Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

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

Blood ◽

10.1182/blood.v114.22.155.155 ◽

2009 ◽

Vol 114 (22) ◽

pp. 155-155

Author(s):

Meghna Ulhas Naik ◽

Timothy J. Stalker ◽

Lawrence F Brass ◽

Ulhas P Naik

Keyword(s):

Platelet Aggregation ◽

Adhesion Molecule ◽

Wild Type ◽

Clot Retraction ◽

Vessel Occlusion ◽

Platelet Surface ◽

Pulmonary Vessel ◽

Integrin Αiibβ3 ◽

Signaling Events

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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Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.2704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713 ◽

Cited By ~ 53

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Blood ◽

10.1182/blood.v104.11.326.326 ◽

2004 ◽

Vol 104 (11) ◽

pp. 326-326

Author(s):

Wolfgang Bergmeier ◽

Jill R. Crittenden ◽

Crystal L. Piffath ◽

Denisa D. Wagner ◽

David E. Housman ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Knockout Mice ◽

Thrombus Formation ◽

Es Cells ◽

Embryonic Stem ◽

Premature Stop Codon ◽

Small Gtpase ◽

Integrin Αiibβ3

Abstract Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.

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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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In Vivo Effects of Eltrombopag on Human Platelet Function.

Blood ◽

10.1182/blood.v110.11.1301.1301 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1301-1301 ◽

Cited By ~ 2

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

You Fu Li ◽

Marc R. Barnard ◽

...

Keyword(s):

Flow Cytometry ◽

Blood Flow ◽

Platelet Count ◽

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Adult Patients ◽

Platelet Surface ◽

Integrin Αiibβ3

Abstract Eltrombopag, an orally-administered small-molecule agonist of the thrombopoietin receptor (c-Mpl), is under investigation as a treatment for immune thrombocytopenic purpura (ITP). Studies have indicated that eltrombopag does not ‘prime’ platelets for activation in vitro, and eltrombopag administration to healthy volunteers does not increase platelet surface P-selectin or activated integrin αIIbβ3 (Jenkins J. Blood 2007). However, the effects of eltrombopag on platelet function in thrombocytopenic patients in vivo, either by direct binding to c-Mpl receptors on platelets or indirectly by altering the dynamics of platelet production and causing an influx of young, large platelets, is unknown. Whole blood flow cytometry, unlike other assays of platelet function, enables measurement of platelet function in the setting of marked thrombocytopenia (Michelson. Platelets, Elsevier, 2007). As a substudy of larger treatment studies, 17 adult patients with chronic ITP received eltrombopag at a starting dose of 50 mg daily, with the possibility of an increase to 75 mg daily after 3 weeks. Blood samples were drawn pre-treatment, and after 7 and 28 days of therapy. Platelet count, mean platelet volume (MPV), and the immature platelet fraction (IPF, or reticulated platelet count) were measured using a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μM thrombin receptor activating peptide (TRAP), or 20 μM TRAP. Bleeding was quantified by a comprehensive scale that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomical sites according to physical examination and/or history (Page, L.K. Br J Haematol 2007). Eleven of the 17 patients responded to eltrombopag with a rise in platelet count of >30 x 109/L. The IPF increased in responders but not non-responders (table 1). Response to eltrombopag was not predicted by pretreatment MPV or IPF. The ITP bleeding score decreased in responders over the study period in parallel with the increases in platelet count (table 1). As determined by platelet surface P-selectin and activated integrin αIIbβ3, eltrombopag did not result in platelet activation or augment ADP- or TRAP-induced platelet activation (table 2). In summary, eltrombopag increases the platelet count and reduces bleeding in responding adult patients with chronic ITP through the release of new platelets into the circulation. While bleeding is reduced in responders, eltrombopag does not result in platelet activation or augmentation of platelet activation by ADP or TRAP. This suggests that the newer platelets released by eltrombopag stimulation are not hyper-functional (or are only transiently so prior to day 7). Table 1 IPF (maximum absolute change, mean ± SEM x 109/L) Number in whom bleeding decreased Responders 57.0 ± 22.4 8/11 Non-responders 3.3 ± 1.5 1/6 Table 2 Study Day 0 7 28 MFI, mean fluorescence intensity, *P <0.05 compared with day 0 Activated αIIbβ3 MFI No agonist 11.4 11.3 9.2 Low ADP 180.3 159.4 98.4 High ADP 451.2 348.2* 251.8* Low TRAP 158.1 175.5 143.9 High TRAP 385.2 347.0 299.6 P-selectin MFI No agonist 5.5 6.6 6.2 Low ADP 48.6 43.4 38.8 High ADP 144.5 109.0 96.8 Low TRAP 113.8 114.9 107.8 High TRAP 457.3 396.3 330.9

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The GPIIbIIIa Antagonist Drugs Eptifibatide and Tirofiban Inhibit Caspase-3 Activation in Thrombin- and Calcium Ionophore-Stimulated Human Platelets.

Blood ◽

10.1182/blood.v114.22.2998.2998 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2998-2998

Author(s):

Valery Leytin ◽

Asuman Mutlu ◽

Sergiy Mykhaylov ◽

David J. Allen ◽

Armen V. Gyulkhandanyan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Calcium Ionophore ◽

Human Platelets ◽

Shear Stresses ◽

Ionophore A23187 ◽

Platelet Surface ◽

Apoptotic Effects

Abstract Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P<0.01, n=7). However, when platelets were preincubated with eptifibatide and tirofiban before agonist treatment, these drugs significantly inhibited agonist-induced caspase-3 activation by an average of 44-50% (P<0.05, n=7). The fact that eptifibatide and tirofiban do not promote caspase-3 activation in unstimulated platelets suggests that these GPIIbIIIa antagonists do not induce transmission of pro-apoptotic transmembrane signals inside platelets through inactive GPIIbIIIa integrin. The inhibitory effect of eptifibatide and tirofiban on thrombin- and A23187-induced caspase-3 activation suggests a role of GPIIbIIIa integrin in caspase-3 activation induced by these platelet agonists. Conclusions: We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

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Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood-2018-99-114418 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2418-2418

Author(s):

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Tannic Acid ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Platelet Surface ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation. To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets. To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation. To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. Disclosures No relevant conflicts of interest to declare.

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Protective mechanisms of adenosine 5′-monophosphate in platelet activation and thrombus formation

Thrombosis and Haemostasis ◽

10.1160/th13-05-0386 ◽

2014 ◽

Vol 111 (03) ◽

pp. 491-507 ◽

Cited By ~ 34

Author(s):

Eduardo Fuentes ◽

Lina Badimon ◽

Julio Caballero ◽

Teresa Padró ◽

Gemma Vilahur ◽

...

Keyword(s):

Platelet Activation ◽

Adenosine Receptor ◽

Thrombus Formation ◽

Antiplatelet Agent ◽

Binding Pocket ◽

In Vivo Model ◽

Platelet Surface ◽

Camp Levels

SummaryPlatelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5′-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

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An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

Journal of Clinical Medicine ◽

10.3390/jcm7110440 ◽

2018 ◽

Vol 7 (11) ◽

pp. 440 ◽

Cited By ~ 3

Author(s):

Wan Lu ◽

Chi Chung ◽

Ray Chen ◽

Li Huang ◽

Li Lien ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Phospholipase D ◽

Thrombus Formation ◽

Human Platelets ◽

Platelet Activity ◽

Clot Retraction ◽

First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

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