Potentiate Platelet Activation Research Articles

AGK (Acylglycerol Kinase) Potentiates Arterial Thrombosis by Affecting Talin-1 and αIIbβ3-Mediated Bidirectional Signaling Pathway.

AGK (acylglycerol kinase) was first identified as a mitochondrial transmembrane protein that exhibits a lipid kinase function. Recent studies have established that AGK promotes cancer growth and metastasis, enhances glycolytic metabolism and function fitness of CD8+ T cells, or regulates megakaryocyte differentiation. However, the role of AGK in platelet activation and arterial thrombosis remains to be elaborated. We performed hematologic analysis using automated hematology analyzer and investigated platelets morphology by transmission electron microscope. We explored the role of AGK in platelet activation and arterial thrombosis utilizing transgenic mice, platelet functional experiments in vitro, and thrombosis models in vivo. We revealed the regulation effect of AGK on Talin-1 by coimmunoprecipitation, mass spectrometry, immunofluorescence, and Western blot. We tested the role of AGK on lipid synthesis of phosphatidic acid/lysophosphatidic acid and thrombin generation by specific Elisa kits. In this study, we found that AGK depletion or AGK mutation had no effect on the platelet average volumes, the platelet microstructures, or the expression levels of the major platelet membrane receptors. However, AGK deficiency or AGK mutation conspicuously decreased multiple aspects of platelet activation, including agonists-induced platelet aggregation, granules secretion, JON/A binding, spreading on Fg (fibrinogen), and clot retraction. AGK deficiency or AGK mutation also obviously delayed arterial thrombus formation but had no effect on tail bleeding time and platelet procoagulant function. Mechanistic investigation revealed that AGK may promote Talin-1Ser425 phosphorylation and affect the αIIbβ3-mediated bidirectional signaling pathway. However, AGK does not affect lipid synthesis of lysophosphatidic acid/lysophosphatidic acid in platelets. AGK, through its kinase activity, potentiates platelet activation and arterial thrombosis by promoting Talin-1 Ser425 phosphorylation and affecting the αIIbβ3-mediated bidirectional signaling pathway.

Severe fever with thrombocytopenia syndrome virus replicates in platelets and enhances platelet activation

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) infection causes an emerging hemorrhagic fever in East Asia with a high mortality rate. Thrombocytopenia is a consistent feature of SFTS illness, but the mechanism remains elusive. ObjectivesWe aimed to better understand the role of platelets in the pathophysiology of SFTSV infection, including the development of thrombocytopenia. MethodsUsing platelets from healthy volunteers and patients with SFTS, we evaluated the functional changes in platelets against SFTSV infection. We investigated the direct effect of glycoprotein VI on platelet-SFTSV interaction by quantitative real-time PCR, molecular docking, surface plasmon resonance spectrometry, flow cytometry, western blot, and platelet functional studies in vitro. Interactions of SFTSV and platelet-SFTSV complexes with macrophages were also determined by scanning electron microscope, quantitative real-time PCR, and flow cytometry. ResultsThis study is the first to demonstrate that platelets are capable of harboring and producing SFTSV particles. Structural and functional studies found that SFTSVs bind platelet glycoprotein VI to potentiate platelet activation, including platelet aggregation, adenosine triphosphate release, spreading, clot retraction, coagulation, phosphatidylserine exposure, thrombus formation, and adherence. In vitro mechanistic studies highlighted that the interaction of platelets with human THP-1 cells promoted SFTSV clearance and suppressed cytokine production in macrophages. However, unwanted SFTSV replication in macrophages reciprocally aggravated SFTSV persistence in the circulation, which may contribute to thrombocytopenia and other complications during SFTSV infection. ConclusionThese findings together highlighted the pathophysiological role of platelets in initial intrinsic defense against SFTSV infections, as well as intertwined processes with host immunity, which can also lead to thrombocytopenia and poor prognosis.

Circadian nuclear receptor Rev-erbalpha is expressed by platelets and potentiates platelet activation and thrombus formation

Abstract Background Adverse cardiovascular events have day/night patterns with peaks in the morning, potentially related to endogenous circadian clock control of platelet activation. Circadian nuclear receptor Rev-erbα is an essential and negative component of the circadian clock. Purpose We aim to investigate the expression profile and biological function of Rev-erbα in platelets. Methods and results Here we report the presence and functions of circadian nuclear receptor Rev-erbα in human and mouse platelets. Both human and mouse platelet Rev-erbα showed a circadian rhythm that positively correlated with platelet aggregation. Global Rev-erbα knockout and platelet-specific Rev-erbα knockout mice exhibited defective in hemostasis as assessed by prolonged tail-bleeding times. Rev-erbα deletion also reduced ferric chloride-induced carotid arterial occlusive thrombosis, prevented collagen/epinephrine-induced pulmonary thromboembolism, and protected against microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. In vitro thrombus formation assessed by CD41-labeled platelet fluorescence intensity was significantly reduced in Rev-erbα knockout mouse blood. Platelets from Rev-erbα knockout mice exhibited impaired agonist-induced aggregation responses, integrin αIIbβ3 activation and α-granule release. Consistently, pharmacological inhibition of Rev-erbα by specific antagonists decreased platelet activation markers in both mouse and human platelets. Mechanistically, mass spectrometry and co-immunoprecipitation analyses revealed that Rev-erbα potentiated platelet activation via oligophrenin-1-mediated RhoA/ERM (ezrin/radixin/moesin) pathway. Conclusion We provide the first evidence that circadian protein Rev-erbα is functionally expressed in platelets and potentiates platelet activation and thrombus formation. Rev-erbα may serve as a novel therapeutic target for managing thrombosis-based cardiovascular disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by grants from the National Science Fund for Distinguished Young Scholars (81625002), the National Natural Science Foundation of China (81930007).

Circadian nuclear receptor Rev-erbα is expressed by platelets and potentiates platelet activation and thrombus formation.

AimsAdverse cardiovascular events have day/night patterns with peaks in the morning, potentially related to endogenous circadian clock control of platelet activation. Circadian nuclear receptor Rev-erbα is an essential and negative component of the circadian clock. To date, the expression profile and biological function of Rev-erbα in platelets have never been reported.Methods and resultsHere, we report the presence and functions of circadian nuclear receptor Rev-erbα in human and mouse platelets. Both human and mouse platelet Rev-erbα showed a circadian rhythm that positively correlated with platelet aggregation. Global Rev-erbα knockout and platelet-specific Rev-erbα knockout mice exhibited defective in haemostasis as assessed by prolonged tail-bleeding times. Rev-erbα deletion also reduced ferric chloride-induced carotid arterial occlusive thrombosis, prevented collagen/epinephrine-induced pulmonary thromboembolism, and protected against microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. In vitro thrombus formation assessed by CD41-labelled platelet fluorescence intensity was significantly reduced in Rev-erbα knockout mouse blood. Platelets from Rev-erbα knockout mice exhibited impaired agonist-induced aggregation responses, integrin αIIbβ3 activation, and α-granule release. Consistently, pharmacological inhibition of Rev-erbα by specific antagonists decreased platelet activation markers in both mouse and human platelets. Mechanistically, mass spectrometry and co-immunoprecipitation analyses revealed that Rev-erbα potentiated platelet activation via oligophrenin-1-mediated RhoA/ERM (ezrin/radixin/moesin) pathway.ConclusionWe provided the first evidence that circadian protein Rev-erbα is functionally expressed in platelets and potentiates platelet activation and thrombus formation. Rev-erbα may serve as a novel therapeutic target for managing thrombosis-based cardiovascular disease.Key questionAdverse cardiovascular events have day/night patterns with peaks in the morning, potentially related to endogenous circadian clock control of platelet activation. Whether circadian nuclear receptor Rev-erba is present in platelets and regulates platelet function remains unknown.Key findingWe provide the first evidence that Rev-erba is functionally expressed in platelets and acts as a positive regulator of platelet activation/thrombus formation through the oligophrenin-1-mediated RhoA/ERM signalling pathway.Take home messageOur observations highlight the importance of circadian clock machinery in platelet physiology and support the notion that Rev-erba may serve as a novel therapeutic target for managing thrombosis-based cardiovascular diseases.

Galectin 3 enhances platelet aggregation and thrombosis via Dectin-1 activation: a translational study.

Galectin-3, a β-galactoside-binding lectin, is abnormally increased in cardiovascular disease. Plasma Galectin-3 receives a Class II recommendation for heart failure management and has been extensively studied for multiple cellular functions. The direct effects of Galectin-3 on platelet activation remain unclear. This study explores the direct effects of Galectin-3 on platelet activation and thrombosis. A strong positive correlation between plasma Galectin-3 concentration and platelet aggregation or whole blood thrombus formation was observed in patients with coronary artery disease (CAD). Multiple platelet function studies demonstrated that Galectin-3 directly potentiated platelet activation and in vivo thrombosis. Mechanistic studies using the Dectin-1 inhibitor, laminarin, and Dectin-1-/- mice revealed that Galectin-3 bound to and activated Dectin-1, a receptor not previously reported in platelets, to phosphorylate spleen tyrosine kinase and thus increased Ca2+ influx, protein kinase C activation, and reactive oxygen species production to regulate platelet hyperreactivity. TD139, a Galectin-3 inhibitor in a Phase II clinical trial, concentration dependently suppressed Galectin-3-potentiated platelet activation and inhibited occlusive thrombosis without exacerbating haemorrhage in ApoE-/- mice, which spontaneously developed increased plasma Galectin-3 levels. TD139 also suppressed microvascular thrombosis to protect the heart from myocardial ischaemia-reperfusion injury in ApoE-/- mice. Galectin-3 is a novel positive regulator of platelet hyperreactivity and thrombus formation in CAD. As TD139 has potent antithrombotic effects without bleeding risk, Galectin-3 inhibitors may have therapeutic advantages as potential antiplatelet drugs for patients with high plasma Galectin-3 levels.

The JUUL E-Cigarette Elevates the Risk of Thrombosis and Potentiates Platelet Activation.

Background:Smoking is the main preventable cause of death in the United States and worldwide and is associated with serious cardiovascular health consequences, including thrombotic diseases. Recently, electronic cigarettes (e-cigarettes) and, in particular JUUL, have attained wide popularity among smokers, nonsmokers, pregnant females, and even the youth, which is alarming. Interestingly, there is/are no information/studies regarding the effect of JUUL on cardiovascular diseases, specifically in the context of modulation of platelet activation. Thus, it is important to discern the cardiovascular disease health risks associated with JUUL.Methods and Results:We used a passive e-vape vapor inhalation system where C57BL/6J mice (10-12 weeks old) were exposed to JUUL e-cigarette vape. Menthol flavored JUUL pods containing 5% nicotine by weight were used as the e-liquid. Mice were exposed to a total of 70 puffs daily for 2 weeks; 3-second puff duration, and 25-second puff interval. The effects of JUUL relative to clean air were analyzed, on mouse platelet function in vitro (eg, aggregation) and in vivo (eg, FeCl3-induced carotid artery injury thrombosis model). Our results indicate that short-term exposure to JUUL e-cigarette causes hyperactivation of platelets and shortens the thrombus occlusion as well as hemostasis/bleeding times, relative to clean air (medians of 14 vs. 200 seconds, P < .01 and 35 vs. 295 seconds, P < .001, respectively).Conclusion:Our findings document—for the first time—that short-term exposure to the JUUL e-cigarette increases the risk of thrombotic events, in part by modulating platelet function, such as aggregation and secretion, in mice.

Enhancement of hemostatic property of plant derived oxidized nanocellulose-silk fibroin based scaffolds by thrombin loading

To combat post-surgical and traumatic bleeding conditions effective hemostasis is of great importance. The study was designed to investigate the effect of thrombin (Th) loading on hemostatic performance of TEMPO-oxidized cellulose nanofiber (TOCN)-silk fibroin (SF) scaffolds. Addition of SF with TOCN significantly (***P < 0.001) increased blood absorption capacity and improved biocompatibility of TOCN. Thrombin loading potentiated platelet activation and hemostatic property of scaffolds (TOCN-SF-Th) compared to samples without thrombin (TOCN-SF). The hemostatic time of TOCN-SF5-Th in rabbit ear artery bleeding model was reduced (*** P < 0.001) to 114 s from 220 s of TOCN-SF5. Reduction in bleeding time and blood loss of TOCN-SF5-Th in rat tail amputation and liver avulsion model was comparable to commercial hemostat (Floseal). Surface morphology (SEM) of samples applied on bleeding site showed that RBCs and fibrin fiber could strongly interact with TOCN-SF and TOCN-SF-Th scaffolds. The result suggests that TOCN-SF-Th can be a promising candidate for designing hemostatic agents.

Nucleic Acids Interfere with PF4 Tetramer Formation and Stabilise PF4 Monomers to Bind Non-Pathogenic HIT Antibodies: Subtypes of Non-Pathogenic Antibodies Emerge

IntroductionThe pathogenesis of heparin-induced thrombocytopenia (HIT) primarily involves anti-platelet factor(PF)4/heparin antibodies engaging FcγRIIa receptors on platelets to trigger a deleterious cascade of activation, thrombosis, and thrombocytopenia. Recent work with murine monoclonal antibodies has shed light on epitopic determinants that potentiate platelet activation (pathogenic KKO vs non-pathogenic RTO antibodies), but these mechanisms may not fully reflect the gamut of polyspecific antibodies seen in human clinical practice. We examined the development and characteristics of anti-PF4/heparin antibodies in patients undergoing cardiac surgery.MethodsAfter obtaining informed consent from patients undergoing cardiac bypass surgery, we performed ELISA using: (1) commercial Diagnostica Stago Asserachrom IgG kit (Cat.Nr 00624); or (2) in-house ELISA with platelet derived native(n)PF4. ELISA performed as per manufacturer instructions and published methods. Bacterial genomic nucleic acids (NA) were extracted from overnight culture of BL21(DE3)pLysS-wtPF4 using Promega kit Wizard® SV Genomic DNA Purification System (#A2361, Promega). Cross linking assays were performed as previously described using Bis(sulfosuccinimidyl)suberate (BS3) obtained commercially (#21585, Thermo Scientific Pierce). Optical density was normalised with controls to assist interassay comparability.All samples were screened with the commercial kit day 7 post-operatively, or the nearest available serum between day 4-10. Positive samples were then examined longitudinally to chart the onset of antibody formation and their duration.ResultsCommercial ELISA identified 30/123 (24%) patients positive for anti-PF4/heparin antibodies following cardiac surgery, but there was no HIT observed in the complete cohort of 123 patients and therefore all 30 were false positive.Surprisingly, in-house ELISA using nPF4/heparin was negative for 22/30, but the addition of NA to PF4 rescued the OD of these to become positive again.In contrast, OD fell for all true HIT controls with the addition of NA, as they did for a subset of the false-positives 7/30 [Figure A].Five patients had persistently raised OD despite the addition of NA, suggesting the presence of both NA/nPF4/heparin antibodies and nPF4/heparin antibodies.Overall, the incidence of false-positive anti-PF4/heparin antibodies was only 12/123 (10%) using the in-house ELISA method with nPF4. SRA was performed on all patients with nPF4/heparin antibodies, but they were all negative.Spiking nPF4 with increasing concentrations of NA caused reduction of tetramer formation and an increasing proportion of dimeric and monomeric forms seen on cross-linkage assays [Figures B and C].Longitudinal studies demonstrated that all 13 patients positive by commercial ELISA with only low range absolute results (OD <0.4), and without increase at later timepoints, possessed only NA/nPF4/heparin antibodies.By contrast, of the 14 patients whose ELISA OD increased over time, seven had evidence of nPF4/heparin antibodies. Three patients were ELISA positive pre-operatively, and only one of these had evidence of nPF4/heparin antibodies.ConclusionsIncreasing amounts of NA inhibit PF4 tetramer formation, and reduce the ability for HIT antibodies to bind. Thus, NA may participate in the preferential presentation of PF4 monomers that are recognised by non-pathogenic NA/PF4 monomer/heparin antibodies, similar to RTO. There is a smaller subset of “true” non-pathogenic antibodies that bind to PF4 tetramers, and an even smaller number of patients who may possess both classes of non-pathogenic antibodies. We speculate that pre-formed antibodies to NA/PF4 monomers are responsible for the majority of false positive results from commercial ELISA kits, and these are unrelated to the pathogenic anti-PF4/heparin antibodies of HIT.Figure Caption ARepresentative example of five patients with increasing ELISA OD in the presence of NA (NA1-5), two patients with OD that fell in the presence of NA (TFP1-2), and known HIT serum with reduced OD in the presence of NA. B: Reduction in PF4 tetramer formation and increasing PF4 monomers with increasing NA concentration. Lanes from left-right: control PF4 without BS3, and then increasing concentration of NA from 0, 5, 30 and 45ng/μL with BS3. C: Percentage of PF4 as tetramers or monomers with increasing concentration of NA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TRAF3 negatively regulates platelet activation and thrombosis

CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily, binds to CD40, leading to many effects depending on target cell type. Platelets express CD40L and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanisms. A family of proteins called TNF receptor associated factors (TRAFs) plays key roles in mediating CD40L-CD40 signaling. Platelets express several TRAFs. It has been shown that TRAF2 plays a role in CD40L-mediated platelet activation. Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression levels of collagen receptor GPVI and integrin αIIbβ3 in platelets were not affected by deletion of TRAF3, suggesting that increased platelet activation in the TRAF3 knockout mice was not due to increased expression platelet receptors. Time to formation of thrombi in a FeCl3-induced thrombosis model was significantly shortened in the TRAF3 knockout mice. However, mouse tail-bleeding times were not affected by deletion of TRAF3. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.

Abstract 55: Traf3 Negatively Regulates Platelet Activation and Thrombosis

Platelets express CD40 ligand (CD40L) and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanism. CD40L-mediated platelet activation requires the family of TNF receptor-associated factor 2 (TRAF2). Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression of integrin αIIbβ3, GPVI, and PAR4 was similar between TRAF3 knockout platelets and wild type controls, suggesting that increased platelet activation in the TRAF3 knockout mice is not due to increased expression of platelet receptors. TRAF2 expression was not affect by deletion of TRAF3 eitehr. Using the FeCl 3 -induced arterial thrombosis model, we found that time to formation of thrombi in was significantly shortened in TRAF3 knockout mice. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.

The class I phosphoinositide 3-kinases α and β control antiphospholipid antibodies-induced platelet activation.

Antiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies (aPL) associated with increased thrombotic risk and pregnancy morbidity. Although aPL are heterogeneous auto-antibodies, the major pathogenic target is the plasma protein β2-glycoprotein 1. The molecular mechanisms of platelet activation by aPL remain poorly understood. Here, we explored the role of the class IA phosphoinositide 3-kinase (PI3K) α and β isoforms in platelet activation by aPL. Compared to control IgG from healthy individuals, the IgG fraction isolated from patients with APS potentiates platelet aggregation induced by low dose of thrombin in vitro and increases platelet adhesion and thrombus growth on a collagen matrix under arterial shear rate through a mechanism involving glycoprotein Ib (GPIb) and Toll Like Receptor 2 (TLR-2). Using isoforms-selective pharmacological PI3K inhibitors and mice with megakaryocyte/platelet lineage-specific inactivation of class IA PI3K isoforms, we demonstrate a critical role of the PI3Kβ and PI3Kα isoforms in platelet activation induced by aPL. Our data show that aPL potentiate platelet activation through GPIbα and TLR-2 via a mechanism involving the class IA PI3Kα and β isoforms, which represent new potential therapeutic targets in the prevention or treatment of thrombotic events in patients with APS.

P2X1-initiated p38 signalling enhances thromboxane A2-induced platelet secretion and aggregation

ATP released by activated platelets can serve as a positive feedback machinery to amplify platelet responses by activating P2X1 receptors. It has, however, not been defined how P2X1 activities influence thromboxane A2 (TXA2)-stimulated platelet functional responses. Our aim was to elaborate the molecular mechanisms of P2X1 engagements in TXA2-induced platelet secretion and aggregation. P2X1 inhibition by 1 µM NF449 inhibited platelet P-selectin expression induced by a low concentration of the TXA2 analogue U46619 (0.3 µM) (32.0 ± 2.0% vs 43.4 ± 3.0%; n=5; p<0.05). p38 inhibition by SB203580, but not ERK inhibition by U0126, elicited a similar inhibition by NF499. The combination of NF449 and SB203580 provided, however, no additive effects. U46619-induced platelet aggregation was similarly decreased by NF449 and SB203580 alone or in combination, and by P2X1 pre-desensitisation with α,β-Me-ATP. U46619 caused rapid and reversible P2X1-dependent p38 phosphorylation. However, the P2X1-p38 pathway mainly enhanced mild platelet activation by U46619, because α,β-Me-ATP supplementation or p38 blockade had no effect on intense platelet activation induced by a higher concentration of U46619 (3 µM). In conclusion, P2X1 activation, via p38 signalling, potentiates platelet activation initiated by low doses of U46619. Hence, the P2X1-induced p38 signalling promotes more robust platelet activation in response to mild platelet stimuli.

Involvement of nuclear factor κB in platelet CD40 signaling

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Abstract 501: Nuclear Factor-κB Is Involved in Platelet CD40 Signaling and Activation

Introduction and hypothesis: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to influence platelet activation. We have previously shown that upon ligation, CD40 potentiates platelet activation and aggregation via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In B lymphocytes, CD40 induces activation and nuclear translocation of nuclear factor kappa B (NF-κB), which is dependent on the phosphorylation and dissociation of the inhibitor of kappa B α (IκBα). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling. Methods and results: In human platelets, sCD40L induced association of tumor necrosis factor receptor associated factor 2 to CD40, and a time-dependant phosphorylation of IκBα, which is indicative of NF-κB activation. Activation of NF-κB in platelets treated with sCD40L was abolished by CD40L blockade. Pretreatment of platelets with the IκBα inhibitor, BAY 11-7082, reversed IκBα phosphorylation induced by sCD40L, without affecting p38 MAPK activation. On the other hand, pretreatment of platelets with the p38 MAPK phosphorylation inhibitor, SB203580, had no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. Finally, inhibition of IκBα phosphorylation by either BAY 11-7082 or the IκB kinase inhibitor VII reversed sCD40L induced platelet activation, as measured by P-selectin expression, and the potentiation of platelet aggregation induced by a priming dose of collagen. Conclusion: This study demonstrates the implication of NF-κB in platelet signaling downstream of CD40, where it plays a role in platelet activation and aggregation upon sCD40L stimulation.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post-translational modification of beta 2 glycoprotein I.

It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (β2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti-β2GPI antibodies, in complex with β2GPI, elicit effects on blood cells and coagulation-fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against β2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post-translational modifications of β2GPI, which trigger the immune response. The majority of circulating β2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of β2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised β2GPI. These findings hold promise, not only for the delineation of the role of β2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.

Platelets release matrix metalloproteinase-2 in the coronary circulation of patients with acute coronary syndromes: possible role in sustained platelet activation

To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of β-thromboglobulin (β-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A₂ (sPLA₂), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both β-TG and sPLA₂ correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

Dynamics of platelet thrombus formation.

Platelet aggregation and thrombus formation at sites of atherosclerotic plaque rupture is a dynamic process that can lead to intermittent or permanent obstruction to blood flow, resulting in ischemic tissue injury and organ dysfunction. There is a growing body of evidence suggesting that the dynamics of platelet aggregation and initial thrombus development are regulated by two distinct, complementary processes, involving: (i) rheological (biomechanical) and (ii) soluble-agonist-dependent mechanisms. Rheological-dependent platelet aggregation occurs between discoid platelets and requires the biomechanical adhesive and signaling function (mechanotransduction) of the major platelet adhesion receptors, GPIb and integrin alpha(IIb)beta3. Soluble agonists further potentiate platelet activation, stimulating global platelet shape change and degranulation, and play a major role in stabilizing formed aggregates. Unraveling the dynamics of platelet aggregation and thrombus formation in vivo requires consideration of the cooperative interplay between rheological- and soluble agonist-dependent platelet aggregation mechanisms.

Clinically Relevant Concentrations of Dopamine Do Not Amplify Agonist-induced Human Platelet Ca2+ Mobilization or GP IIb IIIa Activation and Do Not Accelerate Acute Coronary Thrombosis in Dogs

Dopamine is an inotrope effective in the short term treatment of acute heart failure - including that caused by coronary artery disease. Catecholamines however can potentiate platelet activation and pre-dispose to coronary thrombosis. Dopamine was studied for effect on agonist induced human platelet Ca mobilization, human platelet GP iib iiia receptor activation and acute coronary thrombosis in dogs. Calcium sensitive indo-1, fluorescent immunostaining and flow cytometry were used for platelet studies while coronary thrombosis was induced in anesthetized dogs via endothelial damage, arterial wall injury and critical stenosis. Dopamine 10 and 10 M had no effect on the amplitude of the platelet Ca signal evoked by thrombin 0.1 U/mL. Likewise, dopamine 10 M had no effect on GP IIb IIIa activation evoked by ADP 10 M and by thrombin 0.1 U/mL. In dogs, intravenous dopamine 8 ug/Kg/min had no effect on repetitive cycles of acute coronary thrombus formation. In positive control studies, epinephrine increased platelet responsiveness and accelerated canine coronary thrombosis. Clinically relevant concentrations of dopamine did not amplify agonist induced human platelet Ca activation, GPiib iiia expression or experimental canine coronary thrombosis--providing a degree of reassurance concerning this versatile inotrope.

Platelets from Sickle Cell Disease Individuals Demonstrate Increased Adhesive Properties That Are Reversed by Hydroxyurea Therapy in Association with Alterations in Intraplatelet cAMP and αIIbα3 Integrin Activation

Sickle cell disease (SCD) is a chronic inflammatory disease that results in hemolytic anemia and vaso-occlusive events, the principal cause of morbidity in SCD patients. Whilst roles for red cells and leukocytes in the vaso-occlusive process in SCD are well established, the contribution of platelets to this process remains unclear. Increased platelet activation has been described in SCD patients, contributing to the hemostatic activation observed in the disease. Since the adhesion of platelets to the vascular endothelium and extracellular matrix (ECM) could potentially contribute to vaso-occulusion, this study compared the adhesive properties of platelets (PLTs) from healthy control subjects (CON), steady-state SCD patients (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day). Washed PLTs were ressuspended in Krebs solution (1.2×108 PLT/ml) and their adhesion to fibrinogen (FB, 50 μg/ml) coated 96-well plates evaluated utilizing static adhesion assays (15 min, 37°C). SCD PLTs demonstrated a significantly greater adhesion to FB than CON PLTs (26.0±4.0%; 15.8±2.0%, respectively, n≥13; p<0.05, Mann-Whitney test). In contrast, SCDHU PLT adhesion was significantly lower than that of SCD PLTs, being similar to that of CON PLTs (11.7±1.5%, n=13; p<0.01 comp. SCD). A thrombin stimulus (50 mU/ml) significantly increased CON, SCD and SCDHU adhesion to FB (26.2±2.1%; 33.8±3.7%; 23.5±3.7%, respectively, n≥13; p<0.001 comp. to basal, paired t test). Levels of the second messenger, cyclic adenosine monophosphate (cAMP), the reduction of which potentiates platelet activation, were found to be significantly decreased in the PLTs of SCD individuals compared to those of CON and SCDHU (5.4±0.6; 8.0±0.7; 7.1±0.4 pmol/108 PLT, respectively, n=7; P<0.05 for SCD comp. to CON and SCDHU). Interestingly, thrombin significantly reduced cAMP levels in CON and SCDHU (6.0±0.9; 5.4±0.5 pmol/108 PLTs, respectively, n=7, P<0.05 comp. to basal), but not SCD PLTs (4.7±0.6 pmol/108 PLT, n=7; P>0.05), providing further evidence that PLTs circulate in an activated state in SCD individuals. Flow cytometry studies demonstrated no alterations in the expressions of CD42b (GPIb molecule), CD62P (P-selectin), CD49b (α2 integrin subunit) and CD41a (αIIb integrin subunit) on the surface of SCD PLTs compared to CON and SCDHU (P<0.05, data not shown). However, binding of the PAC-1 antibody, which specifically recognizes activated αIIbβ3 integrin, was significantly higher on SCD PLTs than on CON and SCDHU PLTs (40.7±8.9%; 17.1±6.5%; 20.3±5.4%, respect., n≥8, p<0.05). The αIIbβ3 integrin, in its activated affinity, can bind to numerous soluble ligands that in turn may bridge the interaction between PLTs, endothelial cells and other cell types. As such, PLTs from SCD individuals in steady state circulate in an activated state and present an increased ability to adhere to the blood component, FB, compared to control PLTs. Increased adhesive properties were found associated with decreased intraplatelet cAMP and high affinity αIIbβ3 integrin expression. Interestingly, HU therapy was associated with a reversal in increased PLT adhesion coupled with augmented intraplatelet cAMP and reduced αIIbβ3 activation. Thus, SCD platelets may have an increased capacity for adhesion to the vascular wall and ECM components, where the presence of adhered and activated platelets may contribute to the vaso-occlusive process by facilitating leukocyte accumulation at the endothelium and the release of platelet factors that may stimulate further inflammation at the site.

Platelets release active matrix metalloproteinase‐2 in vivo in humans at a site of vascular injury: lack of inhibition by aspirin

When stimulated in vitro, human platelets release matrix metalloproteinase-2 (MMP-2) that, in turn, potentiates platelet activation. The present study investigated if MMP-2 is released from activated platelets in vivo in humans and whether aspirin inhibits this release. MMP-2 levels were measured by zymography, immunoblotting, flow-cytometry and an activity assay system, in plasma prepared from blood emerging from a skin wound inflicted for the measurement of the bleeding time (shed blood) and simultaneously from venous blood in 27 healthy human volunteers. In a subgroup, the same measurements were carried out before and 1 h after aspirin intake. MMP-2 was significantly higher in shed blood than in venous blood and increased progressively, consistent with ongoing platelet activation. A significant correlation was evident between platelet number and MMP-2 in shed blood; platelet MMP-2 content in shed blood was lower than that of platelets in venous blood. The level of active MMP-2 released by activated platelets in vivo was within the range of concentrations that potentiate platelet activation. Aspirin did not reduce MMP-2 release in vivo. In conclusion, MMP-2 is released from platelets in vivo in humans at a localised site of vessel wall damage in amounts sufficient to potentiate platelet aggregation; aspirin does not reduce this release.