Platelet Antagonists Research Articles

Abstract TP84: Safety and Effectiveness of Ticagrelor in Patients With Clopidogrel Resistance Undergoing Angioplasty, Stent or Flow Diverter Placement

Background and Purpose: Nearly 30% of the patients have clopidogrel resistance and are at high risk for in-stent thrombosis and adverse ischemic events. Ticagrelor is an allosteric platelet antagonist that reversibly inhibits the P2Y12 platelet receptor more potently than clopidogrel. We report upon the safety and effectiveness of ticagrelor in patients with cerebrovascular diseases and clopidogrel resistance undergoing angioplasty and/or stent or flow diverter placement. Methods: We analyzed data from our prospectively maintained endovascular database and identified patients treated with ticagrelor and underwent treatment with a stent or flow diverter. The patients were divided into an acutely loaded dose group and those converted from clopidogrel due to clopidogrel resistance, defined by platelet aggregation value of 200 PRU VerifyNow P2Y12 test. Patients received standard dual antiplatelet therapy, however, despite repeat clopidogrel loading doses, were found to be clopidogrel non-responders and were then administered ticagrelor 180mg loading dose and maintained on 90mg BID dosing. We ascertained occurrence of any bleeding complications, classified as major (hgb decrease >5 g/dl), minor (hgb decrease 3-5 g/dl), or insignificant. Results: A total of 36 (13.8%) patients (mean age 64.7±13.8 years; 58.3% females) had clopidogrel resistance out of 260 patients screened. Of those, 29 patients were switched from clopidogrel to ticagrelor due to an elevated PRU (avg 294.76±41.32 PRU) and 7 patients without previous antiplatelet use received a bolus and maintenance dose. Only one patient experienced a transient ischemic event within 24 hours after starting ticagrelor. Patients who were acutely loaded did not experience any ischemic events. No patient experienced symptomatic or asymptomatic hemorrhage on ticagrelor in the post-op period. A total of 43 procedures were performed in 36 patients with clopidogrel resistance: 26 pipeline placements, 8 wingspan stent placements, 2 LVIS stents, 1 neuroform stent with pipeline, and 7 angioplasties. Conclusions: Our study demonstrates the safety and effectiveness of loading or switching patients to ticagrelor with cerebrovascular diseases undergoing angioplasty, stent or flow diverter placement.

Impact of Clopidogrel Loading for Coronarography on Bleeding After Urgent First Time CABG.

Background:Excessive bleeding impairs outcome after coronary artery bypass grafting (CABG). Clopidogrel in combination with aspirin, given before percutaneous coronary intervention, have become the standard for stent thrombosis prevention. Some premedicated patients, however, are found to need surgical treatment, thus platelet inhibition caused by clopidogrel becomes a concern for post operation major bleeding.Aims:This study was designed to evaluate the impact of preoperative clopidogrel on bleeding and outcomes after coronary artery bypass graft surgery (CABG).Methods:The study represent a observational retrospective analysis of collected data. The follow up of 223 treated with clopidogrel and aspirin and 77 patients not receiving treatment with platelet antagonist 7 days before CABG are analyzed.Results:The groups were comparable in age, gender, body surface area, preoperative hematocrit, preoperative prothrombin time and prior myocardial infarction. The clopidogrel group had higher12h and 24h mean chest tube output (at 12h mean 519.7ml vs 353.1 ml, p < 0.05, at 24h mean 756.6 ml vs 563.5 ml, p<0.05). Moreover, reoperation for bleeding was 4.5-fold higher in the clopidogrel group (5.9% vs. 1.3%, p <0.01), and more transfusions of red blood cells (3.23U vs 2.6 U, p<0.05), platelets (1.53U vs 1.23U, p<0.01) and fresh frozen plasma (0.84U vs 0.36 U, p<0.01). The clopidogrel group also showed a longer mechanical ventilation time (16.9h vs 12.9 h p = 0.03) and trend towards more prolonged stay in ICU (2.08 days vs 1.7 days p= 0.048).Conclusions:Clopidogrel in combination with aspirin before CABG is associated with higher postoperative bleeding, exposure to blood products and morbidity. These findings raise concern regarding the routine administration of clopidogrel before anticipated but undecided coronary stent implantation.

No Pharmacokinetic Drug–Drug Interaction Between Prasugrel and Vorapaxar Following Multiple‐Dose Administration in Healthy Volunteers

Vorapaxar is a first‐in‐class antagonist of the protease‐activated receptor‐1, the primary thrombin receptor on human platelets, which mediates the downstream effects of thrombin in hemostasis and thrombosis. Prasugrel is a platelet inhibitor that acts as a P2Y12 receptor antagonist through an active metabolite, R‐138727. This study investigated the interaction of these 2 platelet antagonists when coadministered. This was a randomized, open‐label, multiple‐dose study in 54 healthy volunteers consisting of a fixed‐sequence crossover and a parallel group design. In sequence 1, 36 subjects received prasugrel 60 mg on day 1 and then prasugrel 10 mg once daily on days 2 to 7, followed by vorapaxar 40 mg and prasugrel 10 mg on day 8 and then vorapaxar 2.5 mg and prasugrel 10 mg orally once daily on days 9 to 28. In sequence 2, 18 subjects received vorapaxar 40 mg on day 1 and then vorapaxar 2.5 mg once daily on days 2 to 21. The geometric mean ratios (90% confidence intervals) for AUCτ and Cmax of coadministration/monotherapy for vorapaxar (0.93 ng·h/mL[0.85–1.02 ng·h/mL] and 0.95 ng/mL [0.86–1.05 ng/mL]) and R‐138727 (0.91 ng·h/mL [0.85– 0.99 ng·h/mL] and 1.02 ng/mL [0.89–1.17 ng/mL]) were within prespecified bounds, demonstrating the absence of a pharmacokinetic interaction between vorapaxar and prasugrel. There was no specific safety or tolerability risk associated with multiple‐dose coadministration of vorapaxar and prasugrel. In conclusion, in this study in healthy volunteers, there was no pharmacokinetic drug–drug interaction between vorapaxar and prasugrel. Multiple‐dose coadministration of the 2 drugs was generally well tolerated.

TCT-220 Is Inhibition of Platelet Aggregation by Endogenous Endothelial Platelet Antagonists Enhanced by Oral Ticagrelor Mediated P2Y12 Blockade in Patients With Coronary Artery Disease?

P2Y12 receptor antagonists inhibit ADP-induced platelet aggregation and have an important role in the managment of acute coronary syndromes. Nitric oxide and prostacyclin (PGI2) are endogenous endothelial inhibitors of platelet activation. This study evaluated the interaction between P2Y12

Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis.

Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.

Platelet function recovery following exposure to triple anti-platelet inhibitors using an in vitro transfusion model

IntroductionDual anti-platelet therapy (DAPT) with aspirin and a P2Y12 antagonist is standard of care to reduce risk of thrombosis, but does not directly target thrombin-dependent platelet activation. Therefore, PAR-1 antagonist addition to DAPT (i.e., triple anti-platelet therapy; TAPT) may improve the efficacy of treatment, though at the expense of an increase in bleeding risk. Using an in vitro transfusion model, we evaluated if platelet function loss associated with TAPT can be remedied by the addition of drug-naïve platelets. MethodsTo mimic TAPT, platelet-rich plasma (PRP) prepared from consented DAPT patients (DPRP) was incubated with a vorapaxar at therapeutic plasma levels (TPRP). To simulate platelet transfusions, TPRP was mixed with increasing proportions of drug-naïve PRP (NPRP). Platelet function recovery was assessed by light transmission aggregometry (LTA), aggregate morphology, and P-selectin expression. ResultsLTA results demonstrated that 20% NPRP was required to restore the ADP aggregation response in TPRP to the response observed in DPRP and 40% NPRP recovered aggregation to >65%. Higher NPRP fractions (60%) were required to restore the platelet reactivity using TRAP-6 (SFLLRN) or arachidonic acid (AA). PAR-4 aggregation was unaffected by platelet antagonists. A decrease in single, free platelets and incorporation of mepacrine-labeled naïve platelets into aggregates occurred with increasing NPRP portions. Upon agonist activation, the surface density and percent of P-selectin positive platelets increased linearly upon addition of NPRP. ConclusionThis in vitro model demonstrated that administration of drug-naïve platelets can be a useful strategy for reversing overall platelet inhibition observed with TAPT.

The BCR-ABL inhibitor ponatinib inhibits platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling, platelet activation and aggregate formation under shear

BackgroundTreatment of chronic myelogenous leukemia (CML) with the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib significantly improves patient outcomes. As some patients are unresponsive to imatinib, next generation BCR-ABL inhibitors such as nilotinib have been developed to treat patients with imatinib-resistant CML. The use of some BCR-ABL inhibitors has been associated with bleeding diathesis, and these inhibitors have been shown to inhibit platelet functions, which may explain the hemostasis impairment. Surprisingly, a new TKI, ponatinib, has been associated with a high incidence of severe acute ischemic cardiovascular events. The mechanism of this unexpected adverse effect remains undefined. Objective and Methods: This study used biochemical and functional assays to evaluate whether ponatinib was different from the other BCR-ABL inhibitors with respect to platelet activation, spreading, and aggregation. Results and Conclusions: Our results show that ponatinib, similar to other TKIs, acts as a platelet antagonist. Ponatinib inhibited platelet activation, spreading, granule secretion, and aggregation, likely through broad spectrum inhibition of platelet tyrosine kinase signaling, and also inhibited platelet aggregate formation in whole blood under shear. As our results indicate that pobatinib inhibits platelet function, the adverse cardiovascular events observed in patients taking ponatinib may be the result of the effect of ponatinib on other organs or cell types, or disease-specific processes, such as BCR-ABL+cells undergoing apoptosis in response to chemotherapy, or drug-induced adverse effects on the integrity of the vascular endothelium in ponatinib-treated patients.

Abstract 384: The Prothrombotic Profile of BCR-ABL Inhibitors Ponatinib, Nilotinib and Imatinib

Background: Thrombotic complications observed in patients treated with the tyrosine kinase inhibitor ponatinib resulted in the temporary suspension of the drug by the FDA in late 2013. Ponatinib is a pan-BCR-ABL inhibitor designed for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Ponatinib has been shown to inhibit a subset of the class III/IV family of receptor tyrosine kinases. The pathogenic mechanism underlying the prothrombotic phenotype associated with ponatinib remains ill-defined. Hypothesis: We tested the hypothesis that BCR-ABL inhibitors regulate platelet activation, spreading, and aggregation. Methods &amp; Results: Our results show that treatment of platelets with ponatinib (1 μM) abrogated platelet spreading on fibrinogen (50 μg/ml) or collagen (100 μg/ml). Both nilotinib and imatinib inhibited platelet spreading, although not to the same extent as ponatinib at equimolar concentrations. We next examined the effects of BCR-ABL inhibitors on platelet activation and aggregation in response to the GPVI-agonist, CRP, which mediates platelet activation in a receptor tyrosine kinase-dependent manner. Intriguingly, our results show that ponatinib abrogated platelet aggregation in response to CRP (1 μg/ml), whereas equimolar concentrations of nilotinib or imatinib had minimal effects on CRP-induced platelet aggregation. Conclusions: While tyrosine kinase inhibitors are generally associated with bleeding diathesis, thrombotic complications have been observed in patients treated with ponatinib. Our results indicate that ponatinib, nilotinib, and imatinib inhibit platelet spreading and aggregation, with ponatinib having the most significant effect, suggesting that ponatinib may act as a platelet antagonist. Our future work will be focused on identifying platelet proteins affected by the BCR-ABL inhibitors as well as the pathogenic mechanisms underlying the prothrombotic phenotype associated with ponatinib.

Pharmacogenetics of Antiplatelet Therapy

There has been substantial progress toward understanding and investigating the specific genetic factors that influence interindividual variations in platelet-directed therapy. There has also been substantial progress toward better understanding of the pharmacogenetics of drug metabolism and phamacodynamic response to platelet antagonists. We summarize the relationship between genetic polymorphisms, response to platelet antagonists, and clinical impact on patient treatment for the commonly used antiplatelet drugs. The challenge faced in translating genotype identification into improved clinical outcomes reflects the complexity involved in the genomic influence on drug metabolism and activation.

Anticoagulants for Cerebral Venous Thrombosis

See related article, p 8. Evidence relating to efficacy and safety of anticoagulants for CVT is only partially available from studies in the peer reviewed medical literature and from the corresponding authors of those studies. Consequently, this article is an opinion piece rather than a systematic review. Cerebral venous thrombosis (CVT) accounts for ≈0.5% of strokes and may occur at any age.1 Risk factors include pregnancy, oral contraceptives, head trauma, malignancy, infection, anemia, and dehydration. In older reports, the incidence of CVT was 3 to 4 per 1 million people per year in Europe and the United States.2 However, a survey published in 2012 found an incidence of CVT of 13.2 per 1 million people per year.3 With modern brain imaging technology, more cases are being discovered that probably would have resolved without medical intervention. Relative to pre-1990 clinical outcomes data, the average prognosis has dramatically improved.4,5 A Cochrane review of anticoagulant therapy for CVT4 based the recommendation in favor of using anticoagulation treatment (full-dose unfractionated heparin [UFH] or low-molecular-weight heparin [LMWH] followed by a vitamin K antagonist [VKA]) on 2 small randomized placebo-controlled trials (RCTs, n=79). These trials demonstrated a non-statistically significant reduction in deaths with anticoagulation (risk ratio, 0.33; 95% confidence interval [CI], 0.08–1.28). Subsequently, the European Federation of Neurological Sciences6 and the American Heart Association/American Stroke Association7 issued clinical practice guidelines calling for full-dose heparin acutely followed by VKAs for ≥3 months. The efficacy and safety of anticoagulants for CVT have not been comprehensively reviewed, with the inclusion of observational studies and the RCTs excluded from the Cochrane review. This review will evaluate published reports from 1990 to 2013 regarding the efficacy and safety of full-dose heparin (UFH or LMWH) during initial hospitalization and subsequent VKA treatment …

Ticagrelor reduces neutrophil recruitment and lung damage in abdominal sepsis

Platelets play an important role in abdominal sepsis and P2Y12 receptor antagonists have been reported to exert anti-inflammatory effects. Herein, we assessed the impact of platelet inhibition with the P2Y12 receptor antagonist ticagrelor on pulmonary neutrophil recruitment and tissue damage in a model of abdominal sepsis. Wild-type C57BL/6 mice were subjected to cecal ligation and puncture (CLP). Animals were treated with ticagrelor (100 mg/kg) or vehicle prior to CLP induction. Edema formation and bronchoalveolar neutrophils as well as lung damage were quantified. Flow cytometry was used to determine expression of platelet–neutrophil aggregates, neutrophil activation and CD40L expression on platelets. CLP-induced pulmonary infiltration of neutrophils at 24 hours was reduced by 50% in ticagrelor-treated animals. Moreover, ticagrelor abolished CLP-provoked lung edema and decreased lung damage score by 41%. Notably, ticagrelor completely inhibited formation of platelet–neutrophil aggregates and markedly reduced thrombocytopenia in CLP animals. In addition, ticagrelor reduced platelet shedding of CD40L in septic mice. Our data indicate that ticagrelor can reduce CLP-induced pulmonary neutrophil recruitment and lung damage suggesting a potential role for platelet antagonists, such as ticagrelor, in the management of patients with abdominal sepsis.

Potential Role of Endogenous Adenosine in Ticagrelor-Induced Dyspnea

Ticagrelor, a recently approved platelet antagonist indicated for the reduction of thrombotic cardiovascular events in patients with acute coronary syndrome (ACS), has been reported to cause dyspnea in more than 13% of patients. Dyspnea is not a clinically relevant adverse event with other medications indicated for ACS. One suggested mechanism of ticagrelor-induced dyspnea involves an increase in systemic adenosine concentrations through adenosine deaminase inhibition. Dyspnea, a subjective finding resulting from physiologic and sensory mechanisms, may be a consequence of increased systemic adenosine concentrations, leading to amplified and prolonged receptor activity. Current literature suggests, however, that pulmonary status is not compromised, with no reduction of efficacy seen in patients with ticagrelor-induced dyspnea, thus allowing clinicians to continue therapy without reservation. Still, patients with a history of asthma and chronic obstructive pulmonary disease may be more susceptible to ticagrelor-induced dyspnea, potentially leading to nonadherence and exacerbations of morbidity. Therefore, it is paramount that health care providers continually monitor these patients with the aims of maintaining medication therapy adherence and providing relevant options if dyspnea becomes intolerable.

Antiplatelet Agents Can Promote Two-Peaked Thrombin Generation in Platelet Rich Plasma: Mechanism and Possible Applications

BackgroundThrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks.ObjectiveWe sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve.MethodsTissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers.ResultsThe addition of the P2Y12 receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E1 (PGE1), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE1 or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE1 mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one.ConclusionsProcoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types - plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.

Migraine: Possible Role of Shear‐Induced Platelet Aggregation With Serotonin Release

Migraine patients are at an increased risk for stroke, as well as other thromboembolic events. This warrants further study of the role of platelets in a proportion of migraine patients. To extend the "platelet hypothesis" using literature data and observations made in a rat model of shear stress-induced platelet aggregation. Such aggregation causes release of serotonin, leading to vasoconstriction during sufficiently strong aggregation and to long-lasting vasodilation when aggregation diminishes. This vasodilation also depends on nitric oxide and prostaglandin formation. A role for platelet aggregation in a number of migraineurs is indicated by reports of an increased platelet activity during attacks and favorable effects of antiplatelet medication. We hypothesize that in those patients, a migraine attack with or without aura may both be caused by a rise in platelet-released plasma serotonin, albeit at different concentration. At high concentrations, serotonin may cause vasoconstriction and, consequently, the neuronal signs of aura, whereas at low concentrations, it may already stimulate perivascular pain fibers and cause vasodilation via local formation of nitric oxide, prostaglandins, and neuropeptides. Platelet aggregation may be unilaterally evoked by elevated shear stress in a stenotic cervico-cranial artery, by reversible vasoconstriction or by other cardiovascular abnormality, eg, a symptomatic patent foramen ovale. This most likely occurs when a migraine trigger has further enhanced platelet aggregability; literature shows that many triggers either stimulate platelets directly or reduce endogenous platelet antagonists like prostacyclin. New strategies for migraine medication and risk reduction of stroke are suggested.

Platelet Adhesion and Degranulation Induce Pro-Survival and Pro-Angiogenic Signalling in Ovarian Cancer Cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

Blood Component Therapy in Trauma Guided with the Utilization of the Perfusionist and Thromboelastography

25–35% of all seriously injured multiple trauma patients are coagulopathic upon arrival to the emergency department, and therefore early diagnosis and intervention on this subset of patients is important. In addition to standard plasma based tests of coagulation, the thromboelastogram (TEG®) has resurfaced as an ideal test in the trauma population to help guide the clinician in the administration of blood components in a goal directed fashion. We describe how thromboelastographic analysis is used to assist in the management of trauma patients with coagulopathies presenting to the emergency department, in surgery, and in the postoperative period. Indications for the utilization of the TEG® and platelet mapping as point of care testing that can guide blood component therapy in a goal directed fashion in the trauma population are presented with emphasis on the more common reasons such as massive transfusion protocol, the management of traumatic brain injury with bleeding, the diagnosis and management of trauma in patients on platelet antagonists, the utilization of recombinant FVIIa, and the management of coagulopathy in terminal trauma patients in preparation for organ donation. The TEG® allows for judicious and protocol assisted utilization of blood components in a setting that has recently gained acceptance. In our program, the inclusion of the perfusionist with expertise in performing and interpreting TEG® analysis allows the multidisciplinary trauma team to more effectively manage blood products and resuscitation in this population.

A Biotin-Avidin-EL ISA for Studying on Platelet Membrane Surface P-Selectin and Its Clinical Application

AbstractAbstract 5132 IntroductionTo define the abnormalities in surface GPs expression on circulating platelets and provide better biomarker of bleeding and thrombotic disorders, we develop a high-throughput assay using biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) to test platelet responses to key platelet agonists and antagonists. MethodsSZ51(anti-P-selectin mAb) coated-plate was added into 100μ l PRP with 10μ l ADP (27μ mol/L ADP)or 10μ l PBS without ADP and incubated for 1 h. Activated platelets captured were reacted with biotin-SZ-2 for 1 h. Then, the plate was re-incubated with avidin-HRP coiugated for 1h. Binding avidin was detected with OPD. The levels of P-selectin were detected in AMI group, ICH group, DM group or control group using the BA-ELISA method. This BA-ELISA was compared with flow cytometer (FCM). Inhibition of platelet GPs function was evaluated with inhibitory mAb, SZ21 and aspirin respectively. ResultsThe BA-ELISA can detect platelet count as low as 6.25 × 109/L in PRP. Both of the inter-assay and intra-assay coefficient variation (C V) were less than 10%. The OD492nm values of the ADP-induced P-selectin in 30 AMI, 30 ICH and 30 DM platelets in PRP were 1.76± 0.56, 1.95±0.53 and 1.51±0.14 which was significantly higher than that in controls (0.68±0.21, P<0.01). The OD 492nm values of non ADP-induced P-selectin in 30 AMI, 30 ICH or 30 DM platelets in PRP were 0.72±0.25, 1.53±0.61 and 0.61±0.16 significantly higher than that in 30 controls (0.18±0.09, P<0.01). The correlation between the BA-ELISA and FCM was concordant (R=0.86). SZ21 or aspirin could inhibit the expression of P-selectin induced by ADP. ConclusionBA-ELISA is suitable for measuring platelet GP function and is also useful for diagnostic tests and for screening of inhibitors/activators of platelet activation. Disclosures:No relevant conflicts of interest to declare.

Pharmacokinetic, pharmacodynamic and clinical profile of novel antiplatelet drugs targeting vascular diseases

Platelet inhibitors are the mainstay treatment for patients with vascular diseases. The current 'gold standard' antiplatelet agent clopidogrel has several pharmacological and clinical limitations that have prompted the search for more effective platelet antagonists. The candidates include various blockers of the purinergic P2Y12 receptor such as prasugrel, an oral irreversible thienopyridine; two adenosine triphosphate analogues that bind reversibly to the P2Y12 receptor: ticagrelor (oral) and cangrelor (intravenous); elinogrel, a direct-acting reversible P2Y12 receptor inhibitor (the only antiplatelet compound that can be administered both intravenously and orally); BX 667, an orally active and reversible small-molecule P2Y12 receptor antagonist; SCH 530348, SCH 205831, SCH 602539 and E5555, highly selective and orally active antagonists on the protease-activated receptor 1. A number of drugs also hit new targets: terutroban, an oral, selective and specific inhibitor of the thromboxane receptor; ARC1779, a second-generation, nuclease resistant aptamer which inhibits von Willebrand factor-dependent platelet aggregation; ALX-0081, a bivalent humanized nanobody targeting the GPIb binding site of von Willebrand factor and AJW200, an IgG4 monoclonal antibody of von Willebrand factor. The pharmacology and clinical profiles of new platelet antagonists indicate that they provide more consistent, more rapid and more potent platelet inhibition than agents currently used. Whether these potential advantages will translate into clinical advantages will require additional comparisons in properly powered, randomized, controlled trials.

G-protein beta3 subunit polymorphism and bleeding in the orbofiban in patients with unstable coronary syndromes-thrombolysis in myocardial infarction 16 trial.

Variability in platelet response to antiplatelet drugs is heritable. A common single base substitution (825C>T) in the G-protein beta polypeptide 3 (GNB3) gene leads to alternative splicing (41-amino-acid deletion) of the human G-protein beta3 (Gbeta3) subunit. This truncated protein carried by GNB3 T allele carriers is linked to coronary artery disease and implicated as a genetic marker of drug response. Large studies of Caucasians associate T allele carriage with lower platelet reactivity. To evaluate whether the GNB3 genotype would predispose to bleeding in patients treated with a GPIIb/IIIa receptor antagonist. GNB3 genotype distribution was determined in DNA samples from patients in the orbofiban in patients with unstable coronary syndromes-thrombolysis in myocardial infarction (OPUS-TIMI) 16 genetic sub-study. Impact of genotype on the bleeding endpoint and the composite primary endpoint of death, myocardial infarction (MI), re-hospitalization for ischemia and urgent revascularization was estimated in the treatment and placebo arm. Out of 887 patients, 45.1% carried the GNB3 CC genotype, 44.5% CT and 10.4% TT. Interaction between T allele carriership and treatment for bleeding was significant (P = 0.008). This reflects the fact that GNB3 non-T carriers treated with orbofiban had no bleeding effect compared with placebo (RR = 0.92, 95% CI 0.55-1.55) whereas T carriers did (RR = 2.62, 95% CI 1.58-4.35, P < 0.001). Interaction between T allele carriership and treatment was not significant for the primary endpoint (P = 0.18) or MI (P = 0.69). The GNB3 T allele significantly increased bleeding in patients treated with the platelet antagonist orbofiban. Our findings suggest that risk of bleeding associated with an antiplatelet agent is heritable and may be dissociated from risk of thrombosis.

Platelet-active substances in the venom of Bothrops moojeni snake–a novel evaluation method using whole blood aggregometry

The objective of the present study was an investigation of the crude Bothrops moojeni venom, aiming at the identification of new compounds with platelet-activating or -inhibiting activity. The venom was separated by gel filtration chromatography into 18 fractions, which were tested by means of whole blood aggregometry for their activities affecting the aggregation of blood platelets. In order to eliminate interferences caused by prothrombin activators or thrombin like-enzymes, which are frequently present in snake venoms, a test method for screening protein mixtures was developed. To avoid clotting of the blood samples, the thrombin inhibitor hirudin and the synthetic inhibitor of fibrin polymerization Pefabloc® FG were applied. In the present study, a platelet aggregation activator with an activity resembling thrombocytin from B. atrox was identified in one of the examined venom fractions. In addition, a platelet antagonist–most likely a disintegrin–with broad inhibitory activity against aggregation triggered by collagen, adenosine diphosphate and thrombin receptor activating peptide, was identified.