Response to antiplatelet therapy and platelet reactivity to thrombin receptor activating peptide-6 in cardiovascular interventions: Differences between peripheral and coronary angioplasty

Background

The long-term prognosis of patients with peripheral arterial disease (PAD) is significantly worse than the prognosis of coronary artery disease (CAD) patients. Detrimental platelet activation could contribute to the increased rate of adverse cardiovascular events in PAD. We therefore investigated whether response to antiplatelet therapy and thrombin inducible platelet activation differ between patients with best medical therapy undergoing angioplasty and stenting for symptomatic PAD (n = 166) or CAD (n = 104).

Methods

Adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin receptor activating peptide (TRAP)-6 inducible platelet reactivity was measured by multiple electrode aggregometry (MEA). Platelet surface expression of P-selectin and activated glycoprotein IIb/IIIa (GPIIb/IIIa) in response to ADP, AA, and TRAP-6, and the formation of monocyte-platelet aggregates (MPA) in response to ADP and TRAP-6 were assessed by flow cytometry.

Results

Patients with PAD had significantly higher platelet reactivity in response to ADP and AA by MEA compared to CAD patients. Likewise, the expression of P-selectin and GPIIb/IIIa following stimulation with ADP and AA, and MPA formation in response to ADP were significantly higher in PAD patients than in CAD patients. In response to TRAP-6, patients with PAD showed a significantly increased platelet aggregation by MEA, higher expression of activated GPIIb/IIIa, and more pronounced formation of MPA than CAD patients.

Conclusion

Following angioplasty and stenting, PAD patients exhibit a significantly diminished response to dual antiplatelet therapy and an increased susceptibility to TRAP-6 inducible platelet activation compared to CAD patients.     

Prasugrel 5 mg inhibits platelet P-selectin and GPIIb–IIIa expression in very elderly and non elderly: results from the GENERATIONS trial,a pharmacodynamic study in stable CAD patients

Platelet P-selectin and activated glycoprotein IIb–IIIa (GPIIb–IIIa) are markers of platelet activation and mediates platelet aggregation. Prasugrel (Pras) 5 mg may be used in very elderly (VE) acute coronary syndrome (ACS) patients undergoing PCI, but its effect on platelet P-selectin and activated GPIIb–IIIa in those patients is not known. Stable ACS patients, VE (78 ± 5 years, n = 23) and non-elderly (NE) (55 ± 5 years, n = 22) were randomized to Pras (5 or 10 mg) or clopidogrel (Clop) 75 mg during three 12-day periods. Platelet activation markers were measured by flow cytometry on unstimulated or stimulated (adenosine diphosphate (ADP) 20 μM) platelets, before and after each dosing period.Results: At baseline there was no difference in platelet activation markers, either unstimulated or ADP-stimulated, between NE and VE. Pras 5 mg reduced both ADP-stimulated platelet P-selectin and activated GPIIb–IIIa in VE (p < 0.01 for both analyses) and NE (p < 0.001 and p < 0.05, respectively). Clop 75 mg had a similar effect as Pras 5 mg but did not significantly reduce activated GPIIb–IIIa in VE. Prasugrel 10 mg resulted in decreased platelet activation in both age groups compared to Clop 75 mg (p < 0.01).Conclusions: In VE and NE-patients, Pras 5 mg inhibited platelet P-selectin expression similar to Clop 75 mg and Pras 10 mg. Prasugrel 10 mg inhibited platelet P-selectin expression better than Clop 75 mg. Prasugrel 10 mg and 5 mg, but not Clop 75 mg, significantly inhibited activated GPIIb–IIIa in VE. This platelet reactivity data support the use of Pras 5 mg for VE patients.     

Flow cytometric detection of platelet activation in patients undergoing diagnostic and interventional coronary angiography

In 30 patients we investigated the expression of activated GPIIb/IIIa-complex as an indicator of in vivo platelet activation before and after coronary angiography/angioplasty. Patients were divided into three groups according to aspirin treatment: group I: patients having a diagnostic coronary angiography without aspirin (n = 6); group II: patients having a diagnostic coronary angiography with low dose aspirin therapy (n = II); group III: patients having a coronary angioplasty with low dose aspirin therapy plus aspirin i.v. (n = 13). Platelets were identified in a flow cytometer by their characteristic light scatter profile and binding of anti-GP Ib, and activated platelets by an antibody to the activated GP IIb/IIIa. Cardiac catheterization lead to an increase of the mean anti-GP IIb/IIIa-fluorescence of the platelets and of the percentage of platelets with an anti-IIb/IIIa-FL exceeding a fixed threshold value (subpopulation). While aspirin substantially inhibited the increase induced by diagnostic angiography, the increase in the angioplasty group was the greatest of all groups despite aspirin.     

GPIIb/IIIa Inhibitor-Induced Dethrombosis

GPIIb/IIIa inhibitors have demonstrated clinical benefit in patients with acute coronary syndromes and in those undergoing percutaneous coronary intervention. As opposed to the well documented prevention of platelet aggregation by GPIIb/IIIa inhibitors, we here discuss the less well appreciated reversal of platelet aggregation by, and dethrombotic effects of, GPIIb/IIIa inhibitors. In vivo evidence for GPIIb/IIIa inhibitor-induced dethrombosis includes animal models of arterial thrombosis and human studies (both observational and randomized clinical trials) in the setting of acute myocardial infarction. These human studies demonstrated increased coronary perfusion prior to percutaneous coronary intervention in patients receiving GPIIb/IIIa inhibitors as compared to those patients receiving placebo. Mechanisms of GPIIb/IIIa inhibitor-induced dethrombosis include alterations in thrombus size and structure, reduced capacity for subsequent thrombus growth, inhibition of soluble CD40L release from platelets, and a direct platelet disaggregatory effect that parallels fibrinogens interactions with GPIIb/IIIa. In summary, mechanistic studies, animal models, human observational studies and randomized clinical trials all strongly suggest that GPIIb/IIIa inhibitors have a direct dethrombotic effect in addition to their well-described preventative effects on platelet aggregation.     

Nephropathy in Type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 µM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.     

Glycoprotein IIb/IIIa antagonists in acute coronary syndromes: where are we now?

Vascular inflammation, coronary constriction, and thrombus formation are central to all acute coronary syndromes (ACSs). Adhesion and aggregation of activated platelets, initially described during thrombosis, now appear pivotal to all three processes. Several platelet adhesion receptors participate but the integrin glycoprotein (GP) IIb/IIIa occupies a critical role. GPIIb/IIIa antagonists used as an adjunct to percutaneous coronary intervention show clear benefit. However in the setting of ACS results have been disappointing. Indeed, trials of oral GPIIb/IIIa antagonists in patients with ACS were associated with increased mortality. Difficulties with drug dosing and variable pharmacodynamics may contribute to suboptimal receptor occupancy, incomplete inhibition of platelet aggregation, paradoxical partial agonist activity, and proinflammatory effects. Moreover, variable responses of patients to GPIIb/IIIa antagonists may reflect population heterogeneity.     

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.     

Abciximab Pharmacodynamics Are Unaffected by Antecedent Therapy with Other GPIIb/IIIa Antagonists in Non-Human Primates

Background: Tirofiban and eptifibatide are currently approved for the medical stabilization of non-ST segment elevation acute coronary syndromes. In patients undergoing percutaneous coronary intervention (PCI) during infusion of these drugs, conversion to abciximab, which has long term proven clinical efficacy and cost-effectiveness, following PCI may be desirable. The purpose of this study was to determine if the binding or pharmacodynamics of abciximab is affected by a prior infusion of either tirofiban or eptifibatide. Methods: In vitro binding experiments were performed to determine if prior exposure to tirofiban or eptifibatide altered the affinity and extent of binding of abciximab to GPIIb/IIIa. For in vivo experiments, cynomolgus monkeys were pretreated with a bolus and 18 hour infusion of saline, tirofiban, or eptifibatide. At the end of the initial treatment, a bolus and 12 hr infusion of abciximab was started without delay. Inhibition of platelet aggregation, GPIIb/IIIa receptor blockade and abciximab pharmacokinetics were measured during and after both infusions. Results: Equilibrium binding of abciximab in vitro was unaffected by tirofiban or eptifibatide. The extent and duration of abciximab inhibition of ex vivo platelet aggregation, receptor blockade, and abciximab pharmacokinetics in monkeys during and after the abciximab infusion were not affected by prior infusion of the animals with tirofiban or eptifibatide. Conclusions: In vitro and in vivo studies revealed that the molecular interaction of abciximab with the platelet GPIIb/IIIa receptor is not altered by immediate prior exposure of platelets to small molecule GPIIb/IIIa antagonists. These preclinical studies suggest that the efficacy of abciximab should not be impaired if it is initiated following termination of therapy with small molecule GPIIb/IIIa antagonists.     

MK-383 (Tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin α_IIb β₃ (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10^-7 mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

Intracranial Hemorrhages Associated with Intravenous Platelet Glycoprotein IIB/IIIA Receptor Inhibitors in the United States

Summary Objectives: To determine the rates of intracranial hemorrhages associated with GP IIb/IIIa inhibitors in routine practice. Background: Rates of intracranial hemorrhages (ICH) among patients treated with platelet glycoprotein (GP) IIb/IIIa inhibitors for coronary interventions and acute coronary syndromes have been studied within clinical trials but not in routine practice. Methods: We evaluated the rates of ICH in routine practice in United States (US) using national estimates of rates, in-hospital outcomes, and mortality obtained from National Hospital Discharge Survey. Results: There were 367 294 patients aged 18 years or greater who were treated with platelet GP IIb/IIIa inhibitors between 2000 and 2002 in United States. ICH was observed in 479 (0.13%) of the 367 294 patients with a 100% associated mortality. ICHs related to GP IIb/IIIa inhibitors comprised 0.12% of the total number of ICHs (n=411 621) observed in United States between 2000 and 2002. Conclusions: ICH related to platelet GPIIb/IIIa inhibitors is uncommon but associated with high mortality.     

Platelet activation in patients after an acute coronary syndrome: results from the TIMI-12 trial. Thrombolysis in Myocardial Infarction

This study was designed to determine the magnitude and time course of platelet activation during therapy of acute coronary syndromes with an oral platelet antagonist. BACKGROUND: Platelet activation and aggregation are central to the pathogenesis of the acute coronary syndromes (ACS). However, few data are available on levels of platelet activation over time in patients with ACS, especially in the setting of chronic glycoprotein (GP) IIb/IIIa inhibition. METHODS: The Thrombolysis in Myocardial Infarction (TIMI) 12 trial was a phase II, double-blind trial evaluating the effects of sibrafiban, an oral, selective antagonist of the platelet glycoprotein IIb/IIIa receptor in patients stabilized after an ACS. A subset of 90 of the 329 patients in the study had measurement of platelet activation as assessed by the expression of platelet associated P-Selectin on days 0, 7 and 28. Platelet activation was measured in blood samples that were fixed either immediately (spontaneous activation) or after 5 minute incubation with 0, 1 microM or 5 microM ADP in order to assess platelet responsiveness to very low or moderate stimulation. RESULTS: At baseline there was a significant elevation of spontaneous platelet activation as compared to samples obtained from normal donors or from patients who did not have acute coronary syndromes (ACS patients 27.6+/-18.7%, Normal controls 8.5+/-4.4%, Patient controls 10.9+/-7.1%, p < 0.005 for both). In addition, there was a significant decrease in the levels of platelet activation with time during the 28 days of treatment with sibrafiban. Nevertheless, even on day 28, the TIMI-12 patients continued to show elevated platelet activation in comparison to the control groups (p < 0.05 for both). CONCLUSIONS: These results suggest that platelets remain activated long after clinical stabilization post ACS. Although platelet activation decreased after one month of oral GPIIb/IIIa inhibition, levels remained higher than normal, suggesting the need for long-term antiplatelet therapy following ACS.     

Chronic expression of platelet adhesion proteins is associated with severe ischemic heart disease in type 2 diabetic patients: Chronic platelet activation in diabetic heart patients

Cardiac ischemia is a serious complication of type 2 diabetes. However, the pathophysiology underlying the increased severity of myocardial ischemia in diabetes is not clear. This study tested the hypothesis that platelet adhesion protein expression is chronically increased in older type 2 diabetic patients with established ischemic heart disease (IHD) compared to age-matched, nondiabetic patients with IHD. We compared the chronic expression of two platelet adhesion proteins, P-selectin and GPIIb/IIIa, in whole blood and the platelet reactivity to an acute stimulus. We found that the expression of platelet P-selectin was chronically increased in the nondiabetic patients with IHD compared to normal subjects. P-selectin expression was further increased in the diabetic patients with IHD compared to the nondiabetic IHD patients (P<.05). The results were stratified to examine the potential effect of aspirin usage on adhesion protein expression. We found that the expression of the activated GPIIb/IIIa complex was significantly reduced in those diabetic cardiac patients who were taking aspirin (P<.05). These findings indicate that, in patients with IHD, platelet adhesion proteins are chronically expressed and that the level of expression is increased more in IHD patients with type 2 diabetes. This complication of diabetes may exacerbate thrombus formation during a recurrent event, increasing the severity of ischemic injury. The results give further support to the use of aspirin in type 2 diabetics with established cardiac disease.     

P-selectin expression, but not GPIIb/IIIa activation, is enhanced in the inflammatory stage of Takayasu's arteritis.

BACKGROUND: Inflammation and thrombosis are closely related processes, but the association between disease activity and thrombogenicity in Takayasu's arteritis (TA) is poorly understood. To investigate the link between platelet activation and disease activity, flow cytometric analyses of platelet P-selectin and activated GPIIb/IIIa expression were performed in patients with TA. METHODS AND RESULTS: Twenty-two patients with TA, classified into active (Group A, n = 9) and inactive (Group I, n = 13) according to blood-derived inflammatory markers, and 14 healthy age- and gender-matched controls (Group C) were studied. Compared with Group C, the mean fluorescence intensity of P-selectin in response to 0.1-10 micromol/L of ADP was significantly upregulated in Group A, but not in Group I. No differences in platelet GPIIb/IIIa expression in stimulated platelets were seen among the 3 groups. Standard platelet aggregation studies revealed that disease activity did not influence platelet aggregation by ADP. CONCLUSIONS: P-selectin expression, but not activated GPIIb/IIIa, is enhanced in ADP-activated platelets from patients in the inflammatory stage of TA. P-selectin may play a significant role in the inflammatory and thrombotic responses associated with intractable TA, presumably by inducing platelet-leukocyte interactions.     

Efficacy and safety of argatroban with or without glycoprotein IIb/IIIa inhibitor in patients with heparin induced thrombocytopenia undergoing percutaneous coronary intervention for acute coronary syndrome

Background There is limited experience with the use of argatroban in combination with glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitor in acute coronary syndrome (ACS) patients with heparin-induced thrombocytopenia (HIT) undergoing percutaneous coronary intervention (PCI). Materials and methods This single-center, retrospective study evaluated the efficacy (composite of death, myocardial infarction, or urgent revascularization) and safety (evaluated by TIMI major bleeding) of the argatroban with or without a GPIIb/IIIa inhibitor during PCI. Among 102 consecutive ACS patients (71.6% unstable angina or NSTEMI and 28.4% STEMI) who received argatroban (239 ± 104 μg/kg bolus, followed by a 17 ± 11 μg/kg/min infusion) for confirmed or suspected HIT during PCI, 52 patients (51%) received a GPIIb/IIIa inhibitor simultaneously (86% integrilin, 10% tirofiban, 4% abciximab) and 50 patients (49%) did not. Results There was no difference between the groups in the efficacy endpoint, which occurred in nine patients (17.3%) who received GPIIb/IIIa inhibitor and in eight patients (16%) who did not (P = 0.70). TIMI major bleeding occurred in three (5.8%) patients in the GPIIa/IIIb inhibitor group versus 0 (0%) patients in the argatroban alone group (P = 0.085). Conclusion In patients with suspected or confirmed HIT undergoing PCI for ACS, argatroban with or without GPIIb/IIIa appears to provide adequate anticoagulation and is well tolerated with a low rate of bleeding.     

Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes

OBJECTIVES: The study was done to determine the role of partial agonist activity in the lack of effectiveness of the oral GPIIb/IIIa antagonist orbofiban. BACKGROUND: Orbofiban, an oral GPIIb/IIIa antagonist, was found to increase the mortality of patients with acute coronary syndromes (ACS) in the OPUS-TIMI-16 trial, despite the fact that it is a very potent anti-platelet agent and that IV agents have proven very effective. METHODS: Patients (n = 520) with ACS were randomized to orbofiban 30 mg, 40 mg or 50 mg twice daily or 50 mg once daily or placebo. Platelet activity was assessed in 175 patients by examining GPIIb/IIIa receptor conformation, expression of CD63 antigen, and platelet aggregation. RESULTS: Plasma concentrations of orbofiban at the highest dose (74 +/- 6 ng/ml peak, 61 +/- 5 ng/ml trough) exceeded the IC50 for platelet aggregation to adenosine diphosphate (ADP) (29 +/- 6 ng/ml) and thrombin-activating peptide (61 +/- 18 ng/ml). Orbofiban induced a conformational change in GPIIb/IIIa detected as the displacement of the monoclonal antibody mAb2; such conformational changes have been linked to partial agonist activity. Consistent with this, platelet expression of CD63 ex vivo was significantly increased at five time points during the study. In vitro, orbofiban increased platelet aggregation to a submaximal concentration of epinephrine (67 +/- 19% vs. 27 +/- 9%, n = 5) and increased thromboxane formation when the platelet GPIIb/IIIa were clustered using monoclonal antibodies to the receptor. CONCLUSIONS: Orbofiban is both an antagonist and a partial agonist of platelet GPIIb/IIIa. At low concentrations of the drug, this partial agonist activity may enhance platelet aggregation. Along with suboptimal plasma drug levels, these findings may help explain the lack of efficacy seen with orbofiban in patients with ACS.     

In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion

BACKGROUND AND OBJECTIVES: Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. DESIGN AND METHODS: The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. RESULTS: Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. INTERPRETATION AND CONCLUSIONS: Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.     

Platelet function in clopidogrel-treated patients with acute coronary syndrome.

Percutaneous coronary intervention (PCI) in patients presenting with acute coronary syndrome (ACS) is associated with increased risk of thrombotic complications. ACS enhances platelet activation; whether pretreatment with clopidogrel is sufficient to suppress platelet function in patients with ACS is not known. This study assessed platelet function in patients with and without ACS prior to PCI and after pretreatment with a single dose of 600 mg clopidogrel. Blood samples of 402 patients prior to PCI with (n = 119) or without (n = 283) ACS were collected at least 2 h after 600 mg clopidogrel administration. Maximal platelet aggregation in response to ADP (5 and 20 micromol/l), collagen (4 microg/ml) and TRAP (25 micromol/l) was measured with optical aggregometry. Surface expression of glycoprotein IIb/IIIa and P-selectin was assessed with flow cytometry at baseline and after stimulation with 5 and 20 micromol/l ADP. Agonist-induced platelet aggregation did not differ significantly between patients with and without ACS (P > or = 0.15). Parameters of platelet activation (glycoprotein IIb/IIIa and P-selectin surface expression) were significantly higher in ACS patients at baseline and after 5 and 20 micromol/l ADP stimulation (P < 0.0001). Patients with ACS continue to exhibit increased platelet activation after pretreatment with 600 mg clopidogrel. This finding supports the need for additional platelet function inhibition during PCI in patients with ACS.     

Platelet function and blood coagulation system status in childhood essential thrombocythemia

Abstract

Childhood essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder. The quality of life of ET patients may decrease as a result of ischemic and hemorrhagic complications of unclear origin. Our goal was to characterize the hemostatic system in children with ET. We genotyped and investigated blood samples from 20 children with ET in a prospective case series study using platelet aggregation, functional flow cytometry (FC) assay and standard clotting assays. Three children had a JAK2V617F mutation, 4 had mutations in CALR and 13 were triple-negative. Myelofibrosis in stage 1-2 was detected in 3 children. Three patients had bleeding episodes and seven had ischemic events. Aggregation in response to collagen, adenosine diphosphate, and ristomycin was decreased in all patients. In FC, significant changes in the whole patient group compared to the healthy children control group were decrease in the resting forward scatter and PAC1 binding (activated GPIIb/IIIa) level. For the activated platelets, dense granules release (by mepacrine), PAC1, and GPIIb/IIIa levels were significantly decreased. GPIb/V/IX, P-selectin, and phosphatidylserine levels manifested only moderate differences. Forward and side scatter changes in response to stimulation (representing shape change) and dense granules release were significantly lower in the 3 patients with bleeding than in the 17 patients without hemorrhage. Activated partial thromboplastin time was slightly prolonged, prothrombin index was slightly shortened and thrombin time was normal, while fibrinogen was mildly decreased in the ET patients. It could be concluded that the observed platelet function defects could be related to bleeding in ET, and be potentially used as a marker.     

PlA polymorphism and platelet reactivity following clopidogrel loading dose in patients undergoing coronary stent implantation.

The PlA polymorphism (Leu33Pro) of the platelet glycoprotein (GP) IIIa gene has been suggested to play an important role in coronary thrombosis. In vitro studies have shown differences for this polymorphism in platelet sensitivity towards antiplatelet drugs (aspirin and abciximab), suggesting a pharmacogenetic modulation. The aim of the study was to assess the modulatory effect of the PlA polymorphism on clopidogrel-induced antiplatelet effects in 38 patients undergoing coronary stent implantation receiving a 300 mg clopidogrel loading-dose. Platelet reactivity was assessed as GPIIb/IIIa activation and P-selectin expression in platelets stimulated with 2 micromol/l adenosine diphosphate using whole blood flow cytometry. The distribution of the homozygous PlA1/A1 and heterozygous PlA1/A2 genotypes were 74 and 26%, respectively. PlA2 carriers had a higher degree of GPIIb/IIIa activation (P = 0.05) and P-selectin expression (P = 0.02) during the overall study time course and a lower antiplatelet effect to a 300 mg clopidogrel loading-dose up to 24 h following intervention (P < 0.05). In conclusion, the Pl polymorphism of the GPIIIa gene modulates platelet reactivity towards clopidogrel front loading in patients undergoing coronary stenting. This suggests the need for individualized antithrombotic regimens to optimally inhibit platelet reactivity.     

A platelet P-selectin test predicts adverse cardiovascular events in patients with acute coronary syndromes treated with aspirin and clopidogrel

Abstract

There is wide variation in response to antiplatelet therapy and high on-treatment platelet reactivity is associated with adverse cardiovascular events. The objective here was to determine whether the results of a novel strategy for assessing platelet reactivity (based on P-selectin measurement) are associated with clinical outcomes in patients with acute coronary syndromes (ACS). This was a prospective cohort study of 100 ACS patients taking aspirin and clopidogrel. P-selectin tests designed to assess response to P2Y₁₂ antagonists or aspirin were performed alongside light transmission aggregometry. For the P2Y₁₂ P-selectin test, an optimal cutoff for high platelet reactivity was determined by receiver operating characteristic (ROC) curve analysis. Patients were divided into two cohorts based on this value: patients with (n?=?42) or without (n?=?58) high platelet reactivity. The primary endpoint was defined as the composite of cardiovascular death, myocardial infarction and stent thrombosis. After 12 months, the primary endpoint occurred in 12 patients. ROC curve analysis determined that the P2Y₁₂ P-selectin test results were predictive of the primary endpoint (area under curve?=?0.69, p?=?0.046). The primary endpoint occurred more frequently in patients with high on-treatment platelet reactivity compared to those without (21.4% vs. 5.2%; hazard ratio (HR) 4.14; p?=?0.026). The P2Y₁₂ P-selectin test results correlated with light transmission aggregometry (Spearman p?<?0.0001). Using the Aspirin P-selectin test, only two patients demonstrated high on-treatment platelet reactivity. This study suggests that a P2Y₁₂ P-selectin test is capable of detecting high on-treatment platelet reactivity, which is associated with subsequent cardiovascular events.