Human Platelet Activation Research Articles

Role of GPR56 in Platelet Activation and Arterial Thrombosis.

The adhesion G protein-coupled receptor GPR56 mediates cell-cell and cell-extracellular matrix interactions. To examine the function of GPR56 in platelet activation and arterial thrombosis, we generated GPR56-knockout mice and evaluated GPR56 expression in human and mouse platelets. The results revealed that the levels of the GPR56 N-terminal fragment were significantly higher on the first day after myocardial infarction than on the seventh day in the plasma of patients with ST-segment-elevation myocardial infarction. Next, we investigated the effects of GPR56 on platelet function in vitro and in vivo. We observed that collagen-induced aggregation and adenosine triphosphate release were reduced in Gpr56 -/- platelets. Furthermore, P-selectin expression on the Gpr56 -/- platelet surface was also reduced, and the spreading area on immobilized collagen was decreased in Gpr56 -/- platelets. Furthermore, collagen-induced platelet activation in human platelets was inhibited by an anti-GPR56 antibody. Gpr56 -/- mice showed an extended time to the first occlusion in models with cremaster arteriole laser injury and FeCl3-induced carotid artery injury. GPR56 activated the G protein 13 signaling pathway following collagen stimulation, which promoted platelet adhesion and thrombus formation at the site of vascular injury. Thus, our study confirmed that GPR56 regulated the formation of arterial thrombosis. Inhibition of the initial response of GPR56 to collagen could significantly inhibit platelet activation and thrombus formation. Our results provide new insights for research into antiplatelet drugs.

Activated Platelets Mediate Monocyte Killing of Klebsiella pneumoniae.

Platelets are known for essential activities in hemostasis and for their important contribution to protection against infectious pathogens. Klebsiella pneumoniae is an opportunistic pathogen widely known to cause nosocomial infections. Recently, hypervirulent strains of K. pneumoniae have been emerging, which can cause severe infections in immunocompetent individuals. Combined with the increase in antibiotic resistance, it is important to understand how K. pneumoniae affects components of the immune system. We studied the interactions of human platelets with several K. pneumoniae strains (the wild type encapsulated strain, and a nonencapsulated mutant). Thrombin-stimulated whole human and mouse blood significantly inhibited bacterial growth compared to unstimulated whole blood. Furthermore, we investigated the effect of K. pneumoniae on platelet activation. Both strains induced significant increase in activation of both unstimulated and thrombin-stimulated human platelets. Additionally, only the nonencapsulated mutant increased aggregation of platelets in response to ADP. K. pneumoniae killing assays were then performed with washed platelets in the presence or absence of thrombin. Surprisingly, washed platelets failed to exhibit any effects on the growth of K. pneumoniae. We further explored the impact of platelets on monocyte-mediated killing of K. pneumoniae. Importantly, we found that activated platelets significantly enhanced monocyte-mediated killing of K. pneumoniae. This effect was likely due to the formation of platelet-monocyte aggregates in blood upon thrombin stimulation. Overall, this study highlights the role of platelets in mediating a protective response against K. pneumoniae and reinforces the importance of platelets in modulating leukocyte behavior.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbβ3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to μ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 μM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 μM of fluvoxamine, 67.3% decrease, p<0.05; 30 μM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 μM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK.

Modulation of ultralarge immune complexes in heparin–induced thrombocytopenia

BackgroundHeparin–induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti–PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti–thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor–initiated generation of thrombin. ObjectivesTo determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti–PF4 antibody (hRTO). MethodsResults: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor–dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. ConclusionsHIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

Diamond-like carbon films prepared by a low temperature periodic process for application in ventricular assist devices.

Due to the poor tribological properties of titanium (Ti) and its alloy Ti6Al4V (commonly used for ventricular assist devices manufacturing), diamond-like carbon (DLC) films with excellent anti-wear properties are pursued to improve the wear resistance of Ti and its alloys. Considering the effect of temperature on magnets inside pump impellers and workpiece deformation, DLC films are preferred to be prepared under low temperature. In this study, DLC films were prepared on Ti6Al4V alloys by periodic and continuous processes, and the corresponding maximum deposition temperature was 85 and 154°C, respectively. The periodic DLC films exhibited the feature of columnar structure, and the surface hillocks were less uniform than that of continuous DLC films. The periodic DLC films possessed more sp3 -bonded structures, and the accessorial sp3 -bonding mainly existed in the form of CH. Compared to continuous DLC films, the periodic DLC films had lower residual stress and better adhesion with Ti6Al4V substrates. Both DLC films could effectively reduce the friction coefficient and wear rate of Ti6Al4V alloys both in air and fetal bovine serum (FBS), and the periodic DLC films exhibited superior anti-wear properties to that of continuous DLC films in FBS. Haemocompatibility evaluation revealed that both DLC films presented similar levels of more human platelet adhesion and activation as compared with that of bare Ti6Al4V. However, both DLC films significantly prolonged plasma clotting time in comparison to bare Ti6Al4V. This study demonstrates the potential of low-temperature DLC films as wear-resistant surface modification for VADs.

Modulation of Ultralarge Immune Complexes in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is serious prothrombotic disorder in which the target antigen, composed of tetramers of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs), forms ultralarge immune complexes (ULICs) with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses in large part by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful, and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. Therefore, we asked whether HIT antigen and ULIC formation and stability could be modified to attenuate cellular activation by inhibiting PF4-heparin interactions with fondaparinux in conjunction with disrupting PF4 tetramers with a humanized form of a monoclonal anti-PF4 monomer antibody (hRTO, Figure 1). Using dynamic light scattering to measure HIT antigen (PF4 10 µg/ml and heparin 0.2U/ml) and ULIC (PF4 10 µg/ml, heparin 0.2U/ml, KKO 35µg/ml) size, we found that either fondaparinux (FPX 5-20U/ml) or hRTO (400 - 1000 µg/ml) inhibited formation of HIT antigen and ULIC, and promoted HIT antigen and ULIC disassembly. Successively higher concentrations of hRTO were required as PF4 formed large antigenic complexes and then ULICs, likely because the bonds between PF4 and UFH have been strengthened and access to inhibitors lessened (Figure 1A). Combination of fondaparinux and hRTO was effective below the concentration of either alone (FPX 2.5U/ml + hRTO 330 µg/ml). Importantly, fondaparinux and hRTO blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood (Figure 1B). When combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation at concentrations at or below those used clinically to inhibit FXa coagulant activity. Even lower concentrations of inhibitors (FPX < 0.1-0.5 U/ml + hRTO 50 µg/ml) were required to block cell activation than were required to act on ULICs in solution, likely because the affinity of PF4 for GAGs is less than for UFH. These studies show that HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rational, disease-specific intervention that blocks ULIC formation when used in combination with hRTO. Our studies demonstrate that interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, and shorten the duration of anti-thrombotic therapy and help prevent this serious thrombotic disorder. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activation of Human Platelets by Staphylococcus aureus Secreted Protease Staphopain A.

Infection by Staphylococcus aureus is the leading cause of infective endocarditis (IE). Activation of platelets by this pathogen results in their aggregation and thrombus formation which are considered to be important steps in the development and pathogenesis of IE. Here, we show that a secreted cysteine protease, staphopain A, activates human platelets and induces their aggregation. The culture supernatant of a scpA mutant deficient in staphopain A production was reduced in its ability to trigger platelet aggregation. The platelet agonist activity of purified staphopain A was inhibited by staphostatin A, a specific inhibitor, thus implicating its protease activity in the agonism. In whole blood, using concentrations of staphopain A that were otherwise insufficient to induce platelet aggregation, increased binding to collagen and thrombus formation was observed. Using antagonists specific to protease-activated receptors 1 and 4, we demonstrate their role in mediating staphopain A induced platelet activation.

Extraction, Characterization, and Platelet Inhibitory Effects of Two Polysaccharides from the Cs-4 Fungus.

Cardiovascular diseases are associated with platelet hyperactivity, and downregulating platelet activation is one of the promising antithrombotic strategies. This study newly extracted two polysaccharides (purified exopolysaccharides, EPSp and purified intercellular exopolysaccharides, IPSp) from Cordyceps sinensis Cs-4 mycelial fermentation powder, and investigated the effects of the two polysaccharides and their gut bacterial metabolites on platelet functions and thrombus formation. EPSp and IPSp are majorly composed of galactose, mannose, glucose, and arabinose. Both EPSp and IPSp mainly contain 4-Galp and 4-Glcp glycosidic linkages. EPSp and IPSp significantly inhibited human platelet activation and aggregation with a dose-dependent manner, and attenuated thrombus formation in mice without increasing bleeding risk. Furthermore, the EPSp and IPSp after fecal fermentation showed enhanced platelet inhibitory effects. The results have demonstrated the potential value of Cs-4 polysaccharides as novel protective ingredients for cardiovascular diseases.

Glabridin, a Bioactive Flavonoid from Licorice, Effectively Inhibits Platelet Activation in Humans and Mice.

Platelets are crucial for hemostasis and arterial thrombosis, which may lead to severe cardiovascular diseases (CVDs). Thus, therapeutic agents must be developed to prevent pathological platelet activation. Glabridin, a major bioalkaloid extracted from licorice root, improves metabolic abnormalities (i.e., obesity and diabetes) and protects against CVDs and neuronal disorders. To the best of our knowledge, no studies have focused on glabridin’s effects on platelet activation. Therefore, we investigated these effects in humans and mice. Glabridin exhibited the highest inhibitory effects on collagen-stimulated platelet aggregation and moderate effects on arachidonic-acid-stimulated activation; however, no effects were observed for any other agonists (e.g., thrombin or U46619). Glabridin evidently reduced P-selectin expression, ATP release, and intracellular Ca2+ ([Ca2+]i) mobilization and thromboxane A2 formation; it further reduced the activation of phospholipase C (PLC)γ2/protein kinase C (PKC), phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β), mitogen-activated protein kinase (MAPK), and NF-κB. In mice, glabridin reduced the mortality rate caused by acute pulmonary thromboembolism without altering bleeding time. Thus, glabridin effectively inhibits the PLCγ2/PKC cascade and prevents the activation of the PI3K/Akt/GSK3β and MAPK pathways; this leads to a reduction in [Ca2+]i mobilization, which eventually inhibits platelet aggregation. Therefore, glabridin may be a promising therapeutic agent for thromboembolic disorders.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa.

Blood coagulation is an intricate process, and it requires precise control of the activities of pro- and anticoagulant factors and sensitive signaling systems to monitor and respond to blood vessel insults. These requirements are fulfilled by phosphatidylserine, a relatively miniscule-sized lipid molecule amid the myriad of large coagulation proteins. This review limelight the role of platelet membrane phosphatidylserine (PS) in regulating a key enzymatic reaction of blood coagulation; conversion of factor X to factor Xa by the enzyme factor IXa and its cofactor factor VIIIa. PS is normally located on the inner leaflet of the resting platelet membrane but appears on the outer leaflet surface of the membrane surface after an injury happens. Human platelet activation leads to exposure of buried PS molecules on the surface of the platelet-derived membranes and the exposed PS binds to discrete and specific sites on factors IXa and VIIIa. PS binding to these sites allosterically regulates both factors IXa and VIIIa. The exposure of PS and its binding to factors IXa/VIIIa is a vital step during clotting. Insufficient exposure or a defective binding of PS to these clotting proteins is responsible for various hematologic diseases which are discussed in this review.

N-Stearoylethanolamine Inhibits Integrin-Mediated Activation, Aggregation, and Adhesion of Human Platelets.

N-stearoylethanolamine (NSE), a lipid mediator that belongs to the N-acylethanolamine (NAE) family, has anti-inflammatory, antioxidant, and membranoprotective actions. In contrast to other NAEs, NSE does not interact with cannabinoid receptors. The exact mechanism of its action remains unclear. The aim of this study is to evaluate the action of NSE on activation, aggregation, and adhesion of platelets that were chosen as a model of cellular response. Aggregation of platelets was measured to analyze the action of NSE (10^-6-10^-10 M) on platelet reactivity. Changes in granularity and shape of resting platelets and platelets stimulated with ADP in the presence of NSE were monitored by flow cytometry, and platelet deganulation was monitored by spectrofluorimetry. In vivo studies were performed using obese insulin-resistant rats. Binding of fibrinogen to the GPIIb/IIIa receptor was estimated using indirect ELISA and a scanning electron microscopy (SEM). It was found that NSE inhibits the activation and aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. These effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory effect and allows us to prioritize studies of NSE as a potent anti-thrombotic agent. SIGNIFICANCE STATEMENT: N-stearoylethanolamine (NSE) was shown to possess inhibitory action on platelet activation, adhesion, and aggregation. The mechanism of inhibition possibly involves integrin receptors. This finding complements the known anti-inflammatory effects of NSE.

Targeting of a Conserved Epitope in Mouse and Human GPVI Differently Affects Receptor Function.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Metformin Serves as a Novel Drug Treatment for Arterial Thrombosis: Inhibitory Mechanisms on Collagen-Induced Human Platelet Activation

Metformin is widely used as first-line medication for type 2 diabetes (T2D), the main disease comorbid with kidney disease, cardiovascular diseases (CVDs), and retinopathy. Platelets are crucial in platelet-dependent arterial thrombosis, which causes CVDs and cerebrovascular diseases. Research indicates that metformin may improve these diseases; metformin reportedly reduced platelet activation in rats. However, no reports have included human platelets. We investigated the mechanisms underlying metformin’s effects on platelet activation by using human platelets and evaluated its in vivo effectiveness in experimental mice. Metformin inhibited platelet aggregation stimulated by collagen but not by arachidonic acid, U46619, or thrombin. Metformin suppressed ATP release, [Ca2+]i mobilization, and P-selectin expression, as well as phospholipase C (PLC)γ2/protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) phosphorylation. Metformin did not affect vasodilator-stimulated phosphoprotein (VASP) phosphorylation. In the animal studies, metformin reduced acute pulmonary thromboembolism mortality without increasing bleeding times. These results provide insights into the role and mechanisms of metformin in human platelet activation. Metformin decreased platelet activation by interfering with the PLCγ2/PKC, PI3K/Akt/GSK3β, and p38 MAPK pathways through a VASP-independent mechanism. Metformin demonstrates promise as a new class of antiplatelet agent that can inhibit platelet activation.

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

Background CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. Methods ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin–CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and Conclusion We developed a CLEC-2–podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)′2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

Human Blood Platelets Adsorption on Polymeric Materials for Liquid Biopsy.

Platelets are emerging as a promising source of blood biomarkers for several pathologies, including cancer. New automated techniques for easier manipulation of platelets in the context of lab-on-a-chips could be of great support for liquid biopsy. Here, several polymeric materials were investigated for their behavior in terms of adhesion and activation of human platelets. Polymeric materials were selected among the most used in microfabrication (PDMS, PMMA and COC) and commercial and home-made resins for 3D printing technology with the aim to identify the most suitable for the realization of microdevices for human platelets isolation and analysis. To visualize adherent platelets and their activation state scanning, electron microscopy was used, while confocal microscopy was used for evaluating platelets’ features. In addition, atomic force microscopy was employed to further study platelets adherent to the polymeric materials. Polymers were divided in two main groups: the most prone to platelet adhesion and materials that cause few or no platelets to adhere. Therefore, different polymeric materials could be identified as suitable for the realization of microdevices aimed at capturing human platelets, while other materials could be employed for the fabrication of microdevices or parts of microdevices for the processing of platelets, without loss on surfaces during the process.

S298: CS585 IS A FIRST-IN-CLASS COMPOUND TARGETING THE IP RECEPTOR FOR PREVENTION OF THROMBOSIS WITHOUT INCREASED RISK OF BLEEDING

Background: Uncontrolled platelet activation leads to the formation of occlusive thrombi resulting in myocardial infarction, stroke, VTE and critical limb ischemia. There is an unmet need for more efficacious anti-thrombotics with less bleeding in this area. Our group recently identify a novel oxidized lipid in the blood that potently and selectively inhibits platelet activation through activation of the prostacyclin receptor. Aims: Develop a first-in-class antiplatelet drug, CS585, that potently and selectively inhibits platelet reactivity and thrombosis without altering hemostasis and bleeding risk. Methods: We developed a mimetic of the oxidized lipid shown to selectively bind to and activate the prostacyclin receptor. Using human platelets, we assessed the ability of CS585 to inhibit platelet activation by assessing 1)aggregometry, 2)adhesion under arterial flow, and 3) granule secretion and integrin activation using flow cytometry. We additionally assessed thrombosis in vivo in 2 different mouse models as well as bleeding. Finally, we assessed potential off-target effects using thromboelastography (TEG). Results: CS585 potently inhibited both collagen and thrombin-induced platelet aggregation. This inhibition was confirmed by measuring inhibition of αIIbβ3 activation, α-granule secretion, and dense-granule secretion by flow cytometry. Selectivity was confirmed in human platelets by demonstrating a full reversal of inhibition when the IP receptor was pharmacologically blocked or genetically eliminated in IP receptor knockout mice. In vivo inhibition of injury-induced thrombosis in the small (laser-induced cremaster thrombosis model) and large (FeCl3-induced carotid artery thrombosis model) vessels by CS585 was demonstrated and no increased risk for bleeding was observed using the tail vein bleeding model. Finally, TEG experiments in human blood spiked with CS585 demonstrated no delay or decrease in clot strength confirming the tail vein bleeding assay experiments in the mouse. Summary/Conclusion: We have shown for the first time that CS585, a first-in-class analog of an oxidized lipid in the blood potently and selectively activates the prostacyclin receptor resulting in inhibition of human and mouse platelet activation and thrombosis without an increased risk of bleeding. This discovery represents a new class of inhibitors for prevention of platelet activation and thrombosis and protection from myocardial infarction, stroke, VTE, and critical limb ischemia.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12

AbstractThe critical role of G protein–coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5′-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2−/− platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2−/− platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2−/− platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2−/− mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.