Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.     

Which platelet function test is suitable to monitor clopidogrel responsiveness? A pharmacokinetic analysis on the active metabolite of clopidogrel

Summary. Background: Multiple platelet function tests claim to be P2Y12‐pathway specific and capable of capturing the biological activity of clopidogrel. Objectives: The aim of the present study was to determine which platelet function test provides the best reflection of the in vivo plasma levels of the active metabolite of clopidogrel (AMC). Patients/methods: Clopidogrel‐naive patients scheduled for elective percutaneous coronary intervention (PCI) received a 600 mg loading dose of clopidogrel and 100 mg of aspirin. For pharmacokinetic analysis, blood was drawn at 0, 20, 40, 60, 90, 120, 180, 240 and 360 min after clopidogrel loading and peak plasma concentrations (C_max) of the AMC were quantified with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Platelet function testing was performed at baseline and 360 min after the clopidogrel loading. Results: The VASP‐assay, the VerifyNow P2Y12‐assay and 20 μmol L^?1 adenosine diphosphate (ADP)‐induced light transmittance aggregometry (LTA) showed strong correlations with C_max of the AMC (VASP: R² = 0.56, P < 0.001; VerifyNow platelet reactivity units (PRU): R² = 0.48, P < 0.001; VerifyNow %inhibition: R² = 0.59, P < 0.001; 20 μmol L^?1 ADP‐induced LTA: R² = 0.47, P < 0.001). Agreement with C_max of the AMC was less evident for 5 μmol L^?1 ADP‐induced LTA or whole blood aggregometry (WBA), whereas the IMPACT‐R ADP test did not show any correlation with plasmalevels of the AMC. Conclusion: The flow cytometric VASP‐assay, the VerifyNow P2Y12 assay and, although to a lesser extent, 20 μmol L^?1 ADP‐induced LTA correlate best with the maximal plasma level of the AMC, suggesting these may be the preferred platelet function tests for monitoring the responsiveness to clopidogrel.     

Randomized double‐blind placebo‐controlled crossover study to determine the effects of esomeprazole on inhibition of platelet function by clopidogrel

Summary. Background: Pharmacokinetic studies suggest that clopidogrel and esomeprazole are metabolized by similar hepatic enzymes; however, previous studies have not identified a biochemical interaction. Objectives: To determine whether addition of esomeprazole to patients receiving aspirin and clopidogrel reduces the antiplatelet effects of clopidogrel. Patient/Methods: Patients with a history of an acute coronary syndrome who had previously received clopidogrel were recruited. Subjects were commenced on clopidogrel and randomized to one of two treatment arms (esomeprazole or placebo) for 6 weeks. Following a 2‐week washout period for study medications, patients were crossed over onto the alternative treatment arm for a further 6 weeks. Platelet function tests were undertaken at baseline, following the first treatment period, after washout and following the second treatment period. Results: Thirty‐one patients were enrolled. Significant attenuation of clopidogrel’s antiplatelet effects was seen with co‐administration of esomeprazole compared with placebo. Vasodilator stimulated phosphoprotein (VASP), platelet aggregometry (area under the curve (AUC)) and VerifyNow results were 54.7% ± 2.8 platelet reactivity index (PRI), 66.3 ± 2.6 AUC units and 213.1 ± 14.1 platelet reactivity units (PRU) with esomeprazole vs. 47% ± 2.7 PRI, 59.7 ± 3.7 AUC units and 181.4 ± 14.6 PRU with placebo (P < 0.01 esomeprazole vs. placebo for all measures). There was no significant difference in platelet aggregometry (maximal aggregation) between the esomeprazole group (68.9% ± 2.7 units) and placebo‐treated group (64.5% ± 4.1 units; P > 0.05). Conclusion: Esomeprazole when co‐administered with aspirin and clopidogrel results in a significant attenuation of clopidogrel’s antiplatelet effects.     

In the presence of strong P2Y12 receptor blockade,aspirin provides little additional inhibition of platelet aggregation

Summary. Background: Aspirin and antagonists of platelet ADP P2Y₁₂ receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y₁₂ receptors can inhibit thromboxane A₂ (TXA₂)‐dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y₁₂ receptor blockade. Methods: With the use of platelet‐rich plasma from healthy volunteers, determinations were made in 96‐well plates of platelet aggregation, TXA₂ production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP‐6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L^?1), aspirin (30 μmol L^?1), PAM + aspirin or vehicle. Results: PAM concentration‐dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L^?1. In further tests of PAM (3 μmol L^?1), aspirin (30 μmol L^?1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA₂ and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y₁₂ receptors are critical to the generation of irreversible aggregation through the TXA₂‐dependent pathway. As a result, strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.     

Earlier recovery of platelet function after discontinuation of treatment with ticagrelor compared with clopidogrel in patients with high antiplatelet responses

Summary. Background: The rate of recovery of platelet function after discontinuation of P2Y₁₂ inhibitors depends on the reversibility of the antiplatelet effect and the extent of the on‐treatment response. P2Y₁₂ inhibition increases the bleeding risk in patients requiring surgery. Objectives: To evaluate recovery of platelet function after discontinuation of ticagrelor vs. clopidogrel in stable coronary artery disease (CAD) patients with high levels of platelet inhibition (HPI) during the ONSET/OFFSET study. Methods: Patients received aspirin 75–100 mg per day and either ticagrelor 90 mg twice‐daily or clopidogrel 75 mg daily for 6 weeks. This subanalysis included patients with HPI after the last dose of maintenance therapy, defined as: inhibition of platelet aggregation (IPA) > 75% 4 h post‐dose (ADP 20 μm , final extent); < 120 P2Y₁₂ reaction units 8 h post‐dose (VerifyNow P2Y₁₂ assay); or platelet reactivity index < 50% 8 h post‐dose (VASP‐P assay). Results: IPA > 75% was observed in 39 out of 47 ticagrelor‐treated and 17 out of 44 clopidogrel‐treated patients. The rate of offset of IPA over 4–72 h was greater with ticagrelor (IPA %/hour slope: ?1.11 vs. ?0.67 for clopidogrel; P < 0.0001). Mean IPA was significantly lower with ticagrelor than clopidogrel between 48 and 168 h post‐dose (P < 0.01). Similar findings were observed with the other assays. The average time for IPA to decline from 30% to 10% was 50.8 h with ticagrelor vs. 110.4 h with clopidogrel. Conclusions: In patients with HPI, recovery of platelet function was more rapid after discontinuation of ticagrelor than clopidogrel leading to significantly greater platelet reactivity by 48 h after the last dose in the ticagrelor group.     

Cocaine administration enhances platelet reactivity to subendothelial components: studies in a pig model

Myocardial infarction in cocaine abusers may be related to a direct platelet-activating effect. We analysed this possibility in an experimental model. Studies were carried out in eight normal, anaesthetized pigs with a weight of 30.7 ± 3.7 kg. Blood samples were withdrawn before and 20 min after i.v. administration of cocaine (10 mg kg^?1; at 1 mg kg^?1 every 2 min). Modifications in platelet responses to arachidonic acid (AA; 1.4 mmol L^?1), ADP (1–4 μM), synthetic thromboxane endoperoxide analogue (U46619; 1 μM), collagen (2.5–5 μg mL^?1), adrenaline (10 μM) and ristocetin (0.8–1 mg mL^?1) were tested by conventional aggregometry. Changes in the capacity of platelets to form aggregates on damaged subendothelium were assessed by means of an ex vivo perfusion system in which blood was circulated for 10 min at 800 s^?1, a shear rate similar to that found in normal coronary arteries. The interaction of platelets with perfused denuded arterial segments was morphometrically quantified and expressed as a percentage of damaged vessel surface covered by platelets (%CS). Cocaine administration did not influence platelet aggregation patterns in pigs. However, there was a significant increase in the interaction of pig platelets with subendothelial structures after cocaine infusion (%CS = 40 ± 17% vs. 27 ± 16% baseline; mean ± SD; P < 0.01). Cocaine administration in this animal model increases the reactivity of platelets exposed to subendothelium. These results support the concept that the administration of cocaine to pigs has a prothrombotic effect by facilitating the interaction of platelets with damaged arteries.     

Moderate hyperthyroidism reduces liver amino nitrogen conversion,muscle nitrogen contents and overall nitrogen balance in rats

There are conflicting data on the effect of thyroid hormones on nitrogen metabolism. We determined the basal blood amino nitrogen (amino-N) concentrations, the urea nitrogen (urea-N) synthesis rate and the maximum hepatic capacity of urea nitrogen synthesis during saturating infusion of alanine, in moderately acutely (24 h) and chronically (7 days) hyperthyroid rats and compared this with changes in organ nitrogen contents in muscles and kidney, nitrogen excretion and nitrogen balance. Forty-three rats were made acutely hyperthyroid through administration of 5 μg 100 g^?1 triiodothyronine twice daily (T₃: 2.2 ± 0.7 vs. 0.87 ± 0.04 nmol L^?1, P < 0.01). Fifty-one rats were made chronically hyperthyroid through administration of 12.5 μg 100 g^?1 thyroxine twice daily (T₃: 2.63 ± 0.18 vs. 0.87 ± 0.04 nmol L^?1, P < 0.01). Weight gain was halved in this group. Both acute and chronic hyperthyroidism increased basal blood amino-N concentration in both groups by 16% (4.5 ± 0.15 vs. 3.9 ± 0.13 mmol L^?1 and 4.7 ± 0.12 vs. 3.9 ± 0.13 mmol L^?1, respectively, P < 0.01), and decreased basal urea-N synthesis rate in both groups by 30% [2.7 ± 03 vs. 4.1 ± 0.3 μmol (min ×100 g)^?1 and 3.1 ± 0.3 vs. 4.1 ± 0.3μmol (min ×100 g g)^?1, respectively, P < 0.01]. The capacity of urea-N synthesis during saturation fell in both groups by 35% compared with controls [6.5 ± 0.4 vs. 9.3 ± 0.5 μmol (min ×100 g)^?1 and 5.7 ± 0 .5 vs. 9.3 ± 0.6 μmol (min ×100 g)^?1, respectively, P < 0.01]. Nitrogen contents in the muscles, soleus and extensor digitorum longus, of chronically hyperthyroid rats decreased by 22% and 11%, respectively, whereas kidney N-content increased by 12% (P < 0.05). N-balance and urinary urea-N excretion fell by 30%, whereas faeces-N excretion increased by 80% in hyperthyroid rats. Overall liver function assessed by galactose elimination capacity did not differ among groups. Both acute and chronic moderate hyperthyroidism increase blood amino-N and decrease basal and maximum rate of urea formation. Furthermore, chronic hyperthyroidism reduces N-contents of muscles, urinary urea-N excretion and N-balance. Thyroid hormones thus mobilize muscle-N, whereas amino-N in the liver is spared from irretrievable conversion into urea.     

Forearm metabolism during infusion of adrenaline: comparison of the dominant and non‐dominant arm

Human skeletal muscle metabolism is often investigated by measurements of substrate fluxes across the forearm. To evaluate whether the two forearms give the same metabolic information, nine healthy subjects were studied in the fasted state and during infusion of adrenaline. Both arms were catheterized in a cubital vein in the retrograde direction. A femoral artery was catheterized for blood sampling, and a femoral vein for infusion of adrenaline. Forearm blood flow was measured by venous occlusion strain‐gauge plethysmography. Forearm subcutaneous adipose tissue blood flow was measured by the local ¹³³Xe washout method. Metabolic fluxes were calculated as the product of forearm blood flow and a‐v differences of metabolite concentrations. After baseline measurements, adrenaline was infused at a rate of 0·3 nmol kg^?1 min^?1. No difference in the metabolic information obtained in the fasting state could be demonstrated. During infusion of adrenaline, blood flow and lactate output increased significantly more in the non‐dominant arm (8·12 ± 1·24 versus 6·45 ± 1·19 ml 100 g^?1 min^?1) and (2·99 ± 0·60 versus 1·83 ± 0·43 μmol 100 g^?1 min^?1). Adrenaline induced a significant increase in oxygen uptake in the non‐dominant forearm (baseline period: 4·98 ± 0·72 μmol 100 g^?1 min^?1; adrenaline period: 6·63 ± 0·62 μmol 100 g^?1 min^?1) while there was no increase in the dominant forearm (baseline period: 5·69 ± 1·03 μmol 100 g^?1 min^?1; adrenaline period: 4·94 ± 0·84 μmol 100 g^?1 min^?1). It is concluded that the two forearms do not respond equally to adrenaline stimulation. Thus, when comparing results from different studies, it is necessary to know which arm was examined.     

The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet‐mediated fibrinolysis resistance: a thromboelastographic study in whole blood

Summary. Background: The resistance of platelet‐rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI‐1 release. The role of TAFI in platelet‐mediated resistance to lysis is unclear. Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography. Methods: Platelet‐poor (PP‐WB, < 40 × 10³ μL^?1) and platelet‐rich (PR‐WB, > 400 × 10³ μL^?1) blood samples were obtained from normal human blood (N‐WB, 150–220 × 10³ μL^?1). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t‐PA (100 ng mL^?1) and tissue factor (1:1000 Recombiplastin). Results: t‐PA‐induced lysis time increased in parallel with platelet concentration (up to 3‐fold). Neutralization of TAFI, but not of PAI‐1, shortened the lysis time by ～ 50% in PR‐WB and by < 10% in PP‐WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR‐WB than in PP‐WB. A similar TAFI‐dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet‐mediated fibrinolysis resistance was attenuated by an anti‐FXI but not by an anti F‐XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity. Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents.     

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary

Summary. Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL⁻¹) and adenosine diphosphate (ADP 2 and 5 μm ) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL⁻¹ ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL⁻¹. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL⁻¹ in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.     

Reversal of the anti‐platelet effects of aspirin and clopidogrel

Summary. Background: Guidelines recommend stopping aspirin and clopidogrel 7 to 10 days before surgery to allow time for replacement of permanently inhibited platelets by newly released uninhibited platelets.Objectives: The purpose of the present study was to determine the rate of offset of the anti‐platelet effects of aspirin and clopidogrel after stopping treatment and the proportion of untreated donor platelets that are required to reverse their anti‐platelet effects.Methods: Cohort 1 consisted of 15 healthy subjects who received aspirin 81 mg day^?1 or clopidogrel 75 mg day^?1 for 7 days and underwent serial blood sampling until platelet function testing results normalized. Cohort 2 consisted of 36 healthy subjects who received aspirin 325 mg day^?1, clopidogrel 75 mg day^?1, aspirin 81 mg day^?1 plus clopidogrel 75 mg day^?1 or no treatment for 7 days and underwent a single blood sampling.Results: In cohort 1, arachidonic acid (AA)‐induced light transmission aggregation (LTA) returned to baseline levels in all subjects within 4 days of stopping aspirin, coinciding with the partial recovery of plasma thromboxane B₂ concentrations. ADP‐induced LTA did not return to baseline levels until 10 days after stopping clopidogrel. In cohort 2, AA‐induced LTA in patient treated with aspirin reached control levels after mixing with 30% untreated donor platelets whereas ADP‐induced LTA in patients treated with clopidogrel reached control levels only after the addition of 90% or more donor platelets.Conclusions: Platelet aggregation recovers within 4 days of stopping aspirin but clopidogrel must be stopped for 10 days to achieve a normal aggregatory response.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease

Summary. Background: Previous studies have demonstrated considerable variation in the antiplatelet effect of aspirin. Objectives: To investigate the impact of platelet turnover on the antiplatelet effect of aspirin in patients with stable coronary artery disease (CAD) and to identify determinants of platelet turnover. Methods: Platelet turnover was evaluated by measurements of immature platelets and thrombopoietin in 177 stable CAD patients on aspirin monotherapy, including 85 type 2 diabetics and 92 non‐diabetics. Whole blood platelet aggregation was determined using the VerifyNow^® Aspirin test and multiple electrode aggregometry (MEA, Multiplate^®) induced by arachidonic acid (AA) (1.0 mm ), adenosine diphosphate (ADP) (10 μm ) and collagen (1.0 μg mL^?1). Results: Immature platelet levels significantly correlated with MEA (r = 0.31–0.36, P‐values < 0.0001) and the platelet activation marker sP‐selectin (r = 0.19, P = 0.014). Contrary to the VerifyNow^® test, MEA significantly correlated with variations in platelet count (r = 0.45–0.68, P‐values < 0.0001). Among patients with residual platelet reactivity according to AA, there were significantly more diabetics (61% vs. 41%, P = 0.027) and higher levels of sP‐selectin (77.7 ± 29 vs. 70.2 ± 25 ng mL^?1, P = 0.070) and serum thromboxane B₂ (0.81 [0.46; 1.70] vs. 0.56 [0.31; 1.12] ng mL^?1, P = 0.034). In a multivariate regression analysis, immature platelet levels were determined by thrombopoietin levels (P < 0.001), smoking (P = 0.020) and type 2 diabetes (P = 0.042). Conclusions: The antiplatelet effect of aspirin was reduced in CAD patients with an increased platelet turnover. Once‐daily dosing of aspirin might not suffice to adequately inhibit platelet aggregation in patients with an increased platelet turnover.     

Greater inhibition of platelet aggregation and reduced response variability with prasugrel versus clopidogrel: an integrated analysis

Multiple studies report response variability to a 300-mg clopidogrel loading dose (LD). Pooled platelet aggregometry data compared responses (change in maximal platelet aggregation [DeltaMPA] or inhibition of platelet aggregation [IPA]) to clopidogrel 300-mg (n = 131) or prasugrel 60-mg (n = 109) LDs. Poor responder rates were determined using empiric criteria (IPA < 10% and DeltaMPA < 10% for 20 microM and 5 microM adenosine diphosphate [ADP]) and Bayesian model-based criteria (IPA < 20% and DeltaMPA < 15% for 20 microM ADP; IPA < 25% and DeltaMPA < 20% for 5 microM ADP). Prasugrel achieved greater DeltaMPA and IPA from 2 to 24 hours post-LD (P < .001). For 20 microM ADP, poor responder rates for clopidogrel ranged from 17% to 43%; no prasugrel poor responders were observed. Regardless of the criterion, prasugrel 60 mg achieved greater IPA and fewer poor responders than the clopidogrel 300-mg LD.     

Fibrinogen γ‐chain peptide‐coated,ADP‐encapsulated liposomes rescue thrombocytopenic rabbits from non‐compressible liver hemorrhage

Summary. Background: We developed a fibrinogen γ‐chain (dodecapeptide HHLGGAKQAGDV [H12])‐coated, ADP‐encapsulated liposome (H12‐[ADP]‐liposome) that accumulates at bleeding sites via interaction with activated platelets via glycoprotein IIb–IIIa and augments platelet aggregation by releasing ADP. Objective: To evaluate the efficacy of H12‐(ADP)‐liposomes for treating liver hemorrhage in rabbits with acute thrombocytopenia. Methods: Thrombocytopenia (platelets < 50 000 μL^?1) was induced in rabbits by repeated blood withdrawal (100 mL kg^?1 in total) and isovolemic transfusion of autologous washed red blood cells. H12‐(ADP)‐liposomes with platelet‐poor plasma (PPP), platelet‐rich plasma (PRP), PPP, ADP liposomes with PPP or H12‐(PBS)‐liposomes/PPP, were administered to the thrombocytopenic rabbits, and liver hemorrhage was induced by penetrating liver injury. Results: Administration of H12‐(ADP)‐liposomes and of PRP rescued all thrombocytopenic rabbits from liver hemorrhage as a result of potent hemostasis at the liver bleeding site, although rabbits receiving PPP or ADP liposomes showed 20% survival in the first 24 h. Administration of H12‐(ADP)‐liposomes and of PRP suppressed both bleeding volume and time from the site of liver injury. H12‐(phosphate‐buffered saline)‐liposomes lacking ADP also improved rabbit survival after liver hemorrhage, although their hemostatic effect was weaker. In rabbits with severe thrombocytopenia (25 000 platelets μL^?1), the hemostatic effects of H12‐(ADP)‐liposomes tended to be attenuated as compared with those of PRP treatment. Histologic examination revealed that H12‐(ADP)‐liposomes accumulated at the bleeding site in the liver. Notably, neither macrothombi nor microthrombi were detected in the lung, kidney or liver in rabbits treated with H12‐(ADP)‐liposomes. Conclusions: H12‐(ADP)‐liposomes appear to be a safe and effective therapeutic tool for acute thrombocytopenic trauma patients with massive bleeding.     

Bleeding tendency in dual antiplatelet therapy with aspirin/clopidogrel: rescue of the template bleeding time in a single-center prospective study

Background

Patients with heightened platelet reactivity in response to antiplatelet agents are at an increased risk of recurrent ischemic events. However, there is a lack of diagnostic criteria for increased response to combined aspirin/clopidogrel therapy. The challenge is to identify patients at risk of bleeding. This study sought to characterize bleeding tendency in patients treated with aspirin and clopidogrel.

Patients/methods

In a single-center prospective study, 100 patients under long-term aspirin/clopidogrel treatment, the effect of therapy was assayed by template bleeding time (BT) and the inhibition of platelet aggregation (IPA) by light transmission aggregometry (LTA). Arachidonic acid (0.625 mmol/L) and adenosine diphosphate (ADP; 2, 4, and 8 ??mol/L) were used as platelet agonists.

Results

Bleeding episodes (28 nuisance, 2 hematuria [1 severe], 1 severe proctorrhagia, 1 severe epistaxis) were significantly more frequent in patients with longer BT. Template BT ?? 24 min was associated with bleeding episodes (28 of 32). Risk of bleeding increased 17.4% for each 1 min increase in BT. Correlation was found between BT and IPAmax in response to ADP 2 ??mol/L but not to ADP 4 or 8 ??mol/L.

Conclusion

In patients treated with dual aspirin/clopidogrel therapy, nuisance and internal bleeding were significantly associated with template BT and with IPAmax in response to ADP 2 ??mol/L but not in response to ADP 4 ??mol/L or 8 ??mol/L.     

Postprandial chylomicron and plasma vitamin E responses in healthy older subjects compared with younger ones

The effect of ageing on vitamin E bioavailability in humans was assessed by comparing chylomicron and plasma α-tocopherol postprandial concentrations after a dose of vitamin E (432 or 937 IU as dl-α-tocopherol acetate), in eight young (20–30 years old) and eight healthy elderly men (64–72 years old). The fasting plasma α-tocopherol concentration was significantly higher in the elderly (33 ± 2 μmol L^?1) than in the young (22 ± 2 μmol L^?1). In both groups, the plasma and chylomicron α-tocopherol postprandial concentrations were significantly, approximately twofold, higher after the 937-IU meal than after the 432-IU meal. For both test meals, the chylomicron α-tocopherol areas under the curve were significantly lower in the elderly than in the young subjects: 98.9 ± 16.5 (young group) vs. 55.3 ± 7.8 (elderly group) μmol L^?1 h for the 937-IU test meal and 60.4 ± 14.1 (young group) vs. 26.0 ± 7.6 (elderly group) μmol L^?1 h for the 432-IU test meal, whereas the plasma α-tocopherol area under the curve was significantly higher in elderly than in young subjects: 337.56 ± 16.11 (937-IU test meal) vs. 159.81 ± 35.55 (432-IU test meal) μmol L^?1 h in the young group and 709.55 ± 69.33 (937-IU test meal) vs. 436.39 ± 41.08 (432-IU test meal) μmol L^?1 h in the elderly group. We concluded that (a) the amount of vitamin E appearing in plasma is proportional to the dose ingested (up to 937 IU); (b) the intestinal absorption of vitamin E is not increased, even possibly decreased, in the elderly; and (c) the amount of vitamin E transported by non-chylomicron lipoproteins is apparently higher in the elderly. This suggests that vitamin E postprandial transport is affected by ageing, mainly as the consequence of age-related modifications of lipoprotein metabolism.     

Platelet function normalization after a prasugrel loading‐dose: time‐dependent effect of platelet supplementation

Summary. Background: Hemostatic benefits of platelet transfusions in thienopyridine‐treated acute coronary syndrome (ACS) patients may be compromised by residual metabolite in circulation.Objectives: To estimate the earliest time after a prasugrel loading‐dose when added platelets are no longer inhibited by prasugrel's active metabolite.Methods: Baseline platelet reactivity of healthy subjects (n = 25, 30 ± 5 years, 68% male) on ASA 325 mg was tested using maximum platelet aggregation (MPA, ADP 20 μm ) and VerifyNow^® P2Y12 and was followed by a 60 mg prasugrel loading‐dose. At 2, 6, 12 and 24 h post‐dose, fresh concentrated platelets from untreated donors were added ex‐vivo to subjects’ blood, raising platelet counts by 0% (control), 40%, 60% and 80%. To estimate the earliest time when prasugrel's active metabolite's inhibitory effect on the added platelets ceases, platelet function in supplemented samples was compared across time‐points to identify the time when effect of supplementation on platelet function stabilized (i.e. the increase in platelet reactivity was statistically similar to that at the next time‐point).Results: Supplemented samples showed concentration‐dependent increases in platelet reactivity vs. respective controls by both MPA and VerifyNow^® at all assessment time‐points. For each supplementation level, platelet reactivity showed a sharp increase from 2 to 6 h but was stable (P = NS) between 6 and 12 h.Conclusions: The earliest measured time when supplemented platelets were not inhibited by circulating active metabolite of prasugrel was 6 h after a prasugrel loading‐dose. These findings may have important implications for prasugrel‐treated ACS patients requiring platelet transfusions during surgery.     

Effect of glibenclamide on insulin release at moderate and high blood glucose levels in normal man

Insulin release occurs in two phases; sulphonylurea derivatives may have different potencies in stimulating first- and second-phase insulin release. We studied the effect of glibenclamide on insulin secretion at submaximally and maximally stimulating blood glucose levels with a primed hyperglycaemic glucose clamp. Twelve healthy male subjects, age (mean ± SEM) 22.5 ± 0.5 years, body mass index (BMI) 21.7 ± 0.6 kg m^?2, were studied in a randomized, double-blind study design. Glibenclamide 10 mg or placebo was taken before a 4-h hyperglycaemic clamp (blood glucose 8 mmol L^?1 during the first 2 h and 32 mmol L^?1 during the next 2 h). During hyperglycaemic clamp at 8 mmol L^?1, the areas under the Δinsulin curve (AUC_Δinsulin , mean ± SEM) from 0 to 10 min (first phase) were not different: 1007 ± 235 vs. 1059 ± 261 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.81). However, glibenclamide led to a significantly larger increase in AUC_Δinsulin from 30 to 120 min (second phase): 16 087 ± 4489 vs. 7107 ± 1533 pmol L^?1 × 90 min (with and without glibenclamide respectively, P < 0.03). The same was true for AUC_ΔC-peptide: no difference from 0 to 10 min but a significantly higher AUC_ΔC-peptide from 30 to 120 min on the glibenclamide day (P < 0.01). The M/I ratio (mean glucose infusion rate divided by mean plasma insulin concentration) from 60 to 120 min, a measure of insulin sensitivity, did not change: 0.26 ± 0.05 vs. 0.22 ± 0.03 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.64). During hyperglycaemic clamp at 32 mmol L^?1, the AUC_Δinsulin from 120 to 130 min (first phase) was not different on both study days: 2411 ± 640 vs. 3193 ± 866 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.29). AUC_Δinsulin from 150 to 240 min (second phase) also showed no difference: 59 623 ± 8735 vs. 77389 ± 15161 pmol L^?1 × 90 min (with and without glibenclamide, P = 0.24). AUC_ΔC-peptide from 120 to 130 min and from 150 to 240 min were slightly lower on the glibenclamide study day (both P < 0.04). The M/I ratio from 180 to 240 min did not change: 0.24 ± 0.04 vs. 0.30 ± 0.07 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.25). In conclusion, glibenclamide increases second-phase insulin secretion only at a submaximally stimulating blood glucose level without enhancement of first-phase insulin release and has no additive effect on insulin secretion at maximally stimulating blood glucose levels. Glibenclamide did not change insulin sensitivity in this acute experiment.     

Aspirin and the in vitro linear relationship between thromboxane A2‐mediated platelet aggregation and platelet production of thromboxane A2

Summary. Background: Currently, ‘aspirin resistance’, the anti‐platelet effects of non‐steroid anti‐inflammatory drugs (NSAIDs) and NSAID‐aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A₂ formation is non‐linear and TXA₂ generation must be inhibited by at least 95% to inhibit TXA₂‐dependent aggregation. This relationship, however, has never been rigorously tested. Objectives: To characterize, in vitro and ex vivo, the concentration‐dependent relationships between TXA₂ generation and platelet activity. Method: Platelet aggregation, thrombi adhesion and TXA₂ production in response to arachidonic acid (0.03–1 mmol L^?1), collagen (0.1–30 μg mL^?1), epinephrine (0.001–100 μmol L^?1), ADP, TRAP‐6 amide and U46619 (all 0.1‐30 μmol L^?1), in the presence of aspirin or vehicle, were determined in 96‐well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. Results: Platelet aggregation, adhesion and TXA₂ production induced by either arachidonic acid or collagen were inhibited in concentration‐dependent manners by aspirin, with logIC₅₀ values that did not differ. A linear relationship existed between aggregation and TXA₂ production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R² 0.92; n = 224). The same relationships were seen in combinations of aspirin‐treated and naïve platelets, and in blood from individuals taking an anti‐thrombotic dose of aspirin. Conculsions: These studies demonstrate a linear relationship between inhibition of platelet TXA₂ generation and TXA₂‐mediated aggregation. This finding is important for our understanding of the anti‐platelet effects of aspirin and NSAIDs, NSAID–aspirin interactions and ‘aspirin resistance’.