Collagen-induced Platelet Aggregation Research Articles

Hsp47 Inhibitor Col003 Attenuates Collagen-Induced Platelet Activation and Cerebral Ischemic-Reperfusion Injury in Rats.

Ischemic stroke is a major type of stroke worldwide currently without effective treatment, although antiplatelet therapy is an existing option for it. In previous studies, heat shock protein 47 (Hsp47) was found to be expressed on the surface of human and mice platelets and to strengthen the interaction between platelets and collagen. In recent years, Col003 was discovered to inhibit the interaction of Hsp47 with collagen. We evaluated whether the Hsp47 inhibitor Col003 is a promising therapeutic agent for ischemic stroke. Here, we first verified that Hsp47 is also expressed on the surface of rat platelets, and its inhibitor Col003 significantly inhibited thrombus formation in the FeCl3-induced rat carotid arterial thrombus model. Both Col003 and clopidogrel did not alter the bleeding time or coagulation parameters, while aspirin increased the tail-bleeding time (p < 0.05). The low cytotoxicity level of Col003 to rat platelets and human liver cells was similar to those of aspirin and clopidogrel. Col003 inhibited collagen-induced platelet aggregation, adhesion, [Ca2+]i mobilization, P-selectin expression, reactive oxygen species production and the downstream signal pathway of collagen receptors. The results of the middle cerebral artery occlusion model indicated that Col003 has a protective effect against cerebral ischemic–reperfusion injury in rats. The Hsp47 inhibitor Col003 exerted antiplatelet effect and protective effect against brain damage induced by ischemic stroke through the inhibition of glycoprotein VI (GPVI)and mitogen-activated protein kinase (MAPK) signaling events, which might yield a new antiplatelet agent and strategy to treat ischemic stroke.

Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1–116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

Inhibition of human platelet aggregation by Choisyaternatine isolated from Choisya ternata (Rutaceae)

A newly reported alkaloid was isolated from the hexane extract of Choisya ternata Kunth. (Rutaceae) leaves, identified as 6,8-Dimethoxy-7-(3-methylbut-2-enyloxy)quinolin-2-ol (choisyaternatine) (A), together with the known compounds skimmianine (B), lup-20(29)-en-3?-ol (lupeol) (C) and tecleamaniensine A (D). Compounds C and D are reported for the first time in this species. The structures of isolated compounds were elucidated by extensive spectroscopic analyses and comparison with previously reported spectroscopic data. The structure of choisyaternatine was further confirmed based on X-ray data analysis. Anti-platelet activity of hexane extract, choisyaternatine and skimmianine was also evaluated. The extract was able to significantly inhibit collagen-induced platelet aggregation at all concentrations tested (25, 50 and 100 µg/ml). The new compound, choisyaternatine, has shown a significant inhibition of collagen-induced platelet aggregation demonstrated in a concentration dependent manner from 250 µM. The concentrations which inhibit 50% of platelet aggregation (IC50) were found to be 564 µM for skimmianine and 698 µM for choisyaternatine. This is also the first report for the antiplatelet activity for this plant and these alkaloids using human platelets.

A randomised controlled trial to assess the antithrombotic effects of aspirin in type 1 diabetes: role of dosing and glycaemic control

BackgroundThe enhanced thrombotic milieu in diabetes contributes to increased risk of vascular events. Aspirin, a key antiplatelet agent, has inconsistent effects on outcomes in diabetes and the best dosing regimen remains unclear. This work investigated effects of aspirin dose and interaction with glycaemia on both the cellular and protein components of thrombosis.MethodsA total of 48 participants with type 1 diabetes and 48 healthy controls were randomised to receive aspirin 75 or 300 mg once-daily (OD) in an open-label crossover study. Light transmittance aggregometry and fibrin clot studies were performed before and at the end of each treatment period.ResultsAspirin demonstrated reduced inhibition of collagen-induced platelet aggregation (PA) in participants with diabetes compared with controls, although the higher dose showed better efficacy. Higher aspirin dose facilitated clot lysis in controls but not individuals with diabetes. Collagen-induced PA correlated with glycaemic control, those in the top HbA1c tertile having a lesser inhibitory effect of aspirin. Threshold analysis suggested HbA1c levels of > 65 mmol/mol and > 70 mmol/mol were associated with poor aspirin response to 75 and 300 mg daily doses, respectively. Higher HbA1c was also associated with longer fibrin clot lysis time.ConclusionsPatients with diabetes respond differently to the antiplatelet and profibrinolytic effects of aspirin compared with controls. In particular, those with elevated HbA1c have reduced inhibition of PA with aspirin. Our findings indicate that reducing glucose levels improves the anti-thrombotic action of aspirin in diabetes, which may have future clinical implications.Trial registrationEudraCT, 2008-007875-26, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2008-007875-26.

Abstract 9869: Adverse Effects of Smoking/Vaping of Several Types of Tobacco Products and Marijuana on Cardiac Function and Platelet Aggregation

Tobacco and marijuana smoke contains ~7,000 chemicals that cause disorders in cardiac autonomic function, vascular biology, and platelet function. Along with marijuana, e-cigarettes (e-cigs) and heat-not-burn tobacco products (e.g., IQOS) are often viewed as being less harmful than smoking tobacco. However, there are many unknowns about how marijuana, e-cigs, and IQOS impact cardiovascular physiology and platelet function over the long term. To explore cardiovascular effects of smoking/vaping, we exposed conscious rats to single (one time) and multiple (one time daily for 10 days) pulsatile mainstream smoking/vaping of e-cigs (JUUL), IQOS (American and Russian HeatSticks), marijuana (~10% THC), and cannabinoid-depleted (“placebo”) marijuana, relative to regular tobacco (Marlboro Red) smoking and air controls. Cardiac function was assessed by echocardiography pre- and post- single exposure. Platelet aggregation was measured to determine changes in platelet response immediately post-single exposure and one day after the last of multiple exposures. Single exposure induced acute effects on echocardiographic function with reduced ventricular end-diastolic volume (EDV) in IQOS and marijuana groups of both genders (Fig. A), potentially indicating incomplete ventricular relaxation between heart beats after exposure. However, marijuana caused adjustments of end-systolic volume (ESV) and EDV without changes in ejection fraction (EF) in females, with smaller ESV and reduced EDV. Tobacco, marijuana, and placebo marijuana smoking immediately increased collagen-induced platelet aggregation post-single exposure in female rats. Tobacco smoking, JUUL, IQOS, and both kinds of marijuana significantly increased platelet aggregation post-multiple exposure as compared to air controls in mixed genders (Fig. B). P values ≤ 0.1 are shown. Conclusion: smoking/vaping causes clinically relevant adverse effects on cardiac function and platelet aggregation.

COVID-19 adenovirus vaccine triggers antibodies against PF4 complexes to activate complement and platelets

BackgroundVaccine-induced thrombotic thrombocytopenia (VITT) is a rare coagulation disorder reported after administration of COVID-19 adenovirus-vectored vaccines. VITT is mediated by anti-platelet factor 4 (PF4) antibodies activating platelets through the Fcγ-receptor II (FcγRII), and it is associated with strong fibrin turnover. The complement system is involved in several other immunothrombotic entities, but its impact on VITT is not established. ObjectiveTo assess antibodies in interaction with the activation of platelets and complement triggered by VITT. MethodsAntibodies against adenovirus type 2 hexon protein, ChAdOx1 adenoviral vector-specific IgG and PF4 were analyzed by enzyme immunoassays from VITT patients (n = 5). The EDTA plasma samples of the patients and controls were used to measure both terminal complement complexes (TCC) by ELISA and aggregation of healthy donor platelets. We studied the effects of human immunoglobulin (IVIG) and glycoprotein IIb/IIIa inhibitor (GPIIb/IIIa) on spontaneous and collagen-induced platelet aggregation supplemented with VITT plasma. ResultsNone of the patients had experienced a COVID-19 infection. Antibody analyses confirmed the immunogenicity of the adenovirus-vectored ChAdOx1 vaccine. Moreover, VITT plasma had anti-PF4 antibodies and elevated TCC levels as a sign of complement activation. In isolated healthy donor platelets, VITT patient plasma caused marked, spontaneous aggregation of platelets, which was abolished by eptifibatide and high-dose therapeutic IVIG. ConclusionsOur findings suggest that VITT is triggered by antibodies against adenovirus vector and PF4-polyanion complexes which strongly co-activate complement and platelets. The spontaneous platelet aggregation was suppressed by IVIG or eptifibatide, indicating that besides FcγRII, also GPIIb/IIIa receptor exerts platelet procoagulant role in VITT.

Rutaecarpine, an Alkaloid from Evodia rutaecarpa, Can Prevent Platelet Activation in Humans and Reduce Microvascular Thrombosis in Mice: Crucial Role of the PI3K/Akt/GSK3β Signal Axis through a Cyclic Nucleotides/VASP-Independent Mechanism.

The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs. Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1–5 μM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists (e.g., thrombin). Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3β pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.

Sphingosine-1-phosphate receptors modulate platelet activation and thrombin formation

Abstract Background The bioactive lipid sphingosine-1-phosphate (S1P) is known as a modulator of various cell functions, including cell proliferation, migration, adhesion and survival. Hence, S1P plays a crucial role in both inflammatory and hematopoietic regulations. It is also known that S1P affects key processes of hemostasis, as platelets store and upon activation release large amounts of the phospholipid. S1P acts through binding to its five G-coupled receptors (S1PR1–5). Accumulating evidence suggests that S1P is involved in mechanisms of atherogenesis linking platelet activation and thrombus formation to inflammatory responses. However, the precise role of S1P and its receptors during platelet activation and hemostasis are still under debate. Purpose Until today, the effects of S1P in hemostasis are poorly understood. The aim of this study is to investigate the functions of specific agonists and antagonists for each of the S1PRs 1–5 on platelet aggregation and thrombin generation. Methods Blood was obtained from healthy volunteers after written informed consent. For both platelet aggregation studies using light transmission aggregometry (LTA) and thrombin generation measurements quantified by calibrated automated thrombography (CAT), platelet-rich-plasma (PRP) was used. PRP was generated by centrifugation from whole blood. PRP was incubated with either 10 μM S1PR-agonists or -antagonists for 15 minutes. Subsequently, platelets were activated with adenosine diphosphate (ADP) or collagen to evaluate potential synergistic or antagonistic effects. Results Platelet aggregation weakens after 2–3 hours. As described in the literature, this is presumably due to activation effects and partial release of platelet granules. Determined by LTA, we found that pre-incubation with the S1PR1-agonist CYM5442 maintains and even elevates ADP- but not collagen-induced platelet aggregation for up to 1.5 h. This effect was reversed by co-incubation with the S1PR1-antagonist Ex26. We only observed this findings for S1PR1. No effect on platelet aggregation was detected when PRP was incubated with S1PR1–5-agonists or -antagonists (10 μM) alone. In comparison, none of the S1PR-agonists or -antagonists did directly affect thrombin formation in PRP measured by CAT. However, co-incubation with a S1PR4-agonist and ADP (5 μM) shortened the lag time to thrombin formation significantly. Furthermore, co-incubation with collagen (5 μg/mL) and agonists for S1PR1, 3 or 4 did reduce the time-to-peak (ttpeak) of thrombin formation. Conclusion Activation of S1PR1 maintains the ADP-induced platelet aggregation. In addition, co-activation of S1PR1, 3 or 4 and collagen enhances and accelerates thrombin generation. Consequently, S1P and its receptors appear to play an important role in different processes of hemostasis. Thus, S1PRs may be promising future therapeutic targets for diseases involving elevated platelet activation such as coronary heart disease or diabetes. Funding Acknowledgement Type of funding sources: None.

Clopidogrel treatment inhibits P2Y2-Mediated constriction in the rabbit middle cerebral artery

Clopidogrel is an effective purinergic 2Y12 receptor (P2Y12) antagonist used to prevent arterial thrombosis, but its use is associated with adverse bleeding. Clinical studies have demonstrated that clopidogrel users have an increased risk of cerebral microbleeds and intracerebral hemorrhage. Our previous studies suggest that non-platelet mechanisms mediate these adverse bleeding events; we hypothesize that clopidogrel or one of its metabolites interacts with blood vessels directly to cause bleeding. New Zealand white rabbits (1.9–2.7 kg) were treated orally with vehicle or clopidogrel (3 or 10 mg/kg) for three days. On the fourth day, the rabbits were anesthetized for blood collection and then euthanized. The brain was collected, and the middle cerebral arteries were isolated. We used light transmission aggregometry and pressure myography to elucidate the mechanisms of the off-target effects associated with clopidogrel treatment. We confirmed that inhibition of P2Y12 activation by clopidogrel inhibited ADP-induced platelet aggregation but had no impact on P2Y12-independent arachidonic acid- or collagen-induced platelet aggregation. Analysis of middle cerebral arteries from clopidogrel treated rabbits showed that clopidogrel did not affect P2Y4, P2Y6, and P2Y14 receptor-mediated contraction but attenuated the contractile response after P2Y2 receptor activation. Further analysis determined P2Y2-mediated constriction was endothelium-dependent. Vasoconstriction is a primary component of hemostasis, and impaired vasoconstriction can prolong bleeding. These results suggest clopidogrel inhibits the endothelial P2Y2 receptor in the middle cerebral artery, which provides a mechanistic explanation for the adverse cerebral bleeding associated with the drug.

Ponatinib Exerts an Inhibitory Effect on Collagen-induced Platelet Aggregation and Generation of Coated-Platelets

BCR-ABL tyrosine kinase inhibitors (TKIs) are exceptionally effective drugs in the treatment of chronic myeloid leukemia, nevertheless, TKIs have also an effect on platelets. We aimed to investigate the effect of a third-generation TKI, ponatinib on platelet functions. Collagen-induced platelet aggregation and coated-platelet formation were examined using in vitro and in ex vivo samples of patients on ponatinib therapy. In platelet rich plasma of healthy volunteers, ponatinib at a supra-therapeutic concentration (1,000 nM) significantly impaired collagen induced platelet aggregation (p≤0.01) and reduced the formation of coated-platelets at 150 nM ponatinib concentration (p≤0.05). In addition, upon glycoprotein VI (GPVI) receptor activation, a significantly lower percentage of PAC1 binding platelets (p≤0.05) was observed at 1,000 nM final concentration of ponatinib. Platelets, isolated from patients on ponatinib therapy showed impaired collagen elicited aggregation response, already in pre-dose samples compared to healthy donors. The therapeutic concentration of ponatinib impairs platelet activation processes elicited by GPVI receptor agonists.

Antithrombotic Effect of the Ethanol Extract of Angelica gigas Nakai (AGE 232).

Cardiovascular diseases, such as stroke, are the most common causes of death in developed countries. Ischemic stroke accounts for 85% of the total cases and is caused by abnormal thrombus formation in the vessels, causing deficient blood and oxygen supply to the brain. Prophylactic treatments include the prevention of thrombus formation, of which the most used is acetylsalicylic acid (ASA); however, it is associated with a high incidence of side effects. Angelica gigas Nakai (AG) is a natural herb used to improve blood circulation via anti-platelet aggregation, one of the key processes involved in thrombus formation. We examined the antithrombotic effects of AGE 232, the ethanol extract of A. gigas Nakai. AGE 232 showed a significant reduction in death or paralysis in mice caused by collagen/epinephrine-induced thromboembolism in a dose-dependent manner and inhibition of collagen-induced human platelet aggregation in a concentration-dependent manner. Additionally, AGE 232-treated mice did not show severe bleeding in the gut compared to ASA-treated mice. AGE 232 resulted in a decrease in the number of neutrophils attached to the human umbilical vein endothelial cells (HUVECs) and lower inhibition of COX-1 in response to bleeding and damage to blood vessels, a major side effect of ASA. Therefore, AGE 232 can prevent thrombus formation and stroke.

Dose-dependent effects of anthocyanin supplementation on platelet function in subjects with dyslipidemia: A randomized clinical trial.

BackgroundDyslipidemia induces platelet hyperactivation and hyper-aggregation, which are linked to thrombosis. Anthocyanins could inhibit platelet function in vitro and in mice fed high-fat diets with their effects on platelet function in subjects with dyslipidemia remained unknown. This study aimed to investigate the effects of different doses of anthocyanins on platelet function in individuals with dyslipidemia.MethodsA double-blind, randomized, controlled trial was conducted. Ninety-three individuals who were initially diagnosed with dyslipidemia were randomly assigned to placebo or 40, 80, 160 or 320 mg/day anthocyanin groups. The supplementations were anthocyanin capsules (Medox, Norway). Platelet aggregation by light aggregometry of platelet-rich plasma, P-selectin, activated GPⅡbⅢa, reactive oxygen species (ROS), and mitochondrial membrane potential were tested at baseline, 6 weeks and 12 weeks.FindingsCompared to placebo group, anthocyanins at 80 mg/day for 12 weeks reduced collagen-induced platelet aggregation (-3.39±2.36%) and activated GPⅡbⅢa (-8.25±2.45%) (P < 0.05). Moreover, compared to placebo group, anthocyanins at 320 mg/day inhibited collagen-induced platelet aggregation (-7.05±2.38%), ADP-induced platelet aggregation (-7.14±2.00%), platelet ROS levels (-14.55±1.86%), and mitochondrial membrane potential (7.40±1.56%) (P < 0.05). There were dose-response relationships between anthocyanins and the attenuation of platelet aggregation, mitochondrial membrane potential and ROS levels (P for trend <0.05). Furthermore, significantly positive correlations were observed between changes in collagen-induced (r = 0.473) or ADP-induced (r = 0.551) platelet aggregation and ROS levels in subjects with dyslipidemia after the 12-week intervention (P < 0.05).InterpretationAnthocyanin supplementation dose-dependently attenuates platelet function, and 12-week supplementation with 80 mg/day or more of anthocyanins can reduce platelet function in individuals with dyslipidemia.

The Synergistic Effect of Ginkgo biloba Extract 50 and Aspirin Against Platelet Aggregation.

PurposeWe aimed to investigate potential synergistic antiplatelet effects of Ginkgo biloba extract (GBE50) in combination with aspirin using in vitro models.MethodsArachidonic acid (AA), platelet activating factor (PAF), adenosine 5ʹ-diphosphate (ADP) and collagen were used as inducers. The antiplatelet effects of GBE50, aspirin and 1:1 combination of GBE50 and aspirin were detected by microplate method using rabbit platelets. Synergy finder 2.0 was used to analyze the synergistic antiplatelet effect. The compounds in GBE50 were identified by UPLC-Q/TOF-MS analysis and the candidate compounds were screened by TCMSP database. The targets of candidate compounds and aspirin were obtained in TCMSP, CCGs, Swiss target prediction database and drugbank. Targets involving platelet aggregation were obtained from GenCLiP database. Compound-target network was constructed and GO and KEGG enrichment analyses were performed to identify the critical biological processes and signaling pathways. The levels of thromboxane B2 (TXB2), cyclic adenosine monophosphate (cAMP) and PAF receptor (PAFR) were detected by ELISA to determine the effects of GBE50, aspirin and their combination on these pathways.ResultsGBE50 combined with aspirin inhibited platelet aggregation more effectively. The combination displayed synergistic antiplatelet effects in AA-induced platelet aggregation, and additive antiplatelet effects occurred in PAF, ADP and collagen induced platelet aggregation. Seven compounds were identified as candidate compounds in GBE50. Enrichment analyses revealed that GBE50 could interfere with platelet aggregation via cAMP pathway, AA metabolism and calcium signaling pathway, and aspirin could regulate platelet aggregation through AA metabolism and platelet activation. ELISA experiments showed that GBE50 combined with aspirin could increase cAMP levels in resting platelets, and decreased the levels of TXB2 and PAFR.ConclusionOur study indicated that GBE50 combined with aspirin could enhance the antiplatelet effects. They exerted both synergistic and additive effects in restraining platelet aggregation. The study highlighted the potential application of GBE50 as a supplementary therapy to treat thrombosis-related diseases.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified >13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values >0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Pimpinellin Inhibits Collagen-induced Platelet Aggregation and Activation Through Inhibiting Granule Secretion and PI3K/Akt Pathway.

Pimpinellin is a coumarin-like compound extracted from the root of Toddalia asiatica. Its effects on platelet function has not been investigated. This study found that pimpinellin pretreatment effectively inhibited collagen-induced platelet aggregation, but did not alter ADP- and thrombin-induced aggregation. Platelets pretreated with pimpinellin showed reduced α granule (CD62) level and secretion of dense granule (ATP release). Pimpinellin-treated platelets also exhibited decreased clot reaction and TxB2 production. Pimpinellin pretreatment suppressed adhesion and spreading of human platelets on the fibrinogen coated surface. Analysis of tail bleeding time of mice administered with pimpinellin (40 mg/kg) revealed that pimpinellin did not change tail bleeding time significantly, number of blood cells, and APTT and PT levels. Pimpinellin inhibited collagen-induced ex vivo aggregation of mice platelets. Immunoblotting results showed that pimpinellin suppressed collagen-induced phosphorylation of PI3K-Akt-Gsk3β and PKC/MAPK in platelets.

High residual platelet aggregation in patients with coronary artery disease: a new methodological approach to detection

Aim. To develop a new methodological approach to assessment of collagen-induced platelet aggregation in patients with coronary artery disease (CAD) and to determine the quality of various methods for detecting high residual platelet reactivity (HRPR) to predict the risk of myocardial perfusion disturbance. Materials and methods. 36 patients (10 men and 26 women) aged 41–83 years and having stable CAD were examined. All patients had been undergoing continuous antiaggregation therapy for 6 months. We evaluated platelet aggregation using a laser analyzer with collagen as an aggregation inducer by the standard method 1 and our own patented method 2. The degree of platelet aggregation (%) and the size of aggregates in relative units (r.u.) in platelet-rich plasma were estimated. Myocardial perfusion scintigraphy with 99mTc-methoxy-isobutylisonitrile was performed according to a two-day stress-rest protocol. The summed stress score (SSS) values were used for analysis. SSS &lt; 4 was regarded as normal myocardial perfusion. Results . The degree of platelet aggregation according to method 1 was 12 (5; 64)%, the aggregate size was 3 (2; 7) r.u. The degree of platelet aggregation according to method 2 was 44 (13; 78)%, and the aggregate size was 5 (4; 8) r.u. Method 2 allowed to diagnose the presence of myocardial ischemia with an aggregation degree ≥ 44.9% with sensitivity of 84% and specificity of 92% (area under the curve (AUC) = 0.89; p &lt; 0.0001; odds ratio (OR) 2.18; 95% confidence interval (CI) 0.57–0.98) and an increase in aggregate size ≥ 4.80 r.u. with sensitivity of 84% and specificity of 84% (AUC = 0.95; p &lt; 0.00001; OR 5.83; 95% CI 0.72–0.99). Conclusion. In patients with CAD, detection of high rates of collagen- induced platelet aggregation using the patented technique is associated with the risk of impaired myocardial perfusion. The developed new methodological approach to detection of HRPR allowed to determine high risk of atherothrombotic complications in additional 22% of the examined patients.

The protein disulphide isomerase inhibitor CxxCpep modulates oxidative burst and mitochondrial function in platelets

BackgroundWe have previously described CxxCpep, a peptide with anti-platelet properties that inhibits peri/epicellular protein disulphide isomerase (pecPDI) by forming a mixed disulfide bond with Cys400 within the pecPDI active site. ObjectivesHere we sought to determine if pecPDI targeted by CxxCpep is relevant to redox mechanisms downstream of the collagen receptor GPVI in platelets. Methods and resultsRestriction of effects of CxxCpep to the platelet surface was confirmed by LC-MS/MS following cell fractionation. Platelet aggregation was measured in platelet-rich plasma (PRP) incubated with 30 μM CxxCpep or vehicle. CxxCpep inhibited collagen-induced platelet aggregation but exerted no effect in TRAP-6-stimulated platelets. PRP was incubated with DCFDA to measure oxidative burst upon platelet adhesion to collagen. Results showed that CxxCpep decreased oxidative burst in platelets adhered to immobilized collagen while the number of adherent cells was unaffected. Furthermore, flow cytometry studies using a FITC-maleimide showed that the GPVI agonist CRP stimulated an increase in free thiols on the platelet outer membrane, which was inhibited by CxxCpep. Finally, CxxCpep inhibited platelet mitochondrial respiration upon activation with collagen, but not with thrombin. ConclusionsOur data suggest that pecPDI is a potential modulator of GPVI-mediated redox regulation mechanisms and that CxxCpep can be further exploited as a template for new antiplatelet compounds.

International, Practice-Based Recommendations for Prediction of Bleedings and Anti-Platelet/Anti-Coagulant (AP/AC) Co-Medications in CLL Patients Taking Ibrutinib

Introduction: bleeding events (grade 1-2, 50%, grade 3-4 <5%) have been repeatedly reported in patients taking BTK inhibitors, including ibrutinib (IBR) and acalabrutinib. We lack clear recommendations from real-life clinical practice experience, since all existing management decision support systems have been made out from clinical trials (many of which didn't allow the recruitment of patients taking AC). Furthermore in clinical trials, no thorough exploration of platelet aggregation test was available.Methods: we reviewed the in vitro and in vivo evidence collected in 3 different centres from 3 countries (France, Russia, Austria) including: bleeding symptoms with grade, AP/AC co-medications, agonist-induced platelet aggregation tests (ristocetin RIPA, collagen CIPA), platelet counts. In the French series, complete PK evaluation (t0-0.5h-1h-2h-4h-6h) at one month was available for some patients (Cmax, Cmin, AUC).Results: based on our first 121 pts, our aggregation tests monitoring point towards several common considerations:1/ For patients taking IBR alone (without AP/AC):- Half patients' platelets do not demonstrate any collagen-induced platelet aggregation (CIPA) defect with IBR, even sampled at various time points. This 50/50 segregation between IBR-sensitive and IBR-resistant pts is also observed among 47 healthy donors' platelets. Furthermore, acalabrutinib (sparing TEC kinase more than IBR) in vitro induced very similar (in time and size) aggregation defects only in the IBR-sensitive pts, reinforcing the view of a BTK-effect with the two inhibitors.- Among the 50% early bleedings due to ibrutinib, 50% will recover over time (clinical and CIPA test recovery). As a consequence, in our global population 25-30% of patients still present bleeding after 6 months of therapy (100% grade 1-2). No PK parameter was found to explain the discrepancies of bleeding events among patients, suggesting dose reductions of IBR are not mandatory from the start to leverage bleeding risk. The late risk of severe haemorrhage (grade 3-4) seen in 5 patients seems to be linked with AC (not AP) intake.- the CIPA test has a very good sensitivity (85.7%) and a good specificity (62.5%) to discriminate the patients with bleeding diathesis the first 3 months. The specificity (not sensitivity) was however significantly reduced (33.3%) after 3 months, compromising its utility in pts requiring AC therapy while on ibrutinib for >3 months. Yet, the large majority of patients with bleeding syndromes had a weak aggregation response to collagen 3.3 µg/ml. A lower dose of collagen in vitro can be used (2.2 instead of 3.3microg/ml) to increase specificity after 3-6 months of ibrutinib.2/ for patients taking IBR + AP or AC- the percentage of patients with bleeding syndromes before 6 months did not increase significantly compared to patient under IBR treatment alone (the bleeding grade did not increase neither). Conversely, after 6 months, with AP the risk increases a little bit to 40% (all grade 1-2 except 1 grade 3) as compared to 90% bleeding events in the 10 pts with AC (mainly VKA, including 2 grade 3 and 1 grade 4).- the CIPA tests are no longer valid for patients under AP therapy before start of IBR, as their platelet aggregation response is very low, as expected. In vitro, addition of aspirin or clopidogrel to IBR-resistant platelets reduced aggregation to a significantly lesser extent than that observed in IBR-responsive platelets. This is of interest in the investigation of IBR pts requiring a dual AP therapy.- Most patients under AC and IBR with bleedings had also weak CIPA results. This result would suggest that a weak platelet response to collagen could increase the risk of bleeding (inherent to the AC drug), and therefore heparin should be the preferred choice with ibrutinib.- Take home message: our clinical and biological data collectively identify a strong concern with the use of a triplet therapy, IBR+AP+AC, which should be avoided.Conclusion: the monitoring of CIPA testing is not recommended before starting IBR if no AP/AC is added. In clinical situations where AP/AC is mandated, it can predict an “IBR-related” additional risk factor the bleeding risk of the AP drug. Our results are currently being compared with those of RIPA testing (in the Austrian cohort). We will present updated recommendation charts (including direct anticoagulants use) during the meeting. DisclosuresPanteleev:Amgen: Research Funding. Jaeger:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Payrastre:Janssen: Research Funding. Ysebaert:Janssen: Consultancy, Research Funding, Speakers Bureau.

The NLRP3 Inflammasome in Platelets Is Upregulated in Sickle Cell Disease and Promotes Platelet Aggregation and In Vitro Thrombosis

Sickle cell disease (SCD) is a severe monogenic disorder characterized by chronic hemolysis and abnormal coagulation and inflammation. Activated platelets play a critical role at the interphase of thrombosis and inflammation in the disease context. Sensing of free heme, a degradation byproduct of hemoglobin oxidation during hemolysis, involves the pattern recognition receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3), which typically forms multiprotein inflammasome complexes and controls caspase-1 activity and cleavage of IL-1β not only in immune cells but also platelets. We have shown recently that the NLRP3 inflammasome in platelets controls platelet activation, aggregation, and in vitro thrombus formation, which was dependent on platelet bruton's tyrosine kinase (BTK). The role of the platelet NLRP3 inflammasome and BTK in SCD, however, is unexplored.Platelets were isolated from humanized SCD mice (Townes), which express human sickle hemoglobin, and patients with SCD in steady state and acute vasoocclusive crisis. Platelet NLRP3 activity, as monitored by caspase 1 activation (assessed by fluorescent labeled inhibitor of caspase-1, FLICA), and cleavage and secretion of IL1β (assessed by Western blot and ELISA), was significantly elevated in SCD patients in steady state as compared to healthy subjects (FLICA: mean 340.3 ± 30.2 SD vs. mean 262.2 ± 33.9 SD, p<0.001), and further upregulated in SCD patients in crisis (FLICA: mean 390.3 ± 34.0 SD vs. mean 340.3 ± 30.2 SD, p<0.05). NLRP3 activity in platelets was also significantly increased in homozygous SCD mice, as compared to heterozygous and control mice (FLICA: mean 165.1 ± 32.4 SD vs. mean 86.7 ± 20.5 SD, p<0.001), which was significantly reversed by the heme scavenger hemopexin or pharmacological inhibition of the NLRP3 inflammasome/BTK pathway. Moreover, incubation of platelets with heme induced intraplatelet colocalization of NLRP3 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), as evaluated with immunofluorescence staining coupled with confocal laser scanning microscopy, indicating the assembly of the NLRP3 inflammasome in platelets upon exposure to heme. We studied the functional effects of platelet NLRP3 inflammasome upregulation in SCD, and found that collagen-induced platelet aggregation was significantly elevated in homozygous SCD mice as compared to control animals (area under the curve, AUC: mean 65.25 ± 7.99 SD vs. mean 40.60 ± 7.92 SD, p<0.05). In SCD patients in acute crisis, aggregation of circulating platelets was significantly upregulated as compared to patients in steady state (AUC: mean 113.70 ± 16.26 SD vs. mean 78.53 ± 1.93 SD, p<0.05). Moreover, platelet aggregation in steady state patients was significantly elevated as compared to healthy subjects (AUC: 78.53 ± 1.93 SD vs. mean 30.87 ± 4.34 SD, p<0.01). The upregulated platelet aggregation responses in homozygous SCD mice and SCD patients in acute crisis were dependent on heme/NLRP3/BTK pathway, as evaluated by hemopexin (mice: 69.3 ± 5.7% inhibition; patients: 61.6 ± 5.2% inhibition), the NLRP3 inhibitor MCC950 (mice: 75.7 ± 3.9% inhibition; patients: 72.1 ± 3.8% inhibition), and the BTK inhibitor CGI1746 (mice: 65.9 ± 3.6% inhibition; patients: 67.1 ± 4.1% inhibition) (p<0.01). Similar results were obtained with a collagen-coated flow chamber system.Our data show that the platelet NLRP3 inflammasome is upregulated in SCD via heme, which results in elevated platelet aggregation and in vitro thombus formation, and is potentially targetable using BTK inhibitors for affected patients. DisclosuresNo relevant conflicts of interest to declare.

PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura

PRN1008 is an oral, reversible covalent inhibitor of Bruton's tyrosine kinase (BTK) in clinical development for the treatment of multiple autoimmune diseases. BTK is an essential signaling element downstream of the B cell receptor (BCR), Fc-gamma receptor and Fc-epsilon receptor pathways. BTK activation is critical for B cell activation and maturation. BTK also regulates antibody mediated activation of other immune cells, such as macrophages, neutrophils and mast cells through Fc receptor signaling.Treatment with PRN1008 in vitro profoundly inhibited B cell activation and blocked antibody mediated activation of immune cells via Fc receptor signaling. In preclinical studies, PRN1008 demonstrated a significant dose-dependent reduction of platelet-loss in an anti-CD41 induced mouse model of immune thrombocytopenia. In addition, PRN1008 showed rapid and significant anti-inflammatory effects in an antibody driven rat arthus model and in spontaneous autoantibody-mediated canine pemphigus foliaceus. The preclinical data suggest that PRN1008 could reduce platelet destruction via inhibition of autoantibody/Fc-gamma receptor signaling in splenic macrophages, and also affect autoantibody generation through effects on B-cell activation, to diminish platelet loss in ITP.Platelets express high levels of BTK, however alternative signaling pathways exist which bypass BTK signaling to retain normal platelet functions. The effect of PRN1008 on platelet function was assessed in vitro in both normal healthy volunteer and ITP patient platelets using a standard panel of platelet agonists. In contrast to ibrutinib, treatment with PRN1008 at clinically relevant concentrations did not impact collagen-induced platelet aggregation in either normal or ITP patient platelet samples, or interfere with responses to all other platelet agonists tested. In the clinic, PRN1008 has been well tolerated. Thrombocytopenia, bleeding or bruising signals have not been reported. An analysis of platelet counts from the ongoing 12 week study of pemphigus vulgaris (NCT02704429) showed no evidence of an effect of PRN1008 on platelet counts in the normal range.Principia Biopharma is developing PRN1008 for the treatment of autoimmune diseases including ITP, blistering skin diseases such as pemphigus vulgaris and other immunologic disorders where autoantibodies and/or B-cell signaling are pathogenic. DisclosuresLangrish:Principia Biopharma: Employment. Bradshaw:Principia Biopharma: Employment. Owens:Principia Biopharma: Employment. Campbell:Principia Biopharma: Employment. Francesco:Principia Biopharma: Employment. Karr:Principia Biopharma: Employment. Murray:Principia Biopharma: Employment. Quesenberry:Principia Biopharma: Employment. Smith:Principia Biopharma: Consultancy. Taylor:Principia Biopharma: Consultancy. Zhu:Principia Biopharma: Employment. Nunn:Principia Biopharma: Employment. Gourlay:Principia Biopharma: Employment.