Platelet Junctional Adhesion Molecule-A Regulates Thrombosis by Negatively Regulating Outside-in Signaling through Integrin αIIbβ3.

Abstract Abstract 155 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out signaling that leads to the activation of integrin αIIbβ3. Upon ligand binding to integrin αIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How these signaling events are regulated is not well understood. We have previously identified JAM-A, a junctional adhesion molecule, on the platelet surface. Genetic ablation of Jam-A significantly (P<0.00001) shortened tail bleeding times (mean 54 seconds) compared to wild-type littermates (mean 98 seconds), suggesting an enhanced clot formation. FeCl3-induced carotid artery thrombosis and laser-induced cremaster arteriole thrombosis, two well recognized in vivo thrombosis model, both showed greatly shortened time of vessel occlusion and increased thrombus formation compared to wild-type (WT). Since JAM-A is expressed both on the endothelium and on platelets, the observed defect could arise from the lack of JAM-A in either cell type. To identify the involvement of platelet derived JAM-A, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs primarily through platelet thrombus without injury to the endothelium. We found that a significantly increased number of Jam-A−/− mice died within two minutes compared to WT mice. When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, we found a significantly greater extent of thromoembolism in Jam-A−/− mice compared to WT mice. Consistent with this finding, agonist-induced platelet aggregation, but not secretion, was significantly enhanced in Jam-A−/− platelets. Interestingly, however, the expression or activation of integrin αIIbβ;3 was not affected by Jam-A deficiency. When we analyzed the rate of clot retraction, we found that Jam-A−/− platelets showed 85% clot retraction within 90 minutes compared to only a 20% clot retraction in WT platelets, indicating that the absence of Jam-A significantly increases the rate of clot retraction (P<0.0001). JAM-A was found to associate with integrin αIIbβ3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation assay suggesting that JAM-A may suppress signaling through the integrin. To delineate the molecular mechanism influenced by JAM-A, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly enhanced in Jam-A−/− platelets. However, activation of focal adhesion kinase was unaffected in Jam-A−/− platelets compared to WT. Furthermore, outside-in signaling-induced phosphorylation of the myosin light chain was increased in Jam-A−/− platelets. These in vivo and in vitro results clearly show that JAM-A negatively regulates outside-in signaling through integrin αIIbβ;3, thus protecting from thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽

10.1182/blood.v112.11.111.111 ◽

2008 ◽

Vol 112 (11) ◽

pp. 111-111 ◽

Cited By ~ 1

Author(s):

Meghna Ulhas Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas Pandurang Naik

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Adhesion Molecule ◽

Resting State ◽

Human Platelets ◽

Artery Occlusion ◽

In Vivo Model ◽

Platelet Surface ◽

Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

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Junctional Adhesion Molecule-A Is a Novel Csk-Binding Protein Which Recruits Csk to the Integrin aIIbb3 and Suppresses Outside-In Signaling In Platelets

Blood ◽

10.1182/blood.v116.21.2120.2120 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2120-2120

Author(s):

Meghna Ulhas Naik ◽

Ulhas P Naik

Keyword(s):

Adhesion Molecule ◽

Conflicts Of Interest ◽

Human Platelets ◽

Clot Retraction ◽

Negative Regulators ◽

Vessel Occlusion ◽

Platelet Surface ◽

Occlusion Time ◽

Functional Studies ◽

Signaling Events

Abstract Abstract 2120 Platelet activation is regulated by both positive and negative regulators present within the platelets so that unwanted activation is suppressed, but, when needed, occurs rapidly. A significant amount of effort has been devoted towards understanding the positive regulators of platelet function. However, very little is known about the negative regulators. Dysregulation of the endogenous negative regulators may aid the thrombotic complications seen in various diseases. Upon ligand binding to integrin aIIbb3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How endogenous negative regulators suppress these events is not well understood. We have previously identified junctional adhesion molecule A (JAM-A), on the platelet surface. We found that Jam-a knockout mice show a prothrombotic phenotype as assessed by significantly (P<0.00001) shortened tail bleeding time, decreased carotid vessel occlusion time and increased pulmonary thromboembolism. Platelet functional studies revealed that Jam-a null platelets were hyperactive to physiological agonists. Surprisingly, inside-out signaling events were not affected in Jam-a null platelets. It is therefore possible that observed hyper-reactivity of Jam-a null platelets could be due to enhanced outside-in signaling. To test this, we performed a clot retraction assay. The clot retraction in wild type (Wt) occurred normally, which began after 1 h and about 50% was completed by 2 h. On the contrary, in Jam-a null platelets, the process of clot retraction was significantly enhanced (P<0.0001). It was initiated before 1h and was completed within 2 h. When analyzed for outside-in signaling events such as b3 tyrosine phosphorylation and c-Src phosphorylation, we found both significantly enhanced in Jam-a null platelets. To assess the mechanism by which JAM-A suppresses outside-in signaling, we analyzed the phosphorylation status of JAM-A. Interestingly, JAM-A was found to be phosphorylated on Y280 in unactivated platelets and rapidly dephosphorylated upon initiation of outside-in signaling. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated, suggesting that there is a dephosphorylation/phosphorylation switch that may be involved in regulating outside-in signaling. Furthermore, we found that JAM-A associates with integrin aIIbb3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation. We also found Csk, a C-terminal Src kinase, coimmunoprecipitating (IP) with JAM-A from resting, but not activated, platelet lysates, suggesting that JAM-A may be recruiting Csk to unactivated integrins and thus suppressing signaling. To test this, we analyzed association of Csk with integrin in Jam-a null platelets and found that in Wt platelets Csk was abundantly present in the integrin IP, but was completely absent in the integrin IP of the Jam-a null platelet lysates. These results clearly suggest that JAM-A recruits Csk to the integrin and thus suppresses outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

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CIB1 Regulates Thrombosis through Integrin αaIIbβ3 Outside-in Signaling, but Not Inside-out Signaling.

Blood ◽

10.1182/blood.v114.22.2992.2992 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2992-2992

Author(s):

Meghna Ulhas Naik ◽

Ulhas Naik

Keyword(s):

Platelet Aggregation ◽

Tyrosine Phosphorylation ◽

Conflicts Of Interest ◽

P38 Map Kinase ◽

In Vivo Model ◽

Clot Retraction ◽

Pulmonary Vessel ◽

Signaling Events ◽

Inside Out

Abstract Abstract 2992 Poster Board II-969 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out that leads to the activation of integrin αaIIbβ3. Upon ligand binding to integrin αaIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. The regulation of these signaling events is not well understood. Previously, we had identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with integrin αaIIbβ3. Using a novel technique to inhibit interaction of CIB1 with integrin αaIIbβ3 in intact platelets, we have shown that CIB1 regulates outside-in signaling through integrin αaIIbβ3. Recently, using Cib1-/- mice, we confirmed that CIB1 is a key regulator of hemostasis. FeCl3-induced carotid artery thrombosis, a well recognized in vivo model of thrombosis showed a significantly extended time of occlusion in Cib1-/- mice compared to wild type (WT) mice. Since CIB1 is expressed both in the endothelium and in platelets, the observed defect could arise from the lack of CIB1 in either cell types. To identify the specific involvement of platelets, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs due to platelet thrombus formation without injury to the endothelium. We found no difference in the number of Cib1-/- mice and WT mice that died within two minutes (n=20). When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, no difference between Cib1-/- and WT mice was observed. Consistent with this finding, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αaIIbβ3 was also not affected by Cib1 deficiency. When we analyzed the rate of clot retraction, we found that incomplete and significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates. To delineate the molecular mechanism regulated by CIB1, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain during outside-in signaling was blocked in Cib1-/- platelets. Interestingly, agonist-induced tyrosine phosphorylation of integrin β3 chain was unaffected in Cib1-/- platelets, but Mn2+-induced outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced. When analyzed for the candidate tyrosine kinase responsible for β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αaIIbβ3. Disclosures: No relevant conflicts of interest to declare.

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Integrin αIIb cytoplasmic Tail Is Essential for Integrin Outside-in Signaling and Regulation of in Vivo Thrombosis

Blood ◽

10.1182/blood.v124.21.4160.4160 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4160-4160

Author(s):

Meghna Ulhas Naik ◽

Ulhas P Naik

Keyword(s):

Platelet Aggregation ◽

Conflicts Of Interest ◽

Cytoplasmic Tail ◽

Cytoplasmic Domain ◽

P38 Map Kinase ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Signaling Events ◽

Inside Out

Abstract Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of events called inside-out signaling that lead to the activation of integrin αIIbβ3. Fibrinogen binding to the activated integrin relay signals termed as outside-in signaling that regulate thrombus growth and stability. Talin and kindlin bind to the integrin β3 cytoplasmic tail to induce inside-out signaling. Although, integrin αIIb cytoplasmic domain also bind to a number of proteins, its importance in hemostasis or thrombosis is not well understood. Previously, we have identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with the integrin αIIb cytoplasmic tail. Using a novel technique to inhibit interaction of CIB1 with integrin αIIb in intact human platelets, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Recently, using Cib1-/- mice, we showed that CIB1 is a key regulator of thrombosis in vivo. Interestingly, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αIIbβ3 was also not affected by Cib1 deficiency, suggesting that integrin inside-out signaling is not affected in Cib1-/- platelets. However, adhesion and spreading on immobilized fibrinogen (Fg) was severely affected in Cib1-/- platelets. When we analyzed the rate of clot retraction, we found that significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates, suggesting that integrin outside-in signaling is impaired in the absence of Cib1. To delineate the molecular mechanism regulated by CIB1 during platelet spreading and clot retraction, we analyzed the known signaling events activated during outside-in signaling. We found that Fg-dependent activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain was also blocked in Cib1-/- platelets adhered to immobilized Fg. Furthermore, outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced in Cib1-/- platelets. When analyzed for the candidate tyrosine kinase responsible for reduced β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. Furthermore, downstream signaling events such as activation of PAK1, PI3K, PDK1, as well as Akt were significantly affected in Cib1-/- platelets adhered to immobilized Fg. To test if impaired inhibition of GSK3-β is the cause of defective outside in signaling in Cib1-/- platelets we treated Cib1-/- platelets with SB216763, a specific GSK3-β inhibitor. We found that inhibition of GSK3-β rescued defective platelet adhesion and clot retraction observed in Cib1-/- platelets. It also rescued activation of p38 and Erk2 activation as well as MLC phosphorylation. However, activation of FAK, Src, PAK1, PI3K, PDK1, and Akt was not rescued, suggesting that these are upstream of GSK3-β. Furthermore, we found that outside-in dependent recruitment of FAK to the integrin-c-Src complex is greatly reduced in the absence of Cib1 suggesting that integrin αIIb cytoplasmic domain serves as a docking site for CIB1 so that it can recruit FAK to the integrin-c-Src complex and propagate outside-in signaling leading to GSK3-β inhibition, which is crucial for thrombus growth and stability. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αIIbβ3. Our results highlight an essential function to integrin αIIb cytoplasmic tail in regulating integrin outside-in signaling and thus thrombus growth and stability. Disclosures No relevant conflicts of interest to declare.

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TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽

10.1182/blood-2021-152975 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1050-1050

Author(s):

Angela Doerr ◽

Denise Pedrosa ◽

Maria Schander ◽

Yotis A. Senis ◽

Alexandra Mazharian ◽

...

Keyword(s):

Platelet Activation ◽

Type I Collagen ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Type I ◽

Wild Type ◽

Platelet Surface ◽

Knock Out ◽

Integrin Αiibβ3

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

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Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Blood ◽

10.1182/blood-2018-99-120196 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2423-2423

Author(s):

Yan Yang ◽

Xiaohong Ruby Xu ◽

Heyu Ni ◽

Liping Ma ◽

Wenhua Ling ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Granule Secretion ◽

Platelet Spreading ◽

Pka Pathway ◽

Inside Out

Abstract Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro. Methods: In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention. Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion. Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. Disclosures No relevant conflicts of interest to declare.

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Wdr1-Mediated Actin Reorganization Is Essential for Integrin αIIbβ3 Activation in Platelets

Blood ◽

10.1182/blood.v126.23.2231.2231 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2231-2231

Author(s):

Swapan Kumar Dasgupta ◽

Qi Da ◽

Anhquyen Le ◽

Miguel A. Cruz ◽

Perumal Thiagarajan

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Carotid Artery ◽

Bleeding Time ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Thrombosis Model ◽

Actin Turnover ◽

Deficient Mice

Abstract In resting platelets, the heterodimeric integrin αIIbβ3 is present in a low-affinity state. During platelet activation, the intracytoplasmic signals induce conformational changes that results in a swung-out conformation of the extracellular domain competent to bind ligands such as fibrinogen with high affinity to mediate platelet aggregation. Actin turnover is essential for this process and dynamic assembly and disassembly of actin filaments regulate it. We have identified Wdr1, a cofilin and actin binding protein containing WD40 repeats, as an essential component of the machinery that orchestrates actin fiber reorganization that leads to integrin αIIbβ3 activation. Methods: Wdr1-deficient mouse strain, Wdr1rd/rd. was obtained through an N-ethyl-N-nitrosourea mutagenesis screen in Baylor College of Medicine. The mutant mouse has a T>A transversion in the second dinucleotide of the intron 9 splice donor site and it produces a mutant transcript containing a 6-bp in-frame deletion that results in a incorrectly folded, nonfunctional protein. Normal splicing produces a small amount of Wdr1 protein (~2%) resulting in a hypomorphic allele. Wdr1-deficient mice are moderately thrombocytopenic (85± 11 x 106 ml Wdr1 deficient versus 427± 52 x 106/ml for wild-type). Platelets were isolated from Wdr1-deficient and control mice. Platelet aggregation was carried out by standard turbidometric methods. Calcium mobilization was measured by incubating Wdr1-deficient and WT (wild-type) platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. Conformational change in αIIbβ3 was determined by flow cytometry with a conformation-specific anti-αIIbβ3 antibody JON/A. In vivo hemostasis was assessed by tail bleeding time and FeCl3-induced endothelial carotid injury/thrombosis model was used to assess the occlusion in carotid artery of mice. Results: Aggregation response of Wdr1-deficient platelets to different doses of collagen was significantly impaired compared to WT platelets. Under similar conditions, the calcium response was similar to the WT. In a parallel-plate flow chamber assay, WT platelets stably adhered to collagen surface and formed stable thrombus. On the other hand, significantly less number of Wdr1 deficient platelets were stably attach to the collagen surface and it did not form stable thrombus. As expected the tail bleeding time of Wdr1 deficient mice is significantly prolonged (> 10 minutes) compared to WT mice (<2 min). In vivo, in FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1 deficient was prolonged significantly compared wild type mice (15.8 ± 12.6 minutes versus 9.0 ± 10.5 minutes (p=0.041, Mann-Whitney non parametric comparison). To examine directly the activation of αIIbβ3, we used JON/A antibody, which selectively binds to activated αIIbβ3 integrins on mouse platelets. Binding of collagen treated Wdr1-deficient platelets to JON/A as determined by flow cytometry, is significantly less compared to WT platelets (6.1±0.3 fluorescence units (FU) versus 17.4±0.6 FU, p≤0.05) indicating impaired inside-out activation of αIIbβ3. Since, Wdr1 promotes actin disassembly, which is essential for the rearrangement of the actin fibers that occurs during platelet activation, we measured actin turn over by measuring F-actin and G-actin ratios of collagen treated platelets at various time points. Actin turnover is highly impaired in Wdr1 deficient platelets compared to WT platelets. Furthermore, integrin αIIbβ3 association with actin cytoskeleton was markedly impaired in Wdr1 deficient mice compared to their WT controls. These studies show that Wdr1 mediated actin cytoskeleton reorganization is essential for integrin αIIbβ3 activation. Disclosures No relevant conflicts of interest to declare.

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Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo

Blood ◽

10.1182/blood-2005-09-3687 ◽

2006 ◽

Vol 107 (10) ◽

pp. 3902-3906 ◽

Cited By ~ 158

Author(s):

Christophe Dubois ◽

Laurence Panicot-Dubois ◽

Glenn Merrill-Skoloff ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Severe Injury ◽

Wild Type ◽

Vessel Occlusion ◽

Platelet Surface ◽

Glycoprotein Vi ◽

Laser Injury ◽

Platelet Accumulation

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRγ-null mice (FcRγ–/–) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl3 injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl3 injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRγ–/– compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRγ–/–. These data indicate a significant role for GPVI in FeCl3-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl3-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRγ–/– was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl3-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/7071987 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Muhammad Irfan ◽

Tae-Hyung Kwon ◽

Dong-Ha Lee ◽

Seung-Bok Hong ◽

Jae-Wook Oh ◽

...

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Thrombus Formation ◽

Calcium Mobilization ◽

Potential Candidate ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Atp Secretion ◽

Antithrombotic Effects ◽

Epimedium Koreanum Nakai

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin αIIbβ3 activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin αIIbβ3-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin αIIbβ3 activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

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