Receptor GPVI Research Articles

PS1582 DECREASED PLATELET REACTIVITY IS ASSOCIATED WITH AN INFLAMMATORY STATUS IN PATIENTS WITH CANCER

Background:Decreased platelet reactivity was observed in cancer patients at high risk of venous thromboembolism (VTE) and death. Prothrombotic and inflammatory mechanisms have been suggested to cooperate in the development of VTE, representing represent the hallmark of thromboinflammation.Aims:To assess whether platelet reactivity is associated with inflammatory parameters in cancer patients.Methods:Platelet reactivity was determined by whole blood flow cytometry in 62 newly diagnosed cancer patients (median age [interquartile range, IQR]: 63 [54–70] years, 48 % female) at several time points during the first 6 months of chemotherapy (total number of samples analyzed: 179). We measured platelet surface P‐selectin and activated glycoprotein (GP) IIb/IIIa expression in vivo and upon ex vivo stimulation with agonists of protease‐activated receptor (PAR) −1, −4, and GPVI. C‐reactive protein (CRP) and fibrinogen levels were analyzed in plasma samples from the corresponding blood draws.Results:Platelet reactivity, as determined by percentage of platelets expressing activated GPIIb/IIIa on the surface, upon stimulation with different agonists, correlated with significantly lower levels of CRP and fibrinogen (Table 1). Platelet surface P‐selectin expression upon stimulation of the GPVI receptor also showed a significant inverse correlation with CRP and fibrinogen (Table 1). Platelet activation in vivo (i.e. without ex vivo agonist activation) did not correlate with CRP and fibrinogen levels (Table 1).Summary/Conclusion:Our data indicate that there is an inverse relationship between platelet reactivity and inflammatory status in cancer patients, whereby platelets of patients with increased inflammatory parameters are less reactive to stimulation with platelet agonists. Further research is needed to better understand underlying pathophysiological mechanisms.image

Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events.

The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

Device-induced platelet dysfunction in mechanically assisted circulation increases the risks of thrombosis and bleeding.

Thrombotic and bleeding complications are the major obstacles for expanding mechanical circulatory support (MCS) beyond the current use. While providing the needed hemodynamic support, those devices can induce damage to blood, particularly to platelets. In this study, we investigated device-induced alteration of three major platelet surface receptors, von Willebrand factor (VWF) and associated hemostatic functions relevant to thrombosis and bleeding. Fresh human whole blood was circulated in an extracorporeal circuit with a clinical rotary blood pump (CentriMag, Abbott, Chicago, IL, USA) under the clinically relevant operating condition for 4hours. Blood samples were examined every hour for glycoprotein (GP) IIb/IIIa activation and receptor loss of GPVI and GPIbα on the platelet surface with flow cytometry. Soluble P-selectin in hourly collected blood samples was measured by enzyme linked immunosorbent assay to characterize platelet activation. Adhesion of device-injured platelets to fibrinogen, collagen, and VWF was quantified with fluorescent microscopy. Device-induced damage to VWF was characterized with western blotting. The CentriMag blood pump induced progressive platelet activation with blood circulating time. Particularly, GPIIb/IIIa activation increased from 1.1% (Base) to 11% (4hours) and soluble P-selectin concentration increased from 14.1ng/mL (Base) to 26.5ng/mL (4hours). Those device-activated platelets exhibited increased adhesion capacity to fibrinogen. Concurrently, the CentriMag blood pump caused progressive platelet receptor loss (GPVI and GPIbα) with blood circulating time. Specifically, MFI of the GPVI and GPIbα receptors decreased by 17.2% and 16.1% for the 4-hours sample compared to the baseline samples, respectively. The device-injured platelets exhibited reduced adhesion capacities to collagen and VWF. The high molecular weight multimers (HMWM) of VWF in the blood disappeared within the first hour of the circulation. Thereafter the multimeric patterns of VWF were stable. The change in the VWF multimeric pattern was different from the progressive structural and functional changes of platelets with the circulation time. This study suggested that the CentriMag blood pump could induce two opposite effects on platelets and associated hemostatic functions under a clinically relevant operating condition. The device-altered hemostatic function may contribute to thrombosis and bleeding simultaneously as occurring in patients supported by a rotary blood pump. Device-induced damage of platelets may be an important cause for bleeding in patients supported with rotary blood pump MCS systems relative to device-induced loss of HMWM-VWF.

Continuous-flow mechanical circulatory support induces shedding of platelet adhesion receptors GpIb and GpVI

Hemorrhagic complications, especially mucosal bleedings, are very common for using continuous-flow mechanical circulatory supports (CF-MCS) including Extracorporeal Membrane Oxygenation (ECMO). CF-MCS could impact platelets by exposing this key player in primary hemostasis to high shear stress. A proteolytic cleavage (shedding) of platelet adhesion receptors GpIb and GpVI has been described under high shear stress and could contribute to the hemorrhagic complications in patients with CF-MCS. To assess whether CF-MCS promotes GpIb and GpVI shedding in vitro and in vivo and determine the kinetic evolution during the CF-MCS. First platelet shedding was investigated in vitro using a CF-MCS (Impella-CP®, Abiomed) loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma collected from healthy donors in fresh frozen plasma. Sampling was performed before and after 2, 5, 30, 60 and 180 min perfusion of plasma-NPC in the loop model ( n = 4 runs). Platelet shedding was also investigated for the blood samples collected from 20 ECMO patients (WITECMO-h trial). Platelets of 20 healthy volunteers were evaluated as a control. GpIbα and GpVI shedding was analyzed by flow cytometry. A significant time-dependent decrease of GpIbα ( P < 0.001) and GpVI ( P < 0.001) MFI was observed after 180 min in the Impella®-CP loop model. In vivo baseline GpIbα and GpVI MFIs has been significantly decreased before ECMO implantation compared to the healthy subjects ( P < 0.001). After ECMO implantation, no significant variation of GpIbα and GpVI MFI was observed. CF-MCS induces platelet GpIb and GpVI shedding in vitro. Increased GpIb and GpVI shedding is already present before ECMO implantation and remain significantly elevated after the implantation. Loss of the platelet receptors GPIbα and GPVI in patients with CF-MCS may contribute to hemorrhagic complications observed in these patients.

Effect of alteplase on platelet function and receptor expression.

ObjectiveTo investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function.MethodsHuman platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined.ResultsThis study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction.ConclusionsAlteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

Platelet CD36 Promotes ERK5 and Caspase-Dependent Procoagulant Phosphatidylserine Externalization and In Vivo Fibrin Formation in Dyslipidemia

Thrombosis during atherosclerotic plaque erosion or rupture is a major cause of death and disability in individuals with cardiovascular disease. Circulating oxidized lipids are risk factors for this condition and are carried in oxidized low-density lipoprotein particles (oxLDL). CD36 is a pattern recognition receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype. We previously reported that recognition of oxLDL by CD36 promotes activation of redox sensitive signaling pathways. This phenotype promotes phosphatidylserine (PSer) externalization. However, signaling events downstream of CD36 are unclear. We hypothesize that CD36 signaling through the MAP kinase redox sensor ERK5 promotes a procoagulant phenotype that supports arterial thrombosis in dyslipidemia. Platelets isolated and washed from healthy human donors were stimulated with oxLDL and were stained with fluorophore-conjugated Annexin V to detect exposure of procoagulant PSer. OxLDL-stimulated platelets showed rapid PSer externalization with maximal PSer between 5-30 min (11.0±0.35%). To mimic physiological conditions, platelets were sensitized with oxLDL before activating the collagen receptor GPVI with the snake venom convulxin (CVX). OxLDL sensitization showed marked enhancement of PSer externalization up to 78±4.6% compared to oxLDL alone (21.9±2.8%) or CVX alone (23±1.6%). To determine the mechanisms of oxLDL sensitized PSer exposure by CVX, platelets were pre-treated with the CD36 blocking antibody FA6, ERK5 inhibitor BIX02188, and inhibitors of “classic” inducers of PSer exposure, cyclosporin A to cyclophilin D and Z-VAD-FMK to caspases. Pre-treatment with FA6, BIX02188, and Z-VAD-FMK, but not cyclosporin A, prevented oxLDL-induced enhanced PSer exposure by CVX. These studies were complemented by using platelets from mice with genetic deletion of CD36, ERK5, cyclophilin D, and Bak/Bax, all of which showed a 45% reduction in PSer externalization by oxLDL compared to wild type controls. Caspase activation was determined mechanistically by immunoblotting for cleaved caspase products. OxLDL stimulated platelets showed enhanced cleaved caspase formation that was prevented to unstimulated levels by inhibiting CD36 and ERK5. PSer exposure links platelets to tissue factor-mediated fibrin formation. Kinetic analysis of fibrin formation ex vivo showed oxLDL decreased the onset time for fibrin to form by CVX from 5.6±0.1 min to 3.4±0.13 min, which was prevented by inhibiting CD36 with FA6, or ERK5 and Bak/Bax with inhibitors. CD36-mediated fibrin formation in dyslipidemia in vivo was studied using a laser-injury thrombosis model on high fat diet-fed (HFD) atherogenic apoE null mice. These mice showed rapid (within 30 sec of injury) enhancement of fibrin and platelet accumulation that was sustained over time. In contrast, HFD-fed apoE:CD36 double null mice did not show accelerated fibrin and platelet accumulation; levels were similar to that observed in control diet conditions. Furthermore, we employed a second thrombosis model by transplantation of the epigastric artery, which has a surface rich with collagen to mediate thrombosis, into the carotid artery lumen. ApoE null mice were transplanted with bone marrows from ERK5 expressing mice or platelet-specific ERK5 null mice. Compared to control diet-fed apoE null mice, fibrin accumulation was enhanced in HFD-fed apoE null mice expressing platelet ERK5. ApoE null mice lacking platelet ERK5 did not show enhanced fibrin formation; HFD was the same as control diet. In conclusion, oxLDL promotes procoagulant PSer and fibrin formation ex vivo through a CD36, ERK5, and caspase-dependent mechanism. In vivo fibrin formation in dyslipidemia were rescued by targeted genetic deletion CD36 or platelet ERK5 in two models of diet-induced arterial thrombosis. These data suggest that the CD36-ERK5-caspase signaling axis could be a potential target to prevent arterial thrombosis in dyslipidemia. DisclosuresJobe:CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.

Investigating Potential Mechanism(s) By Which ASO-Based Drugs Cause Thrombocytopenia

IntroductionAntisense oligonucleotides (ASOs) are a new class of single-stranded DNA based drugs that hold great therapeutic promise for their disease modifying potential in a wide range of genetic diseases. Preclinical toxicology studies in monkeys, as well as late stage clinical trials in humans, have upon repeated dosing, reported events of ASO sequence-specific lowering of platelet counts (mild to severe thrombocytopenia) (Henry et al. Nucleic acid therapeutics 2017). The underlying cause of this platelet decrease is still unclear in humans. We have investigated if the thrombocytopenia associated with ASOs is due to either impaired platelet production and/or destruction of platelets (clearance) due to increased platelet reactivity (activation/aggregation status). Preliminary data from mouse derived fetal liver megakaryocytes suggest that pro-platelet production does not seem to be reduced by ASOs and hence in the current study we hypothesized that the ASO-induced thrombocytopenia is due to increased clearance of platelets from the circulation.MethodsIn the current study we explored how ASOs affect platelet aggregation in platelet rich plasma (PRP) and platelet-leukocyte aggregates in whole blood (WB) obtained from healthy volunteers after informed consent. PRP or WB was treated with a clinically relevant concentration of ASO (5µM) corresponding to expected maximum plasma concentration levels, or a 20x-supra-therapeutic concentration (100µM). Four ASOs were tested: two CpG-rich phosphorothioate deoxyoligonucleotide (PS ODN) sequences: 818290 and 120704, and two non-CpG 2'-MOE containing sequences: 104838 and 501861. 818290 was included as a positive control since it has been shown to cause direct platelet activation (Flierl et al. JEM 2015). 104838 have been reported to cause moderate, dose dependent drops in platelet counts in monkeys and humans, with platelet sequestration in the liver and spleen (Narayanan PK, et al. Toxicol Sci. 2018). 501861 has triggered sporadic severe thrombocytopenia in select monkeys. ASO treated PRP was analyzed for platelet aggregation using 96-well optimul aggregometry (Lordkipanidzé et al. Blood 2014) in the presence of vehicle (PBS) or 6 concentrations of thrombin receptor activating peptide-6 (0.08-80µM,TRAP6). In a separate experiment, PRP was incubated with ASOs plus the spleen tyrosine kinase (Syk) inhibitor PRT-060318 (10µM). ASO treated WB was incubated with fluorescently labelled CD41/61 antibody to label platelets and a leukocyte-specific antibody CD14, and platelet-leukocyte aggregates were analyzed by FACS according to (Gerrits et al. Curr. Protoc. 2016).ResultsThe two non-CpG rich 2'-MOE ASO sequences 104838 and 501861 did not affect platelet aggregation at either concentration (5µM + 100µM) (Figure 1 A+B). Whilst the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM (Figure 1 C+D), that was normalized by co-incubating these ASOs with a Syk inhibitor (Figure 2). 5µM of ASO treatment triggered a significant increase in platelet-leukocyte aggregates in WB for all the ASOs tested (Figure 3).ConclusionWe have shown that the two CpG-rich PS ODN ASOs (818290 and 120704) triggered spontaneous platelet aggregation in PRP at 100µM. This effect was inhibited by a Syk inhibitor. 818290 has previously been identified to activate platelets through a Syk-dependent, GPVI receptor mediated mechanism (Flierl et al. JEM 2015). Here, we report for the first time that the aggregatory effects of 120704 have been identified to be Syk dependent as well, possibly through a similar interaction with platelet GPVI receptors.We have also presented novel data that therapeutically relevant concentrations of all the ASOs tested cause an increase in platelet-leukocyte aggregates in WB. Based on these data we highlight the importance of screening ASOs in multi-cellular assays, not just PRP, since there was no effect of the ASOs 104838 or 501861 on platelet aggregation. Enhanced formation of platelet-leukocyte aggregates could be one contributing factor for increased platelet clearance, explaining ASO-induced thrombocytopenia. Further investigation into the ASO-induced interactions between platelets and immune cells are warranted. Defining the mechanisms by which ASO-based drugs cause low platelet count may yield strategies to manage this drug-induced thrombocytopenia in patients. [Display omitted] DisclosuresThon:Platelet Biogenesis: Employment, Equity Ownership, Other: Co-founder, Patents & Royalties. Henry:Ionis Pharmaceuticals: Employment. Narayanan:Ionis Pharmaceuticals: Employment. Italiano:Platelet Biogenesis: Equity Ownership, Other: Co-founder, Patents & Royalties.

Platelet heterogeneity in activation-induced glycoprotein shedding: functional effects

AbstractThe platelet receptors glycoprotein Ibα (GPIbα) and GPVI are known to be cleaved by members of a disintegrin and metalloprotease (ADAM) family (ADAM10 and ADAM17), but the mechanisms and consequences of this shedding are not well understood. Our results revealed that (1) glycoprotein shedding is confined to distinct platelet populations showing near-complete shedding, (2) the heterogeneity between (non)shed platelets is independent of agonist type but coincides with exposure of phosphatidylserine (PS), and (3) distinct pathways of shedding are induced by elevated Ca2+, low Ca2+ protein kinase C (PKC), or apoptotic activation. Furthermore, we found that receptor shedding reduces binding of von Willebrand factor, enhances binding of coagulation factors, and augments fibrin formation. In response to Ca2+-increasing agents, shedding of GPIbα was abolished by ADAM10/17 inhibition but not by blockage of calpain. Stimulation of PKC induced shedding of only GPIbα, which was annulled by kinase inhibition. The proapoptotic agent ABT-737 induced shedding, which was caspase dependent. In Scott syndrome platelets that are deficient in Ca2+-dependent PS exposure, shedding occurred normally, indicating that PS exposure is not a prerequisite for ADAM activity. In whole-blood thrombus formation, ADAM-dependent glycoprotein shedding enhanced thrombin generation and fibrin formation. Together, these findings indicate that 2 major activation pathways can evoke ADAM-mediated glycoprotein shedding in distinct platelet populations and that shedding modulates platelet function from less adhesive to more procoagulant.

Generation of procoagulant collagen- and thrombin-activated platelets in platelet concentrates derived from buffy coat: the role of processing, pathogen inactivation, and storage.

Collagen- and thrombin-activated (COAT) platelets (PLTs), generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. There is evidence that higher amounts of procoagulant COAT PLTs are associated with stroke, while a decreased ability to generate them is associated with bleeding diathesis. Our aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates (PCs) from buffy coat. Thus, we investigated the effect of processing, pathogen inactivation treatment (amotosalen-UVA), and PC storage. Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated (n = 5). Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC-1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxin (agonist of the collagen receptor GPVI) and THR. Preparation of PCs resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 5'-diphosphate sodium (ADP). Storage further decreased ADP response. Minor differences were observed between untreated or amotosalen-UVA-treated PCs. Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP.

Reverse correlations of collagen-dependent platelet aggregation and adhesion with GPVI shedding during storage.

Platelet receptor GPVI plays an important role in platelet firm adhesion to site of vascular injury. Receptor ligation with collagen, in company with other agonist/receptor interactions, augments inside out signaling pathways leading to platelet aggregation and thrombus formation. As GPVI expression is significantly modulated by ectodomain shedding, this study aimed to examine whether GPVI shedding functionally affects collagen-mediated platelet activation during storage. 6 PRP-platelet concentrates were subjected to adhesion analysis on collagen matrix under mild stirring condition as well as collagen-induced aggregation on day 1, 3 and 5 post-storage. Concurrently, platelet supernatants of same samples were fractionated by ultra-centrifugation and obtained micro-particle-free samples were subjected to western blot analysis for the evaluation of GPVI shedding. We showed a direct correlation between collagen-dependent platelet aggregation and adhesion (r = 0.8, p = 0.0001). The increasing levels of GPVI shedding during storage were in reverse correlation with collagen-induced platelet aggregation (r = - 0.82, p = 0.0004) which was significantly reducing during storage. Platelet adhesion to collagen matrix significantly decreased post-storage while it was also reversely correlated with the levels of GPVI shedding during 5 days storage of platelets (r = - 0.69, p = 0.002). Data presented here demonstrated that progressive shedding of surface adhesion receptor GPVI can affect its functional activities in stored platelets. Thereby considering the crucial role of GPVI in platelet adhesion to the site of injury, whether the therapeutic efficacy of banked platelet products could be influenced by storage-dependent shedding of this receptor, remains to be answered in future studies.

Association of Oxidative Stress and Platelet Receptor Glycoprotein GPIbα and GPVI Shedding During Nonsurgical Bleeding in Heart Failure Patients With Continuous-Flow Left Ventricular Assist Device Support.

Nonsurgical bleeding (NSB) in heart failure (HF) patients with continuous-flow left ventricular assist device (CF-LVAD) support is the most common clinical complication. The aim of this study was to investigate the association between oxidative stress and platelet glycoproteins GPIbα and GPVI shedding on the incidence of NSB in CF-LVAD patients. Fifty-one HF patients undergoing CF-LVAD implantation and 11 healthy volunteers were recruited. Fourteen patients developed NSB (bleeder group) during 1 month follow-up duration, while others were considered nonbleeder group (n = 37). Several biomarkers of oxidative stress were quantified at baseline and weekly intervals in all patients. Surface expression and plasma elements of platelet receptor glycoproteins GPIbα and GPVI were measured. Oxidative stress biomarkers and platelet GPIbα and GPVI receptor-shedding (decreased surface expression and higher plasma levels) were found to be preexisting conditions in baseline samples of both groups of HF patients when compared with healthy volunteers. Significantly elevated oxidative stress biomarkers and platelet glycoprotein receptor shedding were observed in postimplant bleeder group temporarily when compared with nonbleeder group. Strong significant associations between biomarkers of oxidative stress and platelet glycoprotein receptor shedding were observed, suggesting a possible role of oxidative stress in platelet integrin shedding leading to NSB in CF-LVAD patients. Receiver operating characteristic analyses of GPIbα and GPVI indicated that the likelihood of NSB had a predictive power of bleeding complication in CF-LVAD patients. In conclusion, elevated oxidative stress may play a role in GPIbα and GPVI shedding in the event of NSB. Thus, oxidative stress and GPIbα and GPVI shedding may be used as potential biomarkers for bleeding risk stratification in those patients.

NLRP3 regulates platelet integrin αIIbβ3 outside-in signaling, hemostasis and arterial thrombosis.

In addition to their hemostatic function, platelets play an important role in regulating the inflammatory response. The platelet NLRP3 inflammasome not only promotes interleukin-1β secretion, but was also found to be upregulated during platelet activation and thrombus formation in vitro. However, the role of NLRP3 in platelet function and thrombus formation in vivo remains unclear. In this study, we aimed to investigate the role of NLRP3 in platelet integrin αIIbβ3 signaling transduction. Using NLRP3−/− mice, we showed that NLRP3-deficient platelets do not have significant differences in expression of the platelet-specific adhesive receptors αIIbβ3 integrin, GPIba or GPVI; however, NLRP3−/− platelets transfused into wild-type mice resulted in prolonged tail-bleeding time and delayed arterial thrombus formation, as well as exhibiting impaired spreading on immobilized fibrinogen and defective clot retraction, concomitant with decreased phosphorylation of c-Src, Syk and PLCγ2 in response to thrombin stimulation. Interestingly, addition of exogenous recombinant interleukin-1β reversed the defect in NLRP3−/− platelet spreading and clot retraction, and restored thrombin-induced phosphorylation of c-Src/Syk/PLCγ2, whereas an anti-interleukin-1β antibody blocked spreading and clot retraction mediated by wild-type platelets. Using the direct NLRP3 inhibitor, CY-09, we demonstrated significantly reduced human platelet aggregation in response to threshold concentrations of collagen and ADP, as well as impaired clot retraction in CY-09-treated human platelets, supporting a role for NLRP3 also in regulating human platelet αIIbβ3 outside-in signaling. This study identifies a novel role for NLRP3 and interleukin-1β in platelet function, and provides a new potential link between thrombosis and inflammation, suggesting that therapies targeting NLRP3 or interleukin-1β might be beneficial for treating inflammation-associated thrombosis.

Abstract 532: Shear-induced Release of Platelet Receptors Contributes to Bleeding Outcomes in Patients With Lvads or Exposed to Ecmo

Despite advances in design and materials, as well as pharmacological prophylaxis, hemostatic complications continue to plague device recipients. Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. We examined blood samples from patients with heart failure or in receipt of high fluid shear mechanocirculatory support to assess whether loss of platelet receptors occurs in vivo , and the relationship with acquired von Willebrand syndrome (AVWS) and changes in inflammatory cytokines. Platelet counts, levels of inflammatory cytokines IL-1β, IL-6, TNFα, MCP-1, IL-17A and IFNγ, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 13 continuous flow VAD (CF-VAD), 14 ECMO, and 24 heart failure patients. Levels of platelet receptors were measured by flow cytometry or ELISA. The loss of high molecular weight VWF multimers was observed in 12 of 13 CF-VAD and 7 of 14 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Significant differences in levels of inflammatory cytokines monocyte-chemoattractant protein and tissue necrosis factor-a in a subset of patients with decompensated HF. These data link AVWS and increased platelet receptor shedding in patients with CFVADs or ECMO. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CFVAD and ECMO patients may be linked with extent of inflammation and may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.

MMP‐13 binds to platelet receptors αIIbβ3 and GPVI and impairs aggregation and thrombus formation

Unlabelled Box BackgroundAcute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. ObjectivesHere we sought to elucidate whether matrix metalloproteinase‐13 (MMP‐13), a collagenolytic metalloproteinase up‐regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. ResultsWe demonstrate that MMP‐13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbβ3) and platelet glycoprotein (GP)VI. The interactions between MMP‐13, GPVI and αIIbβ3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. ConclusionsOur data demonstrate a role for MMP‐13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP‐13 in atherothrombotic pathologies.

Oligomeric and fibrillar amyloid beta 42 induce platelet aggregation partially through GPVI

The effects of the Alzheimer’s disease (AD)-associated Amyloid-β (Aβ) peptides on platelet aggregation have been previously assessed, but most of these studies focused on Aβ40 species. It also remains to be determined which distinct forms of Aβ peptides exert differential effects on platelets. In AD, oligomeric Aβ42 species is widely thought to be a major contributor to the disease pathogenesis. We, therefore, examine the ability of oligomeric and fibrillary Aβ42 to affect platelet aggregation. We show that both forms of Aβ42 induced significant platelet aggregation and that it is a novel ligand for the platelet receptor GPVI. Furthermore, a novel binding peptide that reduces the formation of soluble Aβ42 oligomers was effective at preventing Aβ42-dependent platelet aggregation. These results support a role for Aβ42 oligomers in platelet hyperactivity.

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.

High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.

Molecular modelling studies in explaining the higher GPVI antagonistic activity of the racemic 2-(4-methoxyphenylsulfonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxamide than its enantiomers

The GPVI receptor on the platelets plays a major role in inhibiting arterial thrombosis with limited risk of bleeding and is considered a potential anti-thrombotic target for arterial thrombosis. In the reported anti-thrombotics, tetrahydropyridoindoles, the title compound was the best inhibitor of the collagen mediated platelet aggregation by antagonizing the platelet receptor GPVI. Interestingly, the racemic title compound showed better antagonism (IC50 racemate = 6.7 μM) than either of its enantiomers (IC50 S enantiomer = 25.3 μM; IC50 R enantiomer = 126.3 μM). In order to explain this, the molecular modelling approaches viz. site map analysis, protein–protein docking and molecular dynamics simulation were carried out, which led to the identification of a second binding site located near the primary antagonist binding site known to bind losartan. The induced fit docking studies for both the enantiomers at the primary and secondary binding sites showed that the S-enantiomer has better interactions at the primary binding site than the R-enantiomer, while the R-enantiomer has better interactions at the secondary site than the S-enantiomer. Hence, the overall interactions of the racemic compound containing equimolar mixture may be higher than any one of the enantiomers and may explain the higher activity than its enantiomers of the racemic compound.

161 Btk inhibitors: friends or foes?

IntroductionBruton’s tyrosine kinase (BTK) plays a crucial role in the development and maturation of B-cells. A common side effect of Ibrutinib, a BTK inhibitor approved for the treatment of chronic...

An absence of platelet activation following thalidomide treatment in vitro or in vivo

Increased risk of thromboembolism and platelet hyperreactivity has been reported in patients receiving thalidomide therapy. Whether thalidomide induces platelet activation directly or through other factors remains unclear. The aim of this study was to evaluate the effect of thalidomide on platelet activation under resting conditions in vitro and in vivo. Isolated human or mouse platelets were treated with different concentrations of thalidomide (10, 50 and 100 μg/ml) for 60 min at 37°C followed by analysis of platelet surface expression of platelet receptors GPIbα, GPVI, αIIbβ3 and P-selectin, and PAC-1 or fibrinogen binding, by flow cytometry and collagen- or ADP-induced platelet aggregation. In addition, thalidomide (200 mg/kg) was intraperitoneally injected into mice for analysis of the effect of thalidomide on platelet activation in vivo. No increased expression of P-selectin, PAC-1 or fibrinogen binding was observed in either human and mouse platelets after thalidomide treatment in vitro for 60 min at 37°C. Thalidomide treatment also did not affect expression of GPIbα, GPVI or αIIbβ3, nor did it affect collagen- or ADP-induced platelet aggregation at threshold concentrations. However, while mice injected with thalidomide displayed no increased surface expression of platelet P-selectin or αIIbβ3, there was a significantly shortened tail bleeding time, thrombin time, prothrombin time together with higher levels of Factor IX and fibrinogen. In conclusion, thalidomide at therapeutic doses does not directly induce platelet activation under resting conditions in vitro or in vivo, but results in increased procoagulant activity, which could explain the thalidomide-dependent prothrombotic tendency in patients.