ADP-induced P-selectin Expression Research Articles

Decreased Platelet Aggregation in Patients with Decompensated Liver Cirrhosis and TIPS Implantation.

Transjugular intrahepatic portosystemic shunt (TIPS) implantation is an effective treatment of portal hypertension in patients with decompensated liver cirrhosis. However, some patients develop TIPS thrombosis with recurrence of portal hypertension. The role of platelets in TIPS thrombosis and the necessity of antiplatelet therapy is unclear. Therefore, we aimed to study platelet function in patients with liver cirrhosis prior to and after TIPS implantation. Platelet aggregation was tested in peripheral and portal-vein blood patient samples on the day (D) of TIPS implantation (D0), D4 and D30 following the procedure (platelet count above 100 × 103/µL, aspirin starting on D5) using whole-blood impedance aggregometry (WBIA) and light transmission aggregometry (LTA). In addition, surface platelet activation markers (P-selectin, activated GPIIb/IIIa) and platelet-neutrophil complexes (PNCs) were assessed by flow cytometry. Thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP) and arachidonic acid (AA) were used as agonists. Healthy subjects were included as controls. Agonist-induced platelet aggregation was reduced (WBIA: TRAP-6 p < 0.01, ADP p < 0.01, AA p < 0.001; LTA: TRAP-6 p = 0.13, ADP p = 0.05, AA p < 0.01) in patients (D0, n = 13) compared with healthy subjects (n = 9). While surface activation markers at baseline were negligibly low, the percentage of PNCs was higher in patients than in controls (p < 0.05). ADP-induced P-selectin expression was increased (p < 0.001), whereas TRAP-6-induced GPIIb/IIIa activation was impaired (p < 0.001) in patients versus controls. PNC formation in response to agonists was not different between groups. Results did not differ between peripheral and portal-vein blood of patients (D0, n = 11) and did not change over time (D0, D4, D30) following TIPS implantation (n = 9). In summary, patients with decompensated liver cirrhosis display in vitro platelet aggregation defects in response to various agonists. Defective aggregation persists upon TIPS implantation. Therefore, we conclude that antiplatelet treatment to prevent TIPS thrombosis is questionable.

Hemoglobin-bound platelets correlate with the increased platelet activity in hemoglobin E/β-thalassemia.

An increase in platelet activity is a contributing factor to vascular complications in hemoglobin E/β-thalassemia (HbE/β-thal). Plasma-free hemoglobin (Hb) increases in HbE/β-thal patients and correlates with platelet activation, but the levels of Hb-bound platelets have never been reported. In this study, we aimed to investigate the levels of Hb-bound platelets and its association with platelet activity in HbE/β-thal patients. Hb-bound platelets were measured by flow cytometry in 22 healthy subjects and 26 HbE/β-thal patients (16 nonsplenectomized and 10 splenectomized HbE/β-thal patients). Plasma Hb was measured by the chemiluminescence method based on the consumption of nitric oxide (NO) by Hb. Expression of P-selectin and activated glycoprotein (aGP) IIb/IIIa on platelets was measured by flow cytometry as a marker of platelet activity. Both nonsplenectomized and splenectomized HbE/β-thal patients had higher levels of Hb-bound platelets and plasma Hb than healthy subjects. In vitro incubation of dialyzed Hb from patients with platelets of healthy subjects caused an increase in Hb-bound platelets, which was partially inhibited by anti-GPIbα antibody. Plasma Hb positively correlated with Hb-bound platelets. Platelet P-selectin expression at baseline and in response to adenosine diphosphate (ADP, 1µM) stimulation was higher in nonsplenectomized and splenectomized HbE/β-thal patients than healthy subjects. The ADP-induced aGPIIb/IIIa expression on platelets was also higher in HbE/β-thal patients than healthy subjects. Hb-bound platelets correlated with baseline P-selectin expression and ADP-induced P-selectin expression. HbE/β-thal patients have increased Hb-bound platelets, which is associated with increased baseline platelet activation and reactivity.

Decreased nitrite reductase activity of deoxyhemoglobin correlates with platelet activation in hemoglobin E/ß-thalassemia subjects.

Nitric oxide (NO) can be generated from nitrite by reductase activity of deoxygenated hemoglobin (deoxyHb) apparently to facilitate tissue perfusion under hypoxic condition. Although hemoglobin E (HbE) solutions have been shown to exhibit decreased rate of nitrite reduction to NO, this observation has never been reported in erythrocytes from subjects with hemoglobin E/ß-thalassemia (HbE/ß-thal). In this study, we investigated the nitrite reductase activity of deoxyHb dialysates from 58 non-splenectomized and 23 splenectomized HbE/ß-thal subjects compared to 47 age- and sex-matched normal subjects, and examined its correlation with platelet activity. Iron-nitrosyl-hemoglobin (HbNO) was measured by tri-iodide reductive chemiluminescence as a marker of NO generation. HbNO produced from the reaction of nitrite with deoxyHb dialysate from both non-splenectomized and splenectomized HbE/ß-thal subjects was lower than that of normal (AA) hemoglobin subjects. P-selectin expression, a marker of platelet activation, at baseline and in reactivity to stimulation by adenosine diphosphate (ADP), were higher in HbE/ß-thal subjects than normal subjects. HbNO formation from the reactions of nitrite and deoxyHb inversely correlated with baseline platelet P-selectin expression, HbE levels, and tricuspid regurgitant velocity (TRV). Nitrite plus deoxygenated erythrocytes from HbE/ß-thal subjects had a lower ability to inhibit ADP-induced P-selectin expression on platelets than erythrocytes from normal subjects. We conclude that deoxyHb in erythrocytes from HbE/ß-thal subjects has a decreased ability to reduce nitrite to NO, which is correlated with increased platelet activity in these individuals.

Production of platelet-rich plasma gel from elderly patients under antithrombotic drugs: Perspectives in chronic wounds care

Platelet-Rich Plasma (PRP) is an autologous biological therapy obtained by centrifuging the patient’s own blood to concentrate platelets. The addition of autologous thrombin and calcium chloride to PRP allows the production of a semi-solid form called PRP gel. PRP gel is increasingly used in a variety of tissue defects and predominantly in the management of non-healing chronic wounds. The topical application of PRP gel seems promising due to the capability of platelets to store and secrete growth factors (GF), fibrin and cytokines, which are essentials for wound healing. Most patients who suffered from chronic wounds are elderly patients with co-morbidities and polypharmacy including antithrombotic drugs such as antiplatelet agents (AP) or anticoagulants (AC), which could hamper the feasibility of this autologous platelet-derived therapy. To date, no study has investigated PRP gel formation in patients with AP or AC. The aim of this study was to evaluate the influence of AP or AC drugs on the production of PRP gel formation from elderly patients. Different biological characteristics were determined to qualify the production of PRP gel from such patients (Interquartile range (IQR) = 75–92 years) compared to healthy volunteers (IQR = 23–37 years). No significant difference was observed in the volume, composition (quantity of platelets, leukocytes and red blood cells) and functionality of platelets from PRP except a higher ADP-induced P-selectin expression in healthy donors compared with elderly patients. Autologous thrombin characteristics were similar in the two groups. Gel time formation (IQR: 120–195 seconds for controls and 135–210 seconds for elderly patients) and final composition of PRP gel were not significantly modified. Concentrations of theoretical thrombin generated in the serum and in the gel were inversely correlated with the time of formation of PRP gel (r2 = 0.57, p = 0.012). Altogether these data indicate that PRP gel preparation is not impacted by the use of antithrombotic drugs. Such results support the feasibility of using this innovative autologous biotherapy in the management of elderly patients with non-healing chronic wounds.

Impact of immature platelets on platelet response to ticagrelor and prasugrel in patients with acute coronary syndrome.

The influence of reticulated platelets (RPs) on platelet inhibition by ticagrelor when compared with prasugrel is unknown. We aimed to determine the influence of RPs on adenosine diphosphate- (ADP-) induced platelet aggregation in patients with acute coronary syndrome who were randomly assigned to receive either ticagrelor or prasugrel for P2Y12 receptor inhibition. One hundred and twenty-four patients were prospectively enrolled. The immature platelet fraction (IPF) was measured by an automated haematoanalyzer and platelet aggregation was assessed by multiple electrode aggregometry. In a subgroup of patients (n = 28) ADP-induced P-selectin expression was measured in dependence of the time point of study drug intake in RPs that were discriminated from mature platelets by thiazole-orange (TO) staining. The primary endpoint was the correlation of platelet aggregation and IPF in ticagrelor- vs. prasugrel-treated patients. Platelet aggregation significantly correlated with IPF in patients who received prasugrel (r = 0.41, P < 0.001). In contrast, no correlation between IPF and platelet aggregation was observed in ticagrelor-treated patients (r = 0.08, P = 0.51, P for difference of correlation coefficients P = 0.05). Within the TO-positive RP fraction, P-selectin expression was significantly higher in prasugrel when compared with ticagrelor-treated individuals (prasugrel 18.6 ± 16.0% vs. ticagrelor 11.5 ± 6.0%, P = 0.047). A time-dependent P-selectin expression was observed in prasugrel but not in ticagrelor-treated patients (prasugrel early 11.9 ± 9.4% vs. late 26.3 ± 19.0%, P = 0.031, ticagrelor early 9.6 ± 4.9% vs. late 13.5 ± 6.6%, P = 0.127). Reticulated platelets show a greater impact on platelet reactivity in response to prasugrel when compared with ticagrelor.

Comparison of Aggregometry with Flow Cytometry for the Assessment of Agonists´-Induced Platelet Reactivity in Patients on Dual Antiplatelet Therapy

Data on the agreement between aggregometry and platelet activation by flow cytometry regarding the measurement of on-treatment platelet reactivity to arachidonic acid (AA) and adenosine diphosphate (ADP) are scarce. We therefore sought to compare three platelet aggregation tests with flow cytometry for the assessment of the response to antiplatelet therapy. Platelet aggregation in response to AA and ADP was determined by light transmission aggregometry (LTA), the VerifyNow assays, and multiple electrode aggregometry (MEA) in 316 patients receiving aspirin and clopidogrel therapy after angioplasty with stent implantation. AA- and ADP-induced P-selectin expression and activated glycoprotein (GP) IIb/IIIa were determined by flow cytometry. LTA, the VerifyNow P2Y12 assay and MEA in response to ADP correlated significantly (all p<0.001), and the best correlation was observed between LTA and the VerifyNow P2Y12 assay (r = 0.63). ADP-induced platelet reactivity by all aggregation tests correlated significantly with ADP-induced P-selectin expression and activated GPIIb/IIIa (all p<0.001). The best correlation was seen between the VerifyNow P2Y12 assay and activated GPIIb/IIIa (r = 0.68). The platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP were significantly higher in patients with high on-treatment residual platelet reactivity (HRPR) to ADP by all test systems (all p<0.001). A rather poor correlation was observed between AA-induced platelet reactivity by LTA and the VerifyNow aspirin assay (r = 0.15, p = 0.007), while both methods did not correlate with MEA. AA-induced platelet reactivity by all aggregation tests correlated significantly, but rather poorly with AA-induced P-selectin expression (all p<0.05), while only AA-induced platelet reactivity by LTA correlated significantly with AA-induced activated GPIIb/IIIa (r = 0.21, p<0.001). The platelet surface expression of P-selectin in response to AA was significantly higher in patients with HRPR by LTA AA and MEA AA (both p<0.02). In contrast, P-selectin expression in response to AA was similar in patients without and with HRPR by the VerifyNow aspirin assay (p = 0.5), and platelet surface activated GPIIb/IIIa in response to AA did not differ significantly between patients without and with HRPR to AA by all test systems (all p>0.1). In conclusion, ADP-induced platelet reactivity by aggregometry translates partly into flow cytometry. In contrast, AA-induced platelet reactivity correlates poorly between different platelet aggregation tests, and between aggregometry and flow cytometry. Overall, both approaches capture different aspects of platelet function and are therefore not interchangeable in the assessment of agonists´-induced platelet reactivity. Clinical outcome data are needed to determine which test systems and settings are associated with different in vivo consequences.

The novel P2Y 12 antagonist AZD6140 rapidly and reversibly reduces platelet activation in diabetic rats

Introduction Excessive platelet activation fundamentally contributes to cardiovascular events and mortality in patients with diabetes mellitus. Functional resistance has been described for current antiplatelet therapies in broad populations that include patients with diabetes. We investigated acute and chronic effects of AZD6140, a reversible oral rapid-onset P2Y 12 antagonist, on platelet reactivity in diabetic rats. Materials and Methods Diabetes was induced by streptozotocin injection in male Wistar rats. After 4 weeks, AZD6140 was administered (5 mg/kg by gavage) and achieved sufficient plasma levels within 30 minutes. Platelet reactivity was determined by ADP-induced P-selectin expression, aggregation and adhesion on fibrinogen coated membranes under arterial flow conditions. Results At 0.5 hour, AZD6140 strongly reduced ADP-induced P-selectin surface expression, inhibited ADP-induced platelet aggregation, and significantly reduced platelet adhesion to fibrinogen under arterial flow conditions. Chronic treatment with AZD6140 (10 mg/kg bid for 2 weeks, based on data obtained in the acute study) starting at day 14 reduced P-selectin surface expression on circulating platelets, indicating lower in vivo platelet activation. Platelet reactivity was improved 12 hours after the last dose, while basal platelet activity remained reduced. AZD6140 was rapidly absorbed in diabetic rats and inhibited platelet reactivity. Chronic treatment lowered in vivo platelet activation of circulating platelets. Conclusion AZD6140 inhibits platelet reactivity in diabetic rats rapidly and reversibly. Markers of tonic platelet activation, which were increased in diabetic rats, were lowered to levels comparable to non-diabetic rats following chronic treatment with AZD6140.

Lidocaine Priming Reduces ADP-induced P-selectin Expression and Platelet–Leukocyte Aggregation

Activation of platelets, which plays an important role in inflammation, has recently been reported to enhance platelet P-selectin expression and form platelet-leukocyte aggregation (PLA). Platelet P-selectin expression and PLA formation have been reported to be potential markers of inflammatory diseases such as sepsis, thrombosis, myocardial ischemic disorders and stroke. Lidocaine, one of the most commonly used anesthetics, is known to inhibit platelet function, but its effect on platelet P-selectin expression and PLA remains unclear. To determine the effect of lidocaine on platelet activation, and on platelet activation-related septic condition (lipopolysaccharide-induced), we treated platelets with lidocaine (0.03-3 mM) and then measured platelet P-selectin expression and PLA. Whole blood for in vitro study was obtained from healthy men aged 27 to 33 years who had not taken any medication for at least 15 days. All samples were analyzed by flow cytometry. We found that lidocaine produced a concentration-dependent inhibition of P-selectin expression and PLA. Moreover, in lipopolysaccharide-challenged samples, lidocaine at concentrations of 1-3 mM inhibited PLA. Our findings may help to elucidate the inhibitory role of lidocaine on platelet P-selectin expression and PLA and infer possible therapeutic targets in the treatment of inflammatory diseases. However, further investigations are needed to determine whether the observed attenuation of excessive inflammatory responses has clinical implications. These results suggest that lidocaine might have potential clinical application in the modulation of excessive platelet activation, inflammatory response and septic condition.

Effect of Cytochrome P450 Polymorphisms on Platelet Reactivity After Treatment With Clopidogrel in Acute Coronary Syndrome

Genetic polymorphisms of cytochrome P450 (CYP) isoforms may promote variability in platelet response to clopidogrel. This study was conducted to analyze, in 603 patients with non-ST elevation acute coronary syndromes, the effect of CYP3A4, CYP3A5, and CYP2C19 gene polymorphisms on clopidogrel response and post-treatment platelet reactivity assessed by adenosine diphosphate (ADP)-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression. The CYP2C19*2 polymorphism was significantly associated with ADP-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression in recessive (p <0.01, p <0.007, and p <0.06, respectively) and codominant (p <0.08, p <0.0001, and p <0.009, respectively) models, but the CYP3A4*1B and CYP3A5*3 polymorphisms were not. The CYP2C19*2 allele carriers exhibited the highest platelet index levels in multivariate analysis (p = 0.03). After covariate adjustment, the CYP2C19*2 allele was more frequent in clopidogrel nonresponders, defined by persistent high post-treatment platelet reactivity (ADP-induced platelet aggregation >70%; p = 0.03). In conclusion, the present data suggest that the CYPC19*2 allele influences post-treatment platelet reactivity and clopidogrel response in patients with non-ST elevation acute coronary syndromes.

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y1 receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y1 antagonist) and AR-C69931 (a P2Y12 antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y1 but also with partial antagonism at P2Y12 receptors. Antagonism at P2Y12 receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X1 receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

Effects of improved metabolic control on platelet reactivity in patients with type 2 diabetes mellitus following coronary angioplasty

The aim of this study was to investigate the impact of improved metabolic control on platelet reactivity in patients with type 2 diabetes undergoing percutaneous coronary intervention (PCI). Twenty-two patients were randomised to intensive insulin or conventional treatment for diabetes. Platelet P-selectin expression was analysed before and three months after PCI. Metabolic control, as measured by level of glycosylated haemoglobin (HbA1c) and platelet P-selectin expression, was similar in the two treatment groups after three months. However, six of the 12 patients in the intensive group had increased levels of HbA1c after three months and three patients of the 10 in the conventionally treated group showed improved metabolic control. A re-analysis was performed, based on metabolic control. It showed that patients with improved control at three months (HbA1c 6.1% +/- 0.7 at baseline; 5.7% +/- 0.5 at three months; p<0.01; n=9) had lower ADP-induced P-selectin expression (p<0.05) than patients with worsened glycaemic control (HbA1c 5.9% +/- 1.0 at baseline; 6.5% +/- 1.4 at three months; p<0.01; n=13). Levels of HbA1c and fasting glucose were correlated to P-selectin expression (R=0.34 and R=0.31; p<0.05). We conclude from this study that improved glycaemic control reduces platelet reactivity in type 2 diabetes patients following PCI.

Meal‐induced platelet activation in Type 2 diabetes mellitus: effects of treatment with repaglinide and glibenclamide

To compare the effects of treatment with repaglinide and glibenclamide on platelet function and endothelial markers in patients with Type 2 diabetes mellitus, before and after a standardized meal. Fifteen patients with Type 2 diabetes were investigated on three occasions: at baseline without oral hypoglycaemic drug treatment, and after 6 weeks' treatment with repaglinide or glibenclamide, respectively, in an open randomized cross-over study. Agonist-induced platelet P-selectin expression and platelet aggregation, urinary thromboxane, soluble P-selectin, von Willebrand factor (VWF), soluble E-selectin, intercellular adhesion molecule (ICAM-1) and C-reactive protein (CRP) were measured. In addition, pre-meal data were compared with non-diabetic control subjects (n = 15), matched for sex, age and BMI. Adenosine diphosphate (ADP)-induced platelet P-selectin expression increased post-meal in Type 2 diabetic patients both at baseline and after treatment with repaglinide and glibenclamide (P < 0.01 for all; repeated measures anova). Repaglinide treatment reduced fasting ADP-induced P-selectin expression compared with baseline (P = 0.01), but did not influence meal-induced platelet hyper-reactivity (P = 0.32). No significant anti-platelet effects of glibenclamide treatment were found. Plasma concentrations of VWF and ICAM-1 were elevated in patients with Type 2 diabetes compared with control subjects (P < 0.05 for both) and were reduced during treatment with repaglinide (P < 0.01 for both) but did not change during glibenclamide treatment. The post-meal state is associated with enhanced platelet reactivity in patients with Type 2 diabetes mellitus. Pre-meal treatment with repaglinide or glibenclamide does not inhibit postprandial platelet activation, but repaglinide treatment is associated with attenuated platelet and endothelial activity in the fasting state.

Different G protein‐coupled signaling pathways are involved in α granule release from human platelets

Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause alpha granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Galpha(q) and Galpha(i) pathways in ADP-mediated alpha granule release. Consistent with these observations, the combination of serotonin (a Galpha(q) pathway stimulator) and epinephrine (a Galpha(z) pathway stimulator) also caused alpha granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated alpha granule release. Additionally, the Galpha(12/13)-stimulating peptide YFLLRNP potentiated alpha granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Galpha(i) and Galpha(12/13) pathways resulted in a significant dose-dependent increase in alpha granule release. We conclude that ADP-induced alpha granule release in aspirin-treated platelets occurs through costimulation of Galpha(q) and Galpha(i) signaling pathways. The P2Y12 receptor plays an important role in thromboxane A(2)-mediated alpha granule release, and furthermore activation of Galpha(12/13) and Galpha(q) signaling pathway can cause alpha granule release.

Variability in inhibition of platelet aggregation with clopidogrel and correlation with ADP-induced P-selectin expression on platelets

Variability in inhibition of platelet aggregation with clopidogrel and correlation with ADP-induced P-selectin expression on platelets

Inhibition of ADP-induced P-selectin Expression and Platelet-Leukocyte Conjugate Formation by Clopidogrel and the P2Y12 Receptor Antagonist AR-C69931MX but not Aspirin

Platelet-leukocyte interactions are recognised to have pro-inflammatory effects, which may be important in the pathophysiology of ischaemic heart disease. Clopidogrel and the novel intravenous antithrombotic agent AR-C69931MX act at the level of the platelet P2Y12 receptor, which is known to amplify platelet activation, aggregation and other responses induced by numerous platelet agonists. We studied the effects of clopidogrel and aspirin on ADP-induced platelet-leukocyte conjugate formation and P-selectin expression in healthy volunteers. The effects of clopidogrel and AR-C69931MX administered to patients with ischaemic heart disease were also assessed. AR-C69931MX and aspirin were also studied in vitro. Clopidogrel and AR-C69931MX suppressed ADP-induced platelet aggregation, P-selectin expression and platelet-leukocyte conjugate formation whereas aspirin had no inhibitory effect. These effects of clopidogrel and AR-C69931MX may confer therapeutic benefits in the management of acute coronary syndromes.

Paradoxical inhibition of fibrinogen binding and potentiation of alpha-granule release by specific types of inhibitors of glycoprotein IIb-IIIa.

To determine whether glycoprotein (GP) IIb-IIIa inhibitors can paradoxically augment activation of platelets, activation of GP IIb-IIIa, alpha-granule degranulation, and lysosome release were induced after exposure of platelets to GP IIb-IIIa inhibitors. ADP-induced platelet activation was assessed after exposure of platelets to Abciximab, or to a non-peptide ligand, the free acid of Orbofiban (Orbofiban(a)). Activation of GP IIb-IIIa was detected based on binding of fluorochrome labeled fibrinogen or a labeled monoclonal antibody, PAC-1. alpha-Granule degranulation was detected based on surface expression of P-selectin and lysosome release was detected based on surface expression of CD63. Despite significant inter-individual variability in inhibition of fibrinogen binding in response to each of the GP IIb-IIIa inhibitors used, a concentration dependent decrease in fibrinogen binding was seen with each agent in samples from each subject. Binding of PAC-1 was inhibited in a parallel manner. Abciximab increased ADP-induced P-selectin expression. Orbofiban(a) did not alter ADP-induced P-selectin expression. Neither agent altered ADP-induced CD63 expression. When platelets were exposed to Abciximab and Orbofiban(a), both Abciximab and Orbofiban(a) were found in the alpha-granules (by confocal microscopy), consistent with potentiation of agonist-induced release of alpha-granular products associated with uptake of proteins. Specific types of GP IIb-IIIa inhibitors can paradoxically augment agonist-induced release of alpha-granules despite inhibiting agonist-induced fibrinogen binding.

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 μM adenosine diphospate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 μM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.

PLATELET FUNCTION AND PLATELET-LEUKOCYTE ADHESION IN SYMPTOMATIC CORONARY HEART DISEASE. EFFECTS OF INTRAVENOUS MAGNESIUM

Spontaneous P-selectin surface expression on platelets and plateletleukocyte adhesion was increased in patients with symptomatic coronary heart disease (n=12) compared to normal controls (n=10) (p<0.05). ADP-induced P-selectin expression and platelet-leukocyte adhesion was also enhanced in the patient group (p<0.05). Administration of intravenous Mg 2+ significantly reduced both platelet surface expression of P-selectin and platelet-leukocyte adhesion ex vivo (p<0.02). The effect of extracellular Mg 2+ was evaluated in in vitro experiments. Both in whole blood and in isolated neutrophil suspension Mg 2+ inhibited platelet adhesion to neutrophils dose dependently with half maximal effects at 4 mM. Moreover, Mg 2+ inhibited adhesion of isolated platelet membranes to neutrophils. We conclude that platelet function is altered in symptomatic coronary heart disease and can be modulated by administration of intravenous Mg 2+.

ADP-induced P-selectin expression on platelets as a predictor of successful thrombolysis.

Unlike coronary thrombolysis, the role of platelet activity in the outcome of local thrombolytic therapy for peripheral ischaemia is not well understood. In the present study ten patients undergoing local pulse spray thrombolysis (PST) with recombinant tissue-type plasminogen activator (rt-PA), six patients undergoing conventional infusion thrombolysis (CT) with rt-PA and another six patients undergoing arteriography with iopamidol were studied. Venous blood samples obtained before and after the procedure were analysed using a flow cytometric technique for detection of platelet activation after labelling platelets with VH10, a monoclonal antibody against P-selectin. In the present study no significant differences were observed in P-selectin expression before and after any of the procedures, except that P-selectin expression following ADP stimulation was reduced in patients who had received conventional thrombolysis. Unexpectedly, we observed relatively greater P-selectin expression, particularly after ADP stimulation, both before and following thrombolysis in ten patients in whom thrombolysis was successful compared with six patients in whom thrombolysis was unsuccessful. ADP-induced P-selectin expression on platelets may therefore be a useful predictor of outcome of peripheral intra-arterial thrombolysis.

Aspirin does not inhibit adenosine diphosphate-induced platelet alpha- granule release

The involvement of metabolites of arachidonic acid in platelet-dense granule secretion and secondary platelet-platelet interactions is well characterized. However, their role in heterotypic interactions dependent on alpha-granule secretion is less well understood. Using platelet-surface expression of P-selectin as a marker of alpha-granule secretion, we have shown that: (1) aspirin treatment of platelets at doses that block dense granule secretion does not inhibit alpha-granule secretion to adenosine diphosphate (ADP); (2) synergism between epinephrine and ADP in the induction of P-selectin expression is similarly unaffected by aspirin; and (3) the ability of P-selectin to mediate adhesion of activated platelets to monocytes and polymorphonuclear lymphocytes in whole blood is also unchanged by aspirin treatment. To further explore the mechanisms responsible for platelet alpha-granule secretion, we have shown that inhibition of Na+/H+ exchange by either acidification of the extracellular medium or amiloride treatment blocked ADP-induced P-selectin expression. In contrast, incubation with the platelet lipoxygenase inhibitor 5,8,11-eicosatrynoic acid, by itself and with aspirin, did not decrease ADP-induced P-selectin expression. We conclude that platelet alpha-granule secretion in response to ADP is dependent on intact Na+/H+ exchange but is independent of the lipoxygenase- and cyclooxygenase-dependent metabolites of arachidonic acid.