The stratification of platelet reactivity and activation in patients with stable coronary artery disease on aspirin therapy

Heightened platelet reactivity may affect the occurrence of ischemic events in patients with coronary artery disease on aspirin therapy. However, a definition to stratify platelet reactivity in this group of patients has not been previously reported. We studied platelet reactivity and activation by measuring platelet aggregation and the expression of p-selectin, total GP IIb/IIIa and active GP IIb/IIIa (n=96). Patients were divided into quartiles by each of the markers; correlations were made between the markers; and a definition of heightened platelet reactivity was proposed. Marked variability in activation and reactivity were observed despite aspirin therapy. BACKGROUND: Heightened platelet reactivity and activation may affect the occurrence of ischemic events in patients with coronary artery disease on aspirin therapy. However, a definition to stratify platelet reactivity has not been previously reported. METHODS AND RESULTS: Platelet aggregation (5 and 20 micromol/l ADP), total GP IIb/IIIa, active GP IIb/IIIa and the expression of maximally stimulated p-selectin were measured in patients about to undergo elective coronary stenting (n=96). All patients had received aspirin (325 mg). There was marked variability in platelet reactivity and activation as measured by all markers. The highest quartile was defined by 77+/-1% and 98+/-1% aggregation by 5 and 20 micromol/l ADP, respectively; 65+/-2% p-selectin positivity; 508+/-15 MFI for total GP IIb/IIIa; and 23.0+/-1.8 MFI for active GP IIb/IIIa. CONCLUSIONS: There is a wide range in platelet reactivity and activation as measured by multiple markers in stable coronary disease patients on aspirin therapy. From these indices, we can define those patients at the extremes of reactivity and activation and thus, the greatest potential risk of thrombosis and bleeding. These indices will serve as a guide to future studies investigating the relationships of platelet reactivity, activation, drug-induced inhibition and clinical outcomes.     

Clinical utility of the platelet function analyzer (PFA-100) for the assessment of the platelet status in patients with congestive heart failure (EPCOT trial)

Background: Data from small studies have shown the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of platelet function analyzer (PFA-100) in the CHF population. Methods: Blood samples were obtained for measurement of adenosine diphosphate (ADP)/collagen and epinephrine/collagen shear-induced closure time (CT), whole blood aggregation, platelet contractile force, activity of glycoprotein (GP) IIb/IIIa, and P-selectin receptors in 100 consecutive outpatients with CHF. Results: Substantial interindividual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were divided by the incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Aspirin use did not affect instrument readings as well. CT correlates well with whole blood aggregometry (r²=.587) and less with GP IIb/IIIa activity (r²=.326). No correlation has been observed for the CT with the platelet-bound P-selectin (r²=.041) and platelet contractile force measures (r²=.028). Conclusions: PFA-100 is indeed capable to serve as a platelet analyzer and may be successfully used as a screening device. However, patients with heart failure enrolled in the EPCOT trial exhibited a marginal, sometimes oppositely directed changes in the platelet function, challenging the diagnostic utility of PFA-100 to serve as a useful tool for the identification of platelet abnormalities, predicting clinical outcomes, or for the monitoring of antiplatelet strategies in this population.     

Evidence of increased platelet reactivity in the first six months after acute ST segment elevation myocardial infarction

Introduction

Platelets play a crucial role in the pathogenesis of acute coronary syndromes. Accordingly, previous studies showed increased platelet reactivity on admission in these patients. In this study we assessed platelet reactivity at short-medium term follow-up in patients with ST-segment elevation acute myocardial infarction (STEMI).

Materials and methods

Fifty-nine patients (58 ± 11 years, 45 men), treated with primary angioplasty, were studied 1 month after STEMI. Thirty-five patients were retested at 6 months. Twenty matched patients with stable coronary artery disease served as controls. Platelet reactivity was assessed by flow cyometry at rest and at peak exercise, with and without adenosine diphosphate (ADP) stimulation, by measuring monocyte-platelet aggregates (MPAs) and glycoprotein IIb/IIIa (CD41) expression in the MPA gate, and CD41 and fibrinogen receptor (PAC-1) expression in the platelet gate.

Results

Compared to controls, basal MPAs and CD41 in the MPA gate were higher in STEMI patients both at 1 month (p = 0.001 and p = 0.002, respectively) and at 6 months (p = 0.03 and p = 0.01, respectively). Basal CD41 and PAC-1 expression was also higher in STEMI patients at the two assessments compared to controls (P < 0.001 for both). Exercise induced a similar increase in platelet reactivity in patients and controls. ADP induced a higher increase in CD41 platelet expression in STEMI patients compared to controls both at 1 and 6 months (P < 0.001).

Conclusion

Platelet reactivity is increased in the first 6 months after STEMI. The persistence of increased platelet reactivity in this time period may play a role in the early recurrence of coronary events after STEMI.     

Platelet reactivity in response to mental stress in syndrome X and in stable or unstable coronary artery disease

INTRODUCTION: Previous studies showed differences in platelet reactivity in response to exercise in patients with syndrome X (SX) and those with obstructive coronary artery disease (CAD). In this study, we investigated whether similar differences could be detectable in response to a mental stressful stimulus. MATERIALS AND METHODS: We studied 30 SX patients (60+/-8 years, 8 men), 20 patients with stable angina and angiographically documented CAD (SA, 66+/-8 years, 14 men), and 11 patients with unstable angina (UA, 67+/-8 years, 6 men). A control group of 22 healthy controls (50+/-7 years, 5 men) was also studied. All subjects underwent a mathematical mental stress test (MST) consisting of rapid consecutive subtractions of number 7 for a period of 5 min. A venous blood sample was collected at baseline and immediately after MST. Platelet reactivity was measured on flowing blood as time necessary to occlude a ring coated with collagen-adenosine diphosphate (ADP; closure time, CT), using the platelet function analyzer (PFA-100) system. RESULTS: At rest, CT was lower in UA patients (87+/-19 s) compared to controls (109+/-24 s, p=0.02) and SA patients (105+/-37 s, p=0.055), and also tended to be lower in SX patients (96+/-18 s) compared to controls (p=0.07). The CT response to MST differed significantly among groups (p=0.0009). At peak MST, CT did not change in controls (110+/-27 s, p=0.88), whereas it decreased in SA patients (96+/-29 s, p=0.003) and tended to decrease in UA patients (82+/-15 s, p=0.25). In contrast, a significant increase in CT with MST was shown in SX patients (103+/-21 s, p=0.007). CONCLUSIONS: Platelet reactivity is increased by MST in patients with CAD, whereas it paradoxically decreases in SX patients, which may constitute a protective effect against stress-induced events in these patients.     

A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex

Collagen–platelet interaction plays an important role in hemostasis and pathological thrombosis. The proposed mechanism of the interaction was the activation of platelets→releasing of contents from granules→aggregation. The common end point is the platelets and fibrin aggregates. Platelet glycoprotein (GP) IIb/IIIa (the IIbβ3 integrin) complexes serve as a receptor for the binding of fibrinogen to form firmed aggregates. Blockading of GP IIb/IIIa has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. The development of various forms of GP IIb/IIIa inhibitor has resulted in the inhibition of platelet aggregation, although studies of IIbβ3 receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having nonplatelet effects. This study investigated platelet inhibition provided by blocking the GP IIb/IIIa complex formation by using a peptide derived from the GP IIIa molecule. The peptide inhibits both types I and III collagen-induced platelet aggregation in a dose-dependent manner. The defined peptide interferes with the formation of the GP IIb/IIIa complex by inhibiting the binding of FITC–PAC-1 onto ADP-, type I collagen-, and type III collagen-activated platelets. However, P-selectin secretion is not affected by the peptide. In addition, the peptide is not interfering with the binding of FITC–PAC-1 to platelets that were preincubated with indomethacin. Results from this study may suggest that the defined peptide is an effective agent to block the interaction of types I and III collagen with platelets.     

Selective inhibition of the platelet phosphoinositide 3-kinase p110beta as promising new strategy for platelet protection during extracorporeal circulation

Extracorporeal circulation (ECC) is used in cardiac surgery for cardiopulmonary bypass as well as in ventricular assist devices and for extracorporeal membrane oxygenation. Blood contact with the artificial surface and shear stress of ECC activates platelets and leukocytes resulting in a coagulopathy and proinflammatory events. Blockers of the platelet glycoprotein (GP) IIb/IIIa (CD41/CD61) can protect platelet function during ECC, a phenomenon called "platelet anaesthesia", but may be involved in post-ECC bleeding. We hypothesized that the new selective phosphoinositide 3-kinase p110beta inhibitor TGX-221 that inhibits shear-induced platelet activation without prolonging the bleeding time in vivo may also protect platelet function during ECC. Heparinized blood of healthy volunteers (n = 6) was treated in vitro with either the GP IIb/IIIa blocker tirofiban, TGX-221 or as control and circulated in an ECC model. Before and after 30 minutes circulation CD41 expression on the ECC-tubing as measure for platelet-ECC binding and generation of the platelet activation marker beta-thromboglobulin were determined using ELISA. Platelet aggregation and platelet-granulocyte binding were analysed in flow cytometry. After log-transforming the data statistical evaluation was performed using multifactor ANOVA in combination with Tukey's HSD test (global alpha = 5%). Tirofiban and TGX-221 inhibited platelet-ECC interaction, platelet aggregation and platelet-granulocyte binding. Tirofiban also inhibited ECC-induced beta-thromboglobulin release. The observed inhibition of platelet-ECC interaction and platelet activation by tirofiban contributes to explain the mechanism of "platelet anaesthesia". TGX-221 represents a promising alternative to GP IIb/IIIa blockade and should be further investigated for use during ECC in vivo.     

Upregulation of GP IIb/IIIa receptors during platelet activation: influence on efficacy of receptor blockade

INTRODUCTION: GP IIb/IIIa inhibitor doses high enough to inhibit platelet macroaggregation may not completely prevent formation of microaggregates. Platelets contain an internal pool of GP IIb/IIIa receptors which externalizes with activation and supports microaggregation. This study assesses microaggregation in the presence of the two GP IIb/IIIa inhibitors abciximab and tirofiban. METHODS: Citrated whole blood was preincubated with abciximab (5 microg/ml), tirofiban (50 ng/ml), or saline as control and activated with TRAP (5 microM) or ADP (2 microM) at 37 degrees C under constant stirring. Microaggregate formation and receptor expression were determined with flow cytometry. RESULTS: Within few seconds after TRAP-activation the platelet count dropped from 266,000/microl to 20,000/microl, the number of microaggregates increased from 3700/microl to 10,100/microl and the mean number of GP IIb/IIIa receptors increased from 53,000 to 65,000/platelet. With TRAP+abciximab the platelet count dropped from 259,000 to 113,000/microl, microaggregates increased from 2500 to 9300/microl, GP IIb/IIIa receptors from 56,000 to 77,000/platelet. Platelet microaggregate formation was reversible. With TRAP and tirofiban platelet count dropped to only 190,000/microl, there was no increase in platelet microaggregates, receptors increased to 66,000/platelet. Platelet activation with ADP gave similar results. CONCLUSIONS: During the early phase of activation additional GP IIb/IIIa receptors externalize to the platelet surface. Abciximab does not block these new receptors sufficiently to prevent microaggregate formation. However, the number of unblocked receptors is not high enough to maintain a stable aggregate, microaggregation is reversible. With tirofiban there is no microaggregate formation, possibly because the inhibitor rapidly binds to newly externalized receptors.     

Identification of low responders to a 300-mg clopidogrel loading dose in patients undergoing coronary stenting

BACKGROUND: Although patients undergoing coronary stenting routinely receive dual antiplatelet treatment to reduce the risk of stent thrombosis, this undesired event still occurs. A suboptimal response to clopidogrel treatment (low responders) has been suggested to contribute to stent thrombosis. In the present study, platelet function profiles were assessed in patients undergoing coronary stenting receiving a standard 300-mg clopidogrel loading dose with the aim to identify low clopidogrel responders. MATERIALS AND METHODS: Platelet aggregation was assessed by light transmittance aggregometry following 6 microM ADP stimuli in 48 patients before and 10 min, 4 and 24 h after receiving clopidogrel front-loading. Patients having > or =40% inhibition of platelet aggregation 24 h after clopidogrel administration were defined as normal responders, whereas those having <40% inhibition were low responders. Glycoprotein (GP) IIb/IIIa activation and P-selectin expression were assessed by whole blood flow cytometry following 2 microM ADP stimuli at the same time points. Platelet function profiles were compared between normal and low clopidogrel responders. RESULTS: Twenty-seven patients (56%) were normal responders and 21 (44%) low responders. Baseline GP IIb/IIIa activation was higher in low responders (74.6+/-16.6% vs. 58.2+/-24.5%, p=0.03). Although GP IIb/IIIa activation reduced following clopidogrel front-loading in both groups, it remained increased among low responders at 24 h (58.6+/-21.3% vs. 40.2+/-28.7%, p=0.05) and during the overall study time course (p=0.02). There were no differences in P-selectin expression. CONCLUSIONS: A considerable proportion of patients have an early suboptimal response to a 300-mg clopidogrel loading dose. An increased GP IIb/IIIa activation before intervention may identify this group of patients suggesting the use of a more aggressive antithrombotic treatment in these individuals.     

Blood platelet membrane fluidity and the exposition of membrane protein receptors in Alzheimer disease (AD) patients--preliminary Study

We estimated membrane fluidity by the (ESR) spectroscopy and the expression of membrane P-selectin and glycoproteins GP Ib alpha and GP IIb/IIIa by flow cytometry in platelets (plts) from 12 AD sufferers. In AD patients membrane fluidity was significantly increased at two different depths (p < 0.05 or less). Platelet reactivity was significantly decreased, as reflected by reduced expression of GP Ib alpha in the resting (p < 0.0001) and activated (p < 0.005) platelets, as well as the expression of P-selectin and beta 3 subunit of GP IIb/IIIa in activated platelets (p < 0.0001; p < 0.04).     

Abciximab therapy is associated with increased platelet activation and decreased heparin dosage in patients with acute myocardial infarction

The inhibition of the glycoprotein (GP) IIb/IIIa receptor for reducing periprocedural ischemic events in patients undergoing coronary intervention is known to influence platelet reactivity. Suboptimal doses of GP IIb/IIIa antagonists have been suggested to be prothrombotic and proinflammatory. This study was performed to observe platelet activation markers, whole blood aggregation and the dosage of unfractionated heparin (UFH) in the presence or absence of the GP IIb/IIIa inhibitor abciximab. Patients with acute myocardial infarction undergoing percutaneous coronary intervention were treated with (n = 15) or without (n = 15) abciximab. Platelet activation markers were flow cytometrically measured before and after PCI. Whole blood platelet aggregation was tested by a platelet function assay. The patients with abciximab showed a significant increase in platelet activation markers (P-selectin: 7.12 +/- 0.36 AU vs 11.05 +/- 0.79 AU) and a lower requirement of UFH to prolong aPTT > 60 sec during the infusion. 12 hours after infusion P-selectin level decreased (7.20 +/- 0.58 AU), whereas whole blood aggregation was increasing again. After stopping abciximab, requirement of UFH to prolong aPTT increased in the treated group to a greater extent to a level similar to the untreated group even when most of the platelets were still inhibited. The increased platelet activation found at the end of abciximab treatment points to a procoaguable condition that should be carefully monitored and treated by adapting anticoagulation and antiplatelet drugs.     

Differential inhibitory effects of platelet glycoprotein IIb/IIIa antagonists on aggregation induced by procoagulant agonists

INTRODUCTION: In addition to mediating the final common pathway of aggregation, the glycoprotein (GP) IIb/IIIa receptor participates in the activation of coagulation on the platelet surface. High-affinity conformation of GP IIb/IIIa in response to collagen-induced inside-out signalling seems to be mediated by GP VI(-FcRgamma) and reinforced by release of soluble mediators. METHODS: We assessed the effects of the three currently available GP IIb/IIIa antagonists--abciximab, tirofiban and eptifibatide--on platelet aggregation induced by various procoagulant and GP VI-related agonists, i.e. collagen-related peptide (CRP), convulxin and collagen fibrils, in PPACK-anticoagulated platelet-rich plasma. RESULTS: At concentrations that equally inhibited 80% of ADP-induced maximal aggregation abciximab-inhibited GP VI-mediated platelet responses to CRP or convulxin significantly more than the low-molecular-weight antagonists (CRP: abciximab 75+/-18%, tirofiban 41+/-7% and eptifibatide 41+/-6%; convulxin: abciximab 90+/-6%, tirofiban 64+/-20%, eptifibatide 61+/-14%, p<0.01 for all). In contrast, aggregation induced by collagen was equally abolished with all antagonists under the similar conditions. During CRP- or convulxin-triggered platelet activation, inhibition of fibrin polymerisation with GPRP potentiated the antiaggregatory effects of tirofiban and eptifibatide to reach that of abciximab. GPRP as such did not affect platelet aggregation. CONCLUSIONS: GP IIb/IIIa antagonists exhibit distinct inhibition profiles in platelet aggregation, depending on fibrin polymerization and calcium. Specifically, the ability of procoagulant platelet agonists to expose pre-activated and ligand-bound GP IIb/IIIa from the internal pool seems important.     

Resistance to aspirin in vitro is associated with increased platelet sensitivity to adenosine diphosphate

BACKGROUND: Platelet activation plays an important role in arterial thrombosis and the widespread use of aspirin has reduced major events by 25% in the secondary prevention of cardiovascular diseases. However, it appears that aspirin antiplatelet effect is not uniform and 8-45% of the population are, in vitro, aspirin resistant, and it is well recognized that platelets can be activated by pathways that are not blocked by aspirin, such as adenosine diphosphate (ADP). OBJECTIVES: To investigate whether aspirin-resistant patients have a modified sensitivity to ADP-induced platelet activation Material AND METHODS: Seventy-two patients were enrolled. Platelet function was measured by the PFA-100(R) analyser; platelet GP IIb-IIIa activation by ADP 10 micro M was assessed by flow cytometry using PAC-1 MoAb. RESULTS: Using a collagen/epinephrine coated cartridge on the PFA-100(R), the prevalence of aspirin resistance was 29.2% (n=21). For aspirin-resistant patients, the collagen/ADP coated cartridge showed a closure time significantly shorter (p=0.004) compared to the sensitive and control groups. Platelets from aspirin-resistant patients bound PAC-1 significantly more (p=0.03) than the aspirin-sensitive patients and controls when activated with 10 micro M ADP. CONCLUSIONS: Platelets from aspirin-resistant patients appear to be more sensitive and activable by ADP. This hypersensitivity could provide a possible explanation for the so-called aspirin resistance, and this could justify therapeutic improvement with alternative antiplatelet agents.     

Flow cytometric analysis of platelet activation in hypertensive patients. Effect of doxazosin

The percentage of spontaneously activated platelets and the platelet response to several agonists were studied in 26 hypertensive patients. The percentage of platelets expressing glycoprotein (GP) IIb/IIIa in its active conformation (GPIIb/IIIa*), P-selectin and phosphatidylserine (PS) was measured by flow cytometry at baseline and 1 and 2 months after treatment with doxazosin (4 mg/day). The response to ADP and Ca2+ ionophore was also evaluated. The results were compared with those of a control group of 71 normotensive volunteers. Spontaneous platelet activation was higher in patients than in controls (P-selectin-positive results in 4.4+/-2.0% patients vs. 2.7+/-1.7 controls, p<0.05; phosphatidylserine-positive results in 0.7+/-0.4% vs. 0.5+/-0.3%, respectively, p<0.05), and higher in response to ionophore action (phosphatidylserine-positive results 51.8+/-11.1% vs. 43.4+/-11.7%, p<0.01). Platelet activation in patients decreased after 2 months of doxazosin administration compared to baseline (P-selectin-positive results 2.7+/-1.4% vs. 4.4+/-2.0%, p<0.05; phosphatidylserine-positive results 0.3+/-0.2% vs. 0.7+/-0.4%, p<0.05). No significant differences were noted in GPIIb/IIIa*. The clinical significance of normalization of platelet activity by doxazosin remains to be established.     

Elevated expression of integrin alpha(IIb) beta(IIIa) in drug-na?ve, first-episode schizophrenic patients.

BACKGROUND: Patients with schizophrenia have an increased risk over the general public of developing cardiovascular illness. It is unknown if there are functional changes in platelet surface receptors in schizophrenia. We therefore analyzed the surface expression of glycoprotein (GP)Ib, the integrin receptor alpha(IIb)beta(IIIa), CD62 (P-selectin), and CD63, and investigated platelet function in schizophrenic patients compared with healthy volunteers. METHODS: Nineteen drug-naive, first-episode patients with a DSM IV diagnosis of paranoid schizophrenia were compared with matched healthy controls. Flow cytometry was used to assess platelet surface expression levels of GPIb, alpha(IIb)beta(IIIa), CD62, and CD63. Adenosine diphosphate-induced platelet aggregation was assayed. RESULTS: The schizophrenic patients had a significantly (p < .0001) increased number of 68,145 +/- 8,260.1 alpha(IIb)beta(IIIa) receptors, platelet compared with 56,235 +/- 8,079.4 receptors, platelet in healthy controls. CONCLUSIONS: Patients with schizophrenia have increased platelet expression of alpha(IIb)beta(IIIa), which may contribute to their increased risk of cardiovascular illness compared with the general population.     

Baseline platelet activity and response after clopidogrel in 257 diabetics among 822 patients with coronary artery disease

The objective was to describe the indices of platelet aggregation and activation in a large cohort of diabetic patients with coronary artery disease (CAD). Recently, a number of observations have indicated that patients with diabetes mellitus (DM) exhibit persistent platelet activation, and low response after antiplatelet therapy, although no randomized data exist. We sought to define the baseline platelet biomarkers, and the patterns of response to aspirin and clopidogrel therapy in DM versus non-diabetic patients. Secondary post-hoc analyses were made of platelet activity biomarkers in the dataset which consisted of patients with documented CAD (n = 822), including those with DM (n = 257). Patients with DM exhibited higher baseline platelet activity by adenosine diphosphate (ADP)- (p = 0.0002), and collagen-induced (p = 0.03) aggregometry; Ultegra- (p = 0.0001), and PFA-100 (p = 0.02) analyzers; and expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (p = 0.01), glycoprotein (GP) IIb/IIIa antigen (p = 0.001), and activity (p = 0.02), vitronectin receptor (p = 0.03), P selectin (p = 0.02), and intact epitope of PAR-1 thrombin receptor (p = 0.02). Antiplatelet response after clopidogrel in diabetics was impaired when compared with non-diabetics. In conclusion, diabetic patients exhibit high pretreatment platelet activity, and do not respond well to the available antiplatelet regimens when compared with similar patients without DM. The clinical implications of these findings are unknown but are potentially important. Considering worsened outcomes in this high-risk population, clinical trials in DM are urgently needed in order to define the optimal degree of platelet inhibition and suitability for more aggressive antiplatelet regimens.     

CD32-dependent platelet activation by a drug-dependent antibody to glycoprotein IIb/IIIa antagonists

Thrombocytopenia is observed with a frequency of up to 2% in patients treated with glycoprotein (GP) IIb/IIIa antagonists. We recently provided evidence that thrombocytopenia is caused by antibody binding to drug-induced conformational changes in GP IIb/IIIa. Here, we report that a murine monoclonal antibody binds to GP IIb/IIIa in an antagonist-dependent manner and activates platelets. Platelet stimulation is associated with a disruption of the phospholipid asymmetry, resulting in the assembly of catalytic active intrinsic Xase and prothrombinase complexes. Further mechanistic studies revealed that this response is (I) mediated in cis, (II) not associated with the formation of prothrombotic microparticles, and (III) requires intact platelet signaling and (IV) is blocked by increases in cAMP. The prothrombotic response is not observed using F(ab')2 fragments and is blocked by incubation of platelets with neutralizing antibodies to the platelet FcgammaRIIa receptor (CD 32).Taken together, these observations suggest that GPIIb/IIIa antagonist-dependent antibody binding to the platelet fibrinogen receptor has the propensity to lead to CD32-mediated platelet activation and accelerated platelet clearance, leading to thrombocytopenia.     

Lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy

INTRODUCTION: Platelets play an important role in the natural history of ischemic stroke, and are known to be activated in the acute phase. Although aspirin reduces risks of myocardial infarction, stroke and cardiovascular death, the extent of platelet action and the effect of aspirin on platelet function in patients recovering from stroke remain unclear. METHODS: We studied 120 individuals divided into three equal groups: aspirin-free patients after ischemic stroke, post-stroke patients receiving aspirin (81-650 mg/daily), and aspirin-free subjects with multiple risk factors for vascular disease. Conventional platelet aggregation induced by 5 microM ADP and 5 microM epinephrine, cartridge-based analyzers (Ultegra, and PFA-100) readings, and expression of CD31, CD41a, CD42b, GPIIb/IIIa activity, CD51/CD61, CD62p, CD63, CD107a, CD154, CD165, formation of platelet-monocyte aggregates, intact (SPAN12), and cleaved (WEDE15) PAR-1 thrombin receptors by flow cytometry were analyzed. RESULTS: There were no differences between aspirin-free post-stroke patients and aspirin-free controls. Although aggregation was slightly higher, 12 out of the 14 receptor analyses, were surprisingly lower in the post-stroke cohort. Aspirin-treated patients exhibited highly significant inhibition of epinephrine-induced aggregation (p=0.0001), prolongation of the closure time (p=0.03), and reduction of the aspirin reactive units (p=0.02) measured by the Ultegra device. In addition, surface platelet expression of thrombospondin (p=0.001), GPIIb/IIIa activity (p=0.04), P-selectin (p=0.03), CD40-ligand (p=0.04), CD165 (p=0.02), the formation of the platelet-monocyte aggregates (p=0.01), and intact epitope of PAR-1 thrombin receptor (p=0.03) were significantly lower in the aspirin-treated group. CONCLUSIONS: Platelets are not activated in aspirin-free patients after ischemic stroke. Platelet function is significantly inhibited in those treated with aspirin when compared with healthy subjects with risk factors for vascular disease. Bleeding complications and hemorrhagic transformations after aggressive antiplatelet regimens could be related to the decreased or normal baseline platelet characteristics in such patients. Further analysis of platelet heterogeneity and its clinical significance remains to be determined in randomized trials.     

Platelet glycoprotein Ia C807T, Ib C3550T, and IIIa Pl(A1/A2) polymorphisms and ischemic stroke in young Taiwanese

Platelet plays a pivotal role in the pathogenesis of thrombotic cardiovascular diseases. Recently, the polymorphism of platelet glycoprotein (GP) genes has been reported to be associated with an increased risk for ischemic stroke. The purpose of this study is to evaluate the association between platelet GP genetic variants and ischemic stroke in young Taiwanese. We conducted a case-control study in 157 young ischemic stroke patients recruited between September 2001 and March 2003 and 157 age- and sex-matched controls. The genotypes of platelet GP Ia C807T, GP Ib C3550T, and GP IIIa Pl(A1/A2) polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Student's t-test, chi-square test, and logistic regression modeling were used for data analyses. The GP Ia C807T CC, CT and TT genotype frequencies were similar between patients (50.3%, 43.9%, 5.7%) and controls (53.5%, 38.9%, 7.6%; p=0.58). There were no significant differences in GP Ib C3550T CC and CT genotype distributions between patients (91.1%, 8.9%) and controls (91.7%, 8.3%; p=0.84). Of all subjects, none carries GP IIIa Pl(A2) mutation. In conclusion, platelet GP Ia C807T and GP Ib C3550T polymorphisms in our population are less common compared with Caucasians, and GP IIIa Pl(A1/A2) genetic mutation is not found, and all of them are not associated with ischemic stroke in young Taiwanese.     

Glycoprotein IIb/IIIa inhibition reduces prothrombotic events under conditions of deep hypothermic circulatory arrest

Deep Hypothermic Circulatory Arrest (DHCA) is employed during thoracic aortic and congenital heart surgery, and can induce postoperative neurological damage probably caused by microthrombembolism. Hypothermia has been reported to induce platelet activation and aggregation. The platelet activation marker P-selectin mediates binding of platelets to leukocytes. Tirofiban and eptifibatide, short-acting inhibitors of the platelet fibrinogen receptor GP IIb/IIIa, have recently been shown to protect platelet function without increasing bleeding during heart surgery using cardiopulmonary bypass. The aim of this study was to investigate the effect of tirofiban and eptifibatide on platelets and platelet-leukocyte interaction under DHCA conditions in vitro. Platelet aggregation, binding of the GP IIb/IIIa activation specific antibody PAC-1, P-selectin expression as well as monocyte and granulocyte content of aggregates were investigated in unstimulated and ADP-stimulated samples using flow cytometry. Tirofiban and eptifibatide inhibited massive platelet aggregation and PAC-1 binding which were induced by DHCA conditions. P-selectin expression was inhibited by tirofiban but increased by eptifibatide at hypothermia. Platelet-bound leukocytes were present in all samples. Eptifibatide increased granulocyte content of aggregates at hypothermia in ADP-stimulated samples. We conclude that under conditions of DHCA both tirofiban and eptifibatide inhibit platelet aggregation but have different effects on platelet P-selectin expression and platelet-leukocyte interaction. Application of a short-acting and non-activating GP IIb/IIIa inhibitor should be considered during DHCA in vivo to prevent occlusion of the microvasculature and subsequent organ damage.     

Platelet anaesthesia during extracorporeal circulation: differential effects of GP IIb/IIIa blockers on platelet activation marker P-selectin expression at hypothermia

INTRODUCTION: Blood contact with artificial surfaces of extracorporeal circulation (ECC) and hypothermia as applied in cardiac surgery cause platelet dysfunction possibly followed by bleeding complications. "Platelet anaesthesia" is a pharmacological strategy to protect platelets against ECC-induced damage using a GP IIb/IIIa blocker, which should be short acting to achieve maximal therapy control thereby avoiding post-ECC haemorrhage. However, GP IIb/IIIa blockers can paradoxically induce platelet activation, which may limit their efficiency as anti-platelet drugs. This in-vitro study investigated potentially platelet-activating effects of short-acting GP IIb/IIIa blockers during normothermic and hypothermic ECC. MATERIALS AND METHODS: Control (untreated) and treated (using either FK633 [half-life: 0.52 h], tirofiban [half-life: 1.5-2 h], or eptifibatide [half-life: 1.5 h]) heparinized blood was circulated in an ECC-model at normothermia (37 degrees C) and hypothermia (18 degrees C). Percentages of platelet aggregates and P-selectin-expressing (activated) platelets, platelet-counts and Thrombin-Antithrombin (TAT) complex formation were determined before (baseline) and after ECC. Statistical analysis was performed using multifactorial ANOVA after log-transforming the data. RESULTS: GP IIb/IIIa blockade inhibited ECC-induced platelet aggregation and platelet loss and decreased P-selectin expression at normothermia. During hypothermic ECC P-selectin was decreased by tirofiban but augmented by FK633 and eptifibatide. TAT formation was only decreased by FK633. CONCLUSIONS: Especially regarding its ultra-short half-life FK633 has the best properties for platelet protection during normothermic ECC. However, at hypothermia FK633 and eptifibatide induce platelet activation. In relation with "platelet anaesthesia" possible hypothermia-associated prothrombotic side effects of GP IIb/IIIa blockers should be considered.