA2 signaling, which may play important preventive roles in cardiovascular diseases.

O-fucosylation of thrombospondin type I repeats is dispensable for trafficking thrombospondin 1 to platelet secretory granules.

Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by β3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.

The relative importance of platelet integrins in hemostasis, thrombosis and beyond.

Integrins are heterodimeric transmembrane receptors composed of α and β chains, with an N-terminal extracellular domain forming a globular head corresponding to the ligand binding site. Integrins regulate various cellular functions including adhesion, migration, proliferation, spreading and apoptosis. On platelets, integrins play a central role in adhesion and aggregation on subendothelial matrix proteins of the vascular wall, thereby ensuring hemostasis. Platelet integrins belong either to the β1 family (α2β1, α5β1 and α6β1) or to the β3 family (αIIbβ3 and αvβ3). On resting platelets, integrins can engage their ligands when the latter are immobilized but not in their soluble form. The effects of various agonists promote an inside-out signal in platelets, increasing the affinity of integrins for their ligands and conveying a modest signal reinforcing platelet activation, called outside-in signaling. This outside-in signal ensures platelet adhesion, shape change, granule secretion and aggregation. In this review, we examine the role of each platelet integrin in hemostatic plug formation, hemostasis and arterial thrombosis and also beyond these classical functions, notably in tumor metastasis and sepsis.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting β-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of β-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb β3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, β-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

A novel association between platelet filamin A and soluble N-ethylmaleimide sensitive factor attachment proteins regulates granule secretion

Background and ObjectiveThe molecular mechanisms that underpin platelet granule secretion remain poorly defined. Filamin A (FLNA) is an actin-crosslinking and signaling scaffold protein whose role in granule exocytosis has not been explored despite evidence that FLNA gene mutations confer platelet defects in humans. Methods and ResultsUsing platelets from platelet-specific conditional Flna-knockout mice, we showed that the loss of FLNA confers a severe defect in alpha (α)- and dense (δ)-granule exocytosis, as measured based on the release of platelet factor 4 (aka CXCL4) and adenosine triphosphate (ATP), respectively. This defect was observed following activation of both immunoreceptor tyrosine-based activation motif (ITAM) signaling by collagen-related peptide (CRP) and G protein–coupled receptor (GPCR) signaling by thrombin and the thromboxane mimetic U46619. CRP–induced spikes in intracellular calcium [Ca2+]i were impaired in FLNA-null platelets relative to controls, confirming that FLNA regulates ITAM-driven proximal signaling. In contrast, GPCR-mediated spikes in [Ca2+]i in response to thrombin and U46619 were unaffected by FLNA. Normal platelet secretion requires complexing of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptosomal-associated protein 23 (SNAP23) and syntaxin-11 (STX11). We determined that FLNA coimmunoprecipitates with both SNAP23 and STX11 upon platelet stimulation. ConclusionFLNA regulates GPCR-driven platelet granule secretion and associates with SNAP23 and STX11 in an activation-dependent manner.

Abstract 10885: Integrin β 3 Directly Inhibits Galpha 13 -p115rhogef Interaction and Its Role in Regulating Platelet Exocytosis

G protein-coupled receptors (GPCRs) and adhesion receptor integrins are fundamental to a cell’s response to extracellular stimuli. GPCRs, through Gα q and Gα i , activate integrins and, through the Gα 13 -p115RhoGEF-RhoA pathway, stimulate contractility and integrin-independent exocytosis (granule secretion). Gα 13 also interacts with integrins and mediates outside-in signaling, cell spreading and integrin-dependent exocytosis. However, it was unknown whether there was crosstalk between integrins and the Gα 13 /p115RhoGEF-mediated RhoA signaling pathway. It was also unclear with the role of Gα 13 -integrin interaction in regulating GPCR- and integrin- mediated platelet exocytosis. In this study, we demonstrate that binding of the integrin β 3 cytoplasmic domain to Gα 13 through its ExE motif, directly disrupts the interaction between p115RhoGEF and Gα 13 , thus inhibiting Gα 13 -p115RhoGEF-mediated RhoA activation and signaling. In platelets, this inhibitory effect of integrins greatly reduces GPCR-mediated integrin-independent granule secretion. On the other hand, the Gα 13 -integrin interaction stimulates integrin outside-in signaling and integrin-dependent granule secretion. Furthermore, we demonstrate that the inhibitor peptide derived from the Gα 13 -binding β 3 ExE motif disrupts the β 3 -Gα 13 interaction to inhibit GPCR-induced integrin-independent granule secretion and also inhibits outside-in signaling and integrin-dependent granule secretion. This dual effect results in robust inhibition of granule secretion without affecting primary platelet aggregation, suggesting a new therapeutic approach for the development of selective inhibitors of platelet granule secretion. In conclusion, we have discovered a mechanism underlying direct integrin-GPCR crosstalk that regulates the Gα 13 -p115RhoGEF-RhoA signaling pathway. Our data also demonstrate an approach to potently inhibit platelet granule secretion without impairing primary platelet adhesion/aggregation and hemostasis.

Notch signaling functions in noncanonical juxtacrine manner in platelets to amplify thrombogenicity.

Notch signaling dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets remains elusive. Transcripts/peptides of Notch1 and Delta-like ligand (DLL)-4 were detected in platelets isolated from human blood by RT-qPCR, Western analysis and flow cytometry. Platelet aggregation, granule secretion and platelet-leukocyte interaction were analyzed by lumi-aggregometry and flow cytometry. Platelet-derived extracellular vesicles were documented with Nanoparticle Tracking Analyzer. Platelet thrombus on immobilized collagen was quantified using microfluidics platform. Intracellular calcium was monitored by fluorescence spectrophotometry. Whole blood coagulation was studied by thromboelastography. Ferric chloride-induced mouse mesenteric arteriolar thrombosis was imaged by intravital microscopy. We demonstrate expression of Notch1, its ligand DLL-4 and their respective transcripts in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated by thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype through cleavage of Notch receptor and release of its intracellular domain (NICD), which was averted by inhibition of γ-secretase and phosphatidylinositol-3-kinase (PI3K). Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin. Our study presents compelling evidence in support of non-canonical juxtacrine Notch signaling within platelet aggregates that synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, Notch pathway can be a potential anti-platelet/anti-thrombotic therapeutic target. Research was supported by grants received by DD from JC Bose Fellowship (JCB/2017/000029), ICMR (71/4/2018-BMS/CAR), DBT (BT/PR-20645/BRB/10/1541/2016) and SERB (EMR/2015/000583). SNC, ME and VS are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively. Funders had no role in design, analysis and reporting of study.

Inhibition of Hypoxia-Inducible Factor Prolyl-Hydroxylase Modulates Platelet Function.

Hypoxia-inducible factor-1α (HIF-1α) involves in redox reactions. Considering the role of reactive oxygen species (ROS) in platelet function, whether it regulates platelet function remains unclear. Using an inhibitor of HIF prolyl-hydroxylase, IOX-2, we intend to investigate its effect on platelet function. Human platelets were treated with IOX-2 (0, 10, 25, and 50 μM) followed by analysis of platelet aggregation, granule secretion, receptor expression, platelet spreading, or clot retraction. Additionally, IOX-2 (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time and arterial thrombosis. IOX-2 significantly inhibited collagen-related peptide (CRP; 0.25 μg/mL) or thrombin (0.03 U/mL)-induced platelet aggregation and ATP release dose dependently without affecting P-selectin expression and the surface levels of glycoprotein (GP)Ibα, GPVI, or αIIbβ3. In addition, IOX-2-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction. Moreover, IOX-2 administration into mice significantly impaired the in vivo hemostatic function of platelets and arterial thrombus formation without affecting the number of circulating platelets and coagulation factors (FVIII and FIX). Further, IOX-2 significantly upregulated HIF-1α in platelets, decreased ROS generation, and downregulated NOX1 expression. Finally, IOX-2 increased the phosphorylation level of VASP (Ser157/239), and inhibited the phosphorylation of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), AKT (Thr308/Ser473), and PKCδ (Thr505) in CRP- or thrombin-stimulated platelets. In conclusion, inhibition of HIF prolyl-hydroxylase modulates platelet function and arterial thrombus formation, possibly through upregulation of HIF-1α expression and subsequent inhibition of ROS generation, indicating that HIF-1α might be a novel target for the treatment of thrombotic disorders.

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.

Human milk oligosaccharide, 2′-Fucosyllactose, attenuates platelet activation in arterial thrombosis

A schematic model of GPVI-mediated platelet activation pathway inhibited by 2′-Fucosyllactose (2′-FL). 2′-FL inhibits multiple aspects of the GPVI activation pathway, including Syk signalosome, granule secretion, calcium mobilization, integrin αIIbβ3 activation and aggregation. GPVI, glycoprotein VI; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; Syk, spleen tyrosine kinase. • 2′-FL effectively reduces the platelet thrombus formation in vitro. • Oral administration of 2′-FL has immediate benefits in thrombus formation and a mouse model of ischemia/reperfusion-induced stroke in vivo. The ingestion of supplemental human milk oligosaccharides (HMOs) is associated with a lower risk of inflammatory diseases and infections. However, whether HMOs play an important role in regulating platelet aggregation and thrombus formation remains unclear. The current study aimed to investigate the antiplatelet and antithrombotic effects of HMOs. Among all HMOs, 2′-fucosyllactose (2′-FL) could significantly inhibit collagen-related peptide-induced platelet aggregation, granule secretion, and intracellular calcium mobilization. Notably, the inhibitory effect of 2′-FL was associated with the inhibition of glycoprotein VI-mediated platelet signal transduction during cell activation. In accordance with antiplatelet activities, the oral administration of 2′-FL was effective in blocking ferric chloride-induced arterial thrombus formation in vivo without prolonging bleeding time. Studies using a mouse model of ischemia-/reperfusion-induced stroke revealed that treatment with 2′-FL reduced infarct volume in stroke. Hence, the therapeutic potential of 2′-FL supplementation may play a protective role against thrombotic diseases.