μM ADP-induced Platelet Aggregation Research Articles

Influence of rabeprazole and famotidine on pharmacodynamic profile of dual antiplatelet therapy in clopidogrel-sensitive patients: The randomized, prospective, PROTECT trial

Although acid suppressants are needed to attenuate gastrointestinal bleeding (GIB) after percutaneous coronary intervention (PCI), pharmacodynamic interaction between clopidogrel and proton pump inhibitor (PPI) can increase the risk of high platelet reactivity (HPR). We sought to evaluate serial changes of platelet measures and influence of rabeprazole on platelet measures. After 600-mg clopidogrel loading for elective PCI, clopidogrel-sensitive patients were recruited and randomly assigned to add rabeprazole of daily 20 mg (n = 40) or famotidine of daily 40 mg (n = 40). Platelet measures were performed with light transmittance aggregometry and VASP-P assay. Primary endpoint was 5 μM ADP-induced platelet aggregation (PA) at 30-day follow-up. HPR was defined as 5 μM ADP-induced PA > 46%. Baseline platelet measures did not differ significantly between the groups. The 30-day level of 5 μM ADP-induced PA was similar between the famotidine vs. rabeprazole group (30.0 ± 16.4% vs. 30.2 ± 13.9%, P= .956). In addition, other platelet measures were comparable between the groups. At 30-day follow-up, the incidence of HPR was similar between the famotidine and rabeprazole groups (20.5% vs. 15.4%; P= .555). In conclusion, adjunctive use of rabeprazole showed the similar antiplatelet effect even in clopidogrel-sensitive patients compared with adjunctive famotidine, which may support the similar effect of rabeprazole and famotidine on the antiplatelet effect of dual antiplatelet therapy with clopidogrel plus aspirin.

Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation.

Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention.

Influence of platelet reactivity on BARC classification in East Asian patients undergoing percutaneous coronary intervention. Results of the ACCEL-BLEED study.

An increasing body of data suggests that East Asian patients have differing risk profiles for both thrombophilia and bleeding compared with Western population. This study was designed to evaluate the relationship of bleeding to platelet function in East Asians undergoing percutaneous coronary intervention (PCI). Patients who had undergone uneventful PCI (n= 301) were prospectively enrolled and bleeding events were evaluated during dual antiplatelet therapy (DAPT) with aspirin and clopidogrel. Platelet function was measured during hospitalisation and at 30-day follow-up by light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) assay. During 30-day follow-up, 29.2 % of patients (n= 88) experienced post-discharge Bleeding Academic Research Consortium (BARC) complications (24.6 % and 7.0 % of BARC type 1 and 2, respectively). Patients presenting with acute myocardial infarction had fewer episodes of type 1 BARC bleeding (odds ratio: 0.41; 95 % confidence interval: 0.22 to 0.76; p= 0.005). The cut-off of low platelet reactivity (LPR) (20 µM ADP-induced platelet aggregation ≤ 46.1 %; platelet reactivity index ≤ 45.1 %) was the independent determinant of type 2 BARC bleeding (odds ratio: 3.55 and 4.44; p= 0.009 and 0.002, respectively). The first 30-day BARC bleeding episodes were associated with an increased rate of subsequent premature DAPT discontinuation during one-year follow-up (4.7 % vs 11.4 %; odds ratio: 2.60; 95 % confidence interval: 1.04 to 6.50; p= 0.035). In conclusion, among East Asians, mild bleeding episodes are common early after PCI and are associated with premature DAPT discontinuation. Type 2 BARC bleeding episodes are associated with LPR cut-offs measured at 30 days post-discharge.

Abstract T P341: Impact Of Cytochrome P450 2C19 Polymorphism On High On-treatment Platelet Reactivity In Stroke Patients Treated With Clopidogrel

BACKGROUND: Clopidogrel is one of the most commonly used anti-platelet drugs. Recent reports suggested the association of allelic variants of the genes modulating metabolic activation (CYP2C19 and paraoxonase-1) and bioavailability (ABCB1) of clopidogrel with high on-treatment platelet reactivity (HTPR), possibly resulting in the recurrence of thrombotic events. However, these studies mostly examined patients with coronary heart disease and the impact of these polymorphisms in stroke patients is largely unknown. METHODS: We conducted a multicenter, prospective cohort study of 518 Japanese patients from 14 hospitals who were administrated clopidogrel. Residual platelet reactivity was determined by a change in light transmission induced by platelet aggregation in platelet-rich plasma after adding ADP. The vasodilator-stimulated phosphoprotein (VASP) index was also measured using flow cytometry. We investigated the association of polymorphisms of aforementioned enzymes with HTPR. RESULTS: In terms of CYP2C19 loss-of-function polymorphisms (*2 and *3), the prevalence of intermediate (IM: *1*2 or *1*3) or poor (PM: *2*2, *2*3, or *3*3) metabolizer was much higher (IM: 47% or PM: 15%) in Japanese than in Caucasian populations. Residual platelet reactivity was significantly higher in PM and IM groups than that in the wild-type extensive metabolizer (EM: *1*1) group assessed by 5 μM ADP-induced platelet aggregation (PM: 69.4% ± 17.1%, IM: 59.7% ± 16.2%, EM: 51.5% ± 17.5%, p < 0.0001) and VASP index (PM: 66.6% ± 14.1%, IM: 52.4% ± 16.0%, EM: 42.9% ± 15.0%, p < 0.0001). Furthermore, HTPR determined by both assays was associated with the ABCB1 polymorphism (rs1045642) but not with the paraoxonase-1 Q192R mutation. CONCLUSIONS: Our results indicate that both CYP2C19 and ABCB1 polymorphisms significantly contribute to HTPR in stroke patients treated with clopidogrel. At present, we are monitoring these patients for a two-year period to determine whether these genetic variants and HTPR affect the recurrence of thrombotic events.

The clinical effect of resistance to antiplatelet therapy and high-dose statin therapy in acute coronary syndrome

Purpose: The aim of this study was to determine whether there is an association between antiplatelet therapy resistance and systemic inflammation in patients with acute coronary syndrome (ACS). Methods: We included 122 patients with ACS hospitalized at Moscow City Hospital 23. We measured platelet function using simultaneous light transmittance aggregometry (spontaneous aggregation and ADP-induced), Thromboelastography Platelet Mapping (TEG-PM), and VerifyNow Aspirin and P2Y12 assays. The following markers of systemic inflammation were estimated: white blood cell count and plasma levels of high sensitivity C-reactive protein (hsCRP), soluble CD40-ligand (sCD40L), sP-selectin, fibrinogen. All patients received aspirin at 250–325 mg and a loading dose of clopidogrel (300–600 mg) on arrival, followed by 125 mg of aspirin and 75 mg of clopidogrel per day. For the following analysis, the patients were separated into two groups. Both groups received atorvastatin: one group at 20 mg per day (N = 18) and the second at 80 mg per day (N = 24) for at least 7 days. Results: We found that patients with elevated levels of plasma von Willebrand factor had increased 5 μM ADP–induced platelet aggregation (p=0.044). High white blood cell counts were associated with increased levels of 10 μM ADP-induced platelet aggregation (p=0.0043). High plasma levels of sP-selectin were associated with high levels of ADPtest (Multiplate) and high percentages of P2Y12-inhibition (VerifyNow) (p=0.046 and 0.047, respectively). Kaplan-Meier survival analysis with a logrank significance test showed an association between a high risk of reinfarction during follow-up (1–22 months after ACS) and high levels both of 10 μM ADP–induced platelet aggregation and of HPR ADP (p = 0.014 and 0.006, respectively). We found that this association remains significant among patients with high levels of hs-CRP (p = 0.021), while in the group of patients with low levels of hs-CRP this relationship was not significant. Among patients receiving atorvastatin at 20 mg, the association between a high risk of reinfarction and high levels of platelet aggregation also remained significant (p = 0.003). In contrast, among patients receiving atorvastatin at 80 mg, this association disappeared. Conclusions: In our study, we found that high on-treatment platelet reactivity is associated with several laboratory signs of systemic inflammation and predicts a poor prognosis in patients with ACS. High-dose statin therapy can overcome the association between high on-treatment platelet reactivity and poor prognosis in patients with ACS.

Effect of rabeprazole versus famotidine on platelet inhibition in patients with normal clopidogrel responsiveness: result of the ACCEL-PROTECT randomized study

Background: Although gastric acid-suppressing therapy is needed to attenuate GI bleeding during antiplatelet therapy, several regimens increase the risk of high on-clopidogrel platelet reactivity (HPR). PPI-clopidogrel interaction appears less evident following rabeprazole than omeprazole. This randomized study aimed to compare the effect of rabeprazole and famotidine on clopidogrel response and the risk of HPR. Methods: Elective PCI-treated patients without HPR were randomized to receive rabeprazole 20 mg daily (n=39) or famotidine 40 mg daily (n = 39). Platelet reactivity was measured at baseline and on day 14 by conventional aggregometry and VASP-P assay. Primary endpoint was 5 μM ADP-induced platelet aggregation (PA) at follow-up. HPR was defined as 5 μM ADP-PA > 46%. Results: Baseline platelet measurements did not differ significantly between the groups. The level of 5 μM ADP-PA on day 14 was similar between the rabeprazole vs. famotidine group (30.2±13.9% vs. 30.0±16.4%, p = 0.956). Other platelet measures including VASP-PRI were also comparable. However, compared with baseline values, follow-up ADP-induced platelet measures were increased by rabeprazole (difference, 7.8%; 95% CI, 2.8-12.7%; p = 0.003) or famotidine (difference, 5.4%; 95% CI, 1.5-9.4%; p = 0.008) co-administration. Furthermore, the risk of HPR was also increased at follow-up (35.9% by rabeprazole and 28.2% by famotidine, respectively). Arachidonic acid- and collagen-induced PAs were similar between baseline and follow-up in both groups. Conclusions: In elective PCI-treated patients, adjunctive rabeprazole and famotidine showed the similar level of platelet reactivity during clopidogrel treatment. When considering GI protective effect and interaction with clopidogrel response together, H2R blocker famotidine may not substitute the position of PPI having low inhibition for CYP2C19 activity.

Pharmacodynamic effect of adjunctive cilostazol vs. high-dos clopidogrel in acute coronary syndrome patients according to the CYP2C19 genotype (ACCEL-GENOTYPE) study

Purpose: Cilostazol use in ACS patients has been reduced the risks of ischemic events and restenosis in East Asians. Compared with Caucasians, East Asians have higher frequency of CYP2C19 loss-of-function allele (*2 and *3). We compared the pharmacodynamic effect of adjunctive cilostazol vs. high-dose clopidogrel in PCI-treated East Asians with ACS according to the CYP2C19 genotype. Methods: ACS patients were assigned to either clopidogrel 150 mg/d (DOUBLE group; n=139) or cilostazol 100 mg twice a day + clopidogrel 75 mg/d (TRIPLE group; n=136) on top of aspirin. Platelet aggregation (PA) was measured at baseline and at least 30-day follow-up with light transmittance aggregometery (20 μM ADP and 6 μg/ml collagen). PAs were adjusted with known clinical covariates. CYP2C19*2 or *3 genotype was determined. Primary endpoint was the level of 20 μM ADP-induced PA at follow-up. HPR was defined as 20 μM ADP-induced PA >59%. Results: During standard-dose clopidogrel treatment, platelet measurements did not differ according to individual CYP2C19 genotype between the groups (Fig. 1A). PA levels and HPR risk increased across the CYP2C19 genotype (p≤0.174). At follow-up, the TRIPLE vs. DOUBLE group showed significantly lower level of PAs irrespective of the CYP2C19 genotype (Fig. 1B). In the DOUBLE group, the risk of HPR was observed in 27.3% of patients and increased across the CYP2C19 genotype (adjusted p=0.042), whereas the effect of TRIPLE regimen was not significantly influenced with the CYP2C19 genotype (adjusted p=0.289). Similar trend was observed for collagen-induced PA. ![Figure][1] Figure 1 Conclusion: In PCI-treated East Asians presented with ACS, adjunctive cilostazol exhibited favorable platelet measurements compared with high-dose clopidogrel irrespective of the CYP2C19 genotype. [1]: pending:yes

Isolation and study of insulin activated nitric oxide synthase inhibitory protein in acute myocardial infarction subjects

Insulin inhibits platelet aggregation through nitric oxide synthesis by stimulating platelet insulin activated nitric oxide synthase. Impaired platelet insulin activated nitric oxide synthase in acute myocardial infarction (AMI) patients had been reported and thus our aim was to identify and isolate the factors impairing insulin activated nitric oxide in acute myocardial infarction patients' plasma and study its effect on platelets aggregation in vitro. The insulin activated nitric oxide synthase inhibitor was identified as a protein and was purified from the plasma of AMI subjects using DEAE cellulose and Sephadex G-50 column, molecular weight determined by SDS-PAGE, nitric oxide quantified by methaemoglobin method, inhibitor protein quantified in plasma by immunoblot and ELISA, platelet aggregation studies done using an aggregometer, thromboxane-A2 in the platelets determined by radioimmunoassay, (125)I-insulin radioligand binding studies done using normal subject platelets. The purified nitric oxide synthase inhibitor protein was ~66kDa, concentration in AMI subjects' plasma varied from 114 to 9,090μM and was undetected in normal subjects' plasma. The inhibitor protein competes with insulin for insulin receptor binding sites. The Incubation of the normal subject PRP with 5.0μM inhibitor for 30min followed by 0.4μM ADP addition caused platelet aggregation in vitro, 130μM aspirin or 400μU insulin/ml addition was able to abrogate 0.4μM ADP induced platelet aggregation even in the presence of 5.0μM inhibitor. A potent inhibitory protein against insulin activated nitric oxide synthase in platelets appears in circulation of AMI subjects impairing nitric oxide production, potentiating ADP induced platelet aggregation and increasing the thromboxane-A2 level in platelets.

A double blind, randomized study on platelet aggregation in patients treated with a daily dose of 150 or 75 mg of clopidogrel for 30 days

In reference to the interesting study by von Beckerath et al. ,1 we would like to offer the following comments. The predicted induced platelet aggregation (IPA) of 50% in the 75 mg clopidogrel group is lower than the actual observations of 65% noted in the study controls. Underestimation of the control IPA impacts effect size and study power, and could limit the validity.2 Further, the 5 and 20 µM ADP IPA observed in …

Platelet reactivity to adenosine diphosphate and long-term ischemic event occurrence following percutaneous coronary intervention: A potential antiplatelet therapeutic target

Platelets play a central role in the genesis of post-percutaneous coronary intervention (PCI) ischemic events. High post-procedural platelet reactivity to adenosine diphosphate (HPRADP) may be a risk factor for ischemic events after PCI. The study was designed to evaluate a cutpoint of platelet reactivity that is associated with the occurrence of ischemic events after PCI. Post-procedural platelet reactivity to ADP was measured by conventional aggregometry in 297 consecutive patients undergoing non-emergent PCI. Patients were prospectively followed for up to 2 years for post-discharge ischemic events. All patients had received clopidogrel and aspirin therapy at the time of aggregation measurements. Eighty-one patients (27%) suffered ischemic events. Patients with ischemic events had higher 5 µM ADP-induced platelet aggregation (46 ± 14% vs. 30 ± 17%, p < 0.001) and 20 µM ADP-induced platelet aggregation (60 ± 13% vs. 43 ± 19%, p < 0.001) compared to patients without ischemic events. Using a combined receiver operator curve analysis, cutpoints of >46% aggregation following 5 µM ADP stimulation and >59% aggregation following 20 µM ADP stimulation (HPRADP) were associated with 58 and 54% of ischemic events, respectively. Multivariate Cox regression demonstrated a significant relation between event occurrence and post-procedural HPRADP cutpoints (5 µM ADP, OR=3.9, and 20 µM ADP, OR=3.8, p < 0.001 for both). High post-procedural platelet reactivity to ADP is an independent risk factor for ischemic events within 2 years of non-emergent PCI. These data support a potential therapeutic target for antiplatelet therapy based on the results of an ex vivo platelet function test. The study is a step towards a personalized medicine approach to guide the intensity of antiplatelet therapy.

Functional impact of high clopidogrel maintenance dosing in patients undergoing elective percutaneous coronary interventions

The currently recommended maintenance dose of clopidogrel is often associated with inadequate platelet inhibition, suggesting the need for a higher dose. The aim of this pilot study was to assess the functional impact of a high (150 mg/day) maintenance dose of clopidogrel in patients undergoing elective percutaneous coronary intervention (PCI). This is a prospective, randomized, platelet function study which was performed in elective PCI patients assigned to treatment with either a 75 mg (n = 20) or 150 mg (n = 20) daily maintenance dose of clopidogrel for 30 days; afterwards, all patients resumed standard dosing. Platelet aggregation was performed using light transmittance aggregometry following 20 microM and 5 microM adenosine diphosphate (ADP) stimuli 30 days after randomization and 30 days after resuming standard dosing. Patients treated with 150 mg/day clopidogrel had lower 20 microM ADP-induced platelet aggregation compared to patients on 75 mg/day (52.1 +/- 9% vs. 64.0 +/- 8%; p < 0.001; primary endpoint). The dose-dependent effect was confirmed by the absolute and relative increase in platelet aggregation after resuming standard dosing (p < 0.001). No changes were observed in patients randomized to standard dosing. Parallel findings were observed following 5 microM ADP stimuli for all assessments. A broad variability in clopidogrel-induced antiplatelet effects was observed irrespective of dosing. In conclusion, a 150 mg/day maintenance dose regimen of clopidogrel is associated with reduced platelet reactivity and enhanced platelet inhibition compared to that achieved with the currently recommended 75 mg/day in patients undergoing elective PCI.

Experimental arterial thrombosis regulated by androgen and its receptor via modulation of platelet activation

Objectives The aim of our study is to elucidate whether experimental arterial thrombosis is regulated by physiological doses of androgen and its receptor via modulation of platelet activation. Methods Surgical castration was performed in male rats and ferric chloride (FeCl 3), as a stimulator, induced the experimental arterial thrombosis. Testosterone was measured directly by chemiluminescent immunoassay on the Bayer ADVIA Centaur analyzer. Dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. A platelet aggregometer was used to assess aggregation, and a platelet adherometer was used to measure adhesion. The contents of TXB 2 and 6-Keto-PGF 1α were assayed by radio-immunoassay using commercially available kits. Results Our data showed that DHT replaced restored circulating DHT of castrated rats to physiological levels, without being altered by treatment with flutamide. Castration caused significant increases in the thrombus area and weight in castrated rats as compared with control group. In PRP diluted with autologous PPP, ADP-induced platelet aggregation rate was only 9.10%. However, in PRP diluted with Tyrode's buffer, 1 μM ADP-induced platelet aggregation rate rose to 63.65%. In PRP diluted with Tyrode's buffer, and pretreated with DHT (1 nM, 2 nM), ADP-induced platelet aggregation was significantly lowered again. Platelet aggregation in PRP diluted with autologous PPP was enhanced in castrated rats as compared with sham-operated rats, and DHT (2 nM) replacement suppressed platelet aggregation in castrated PRP to the level similar to that of sham-operated rats. However, presence of flutamide (3 μM) significantly increased platelet aggregation in PRP diluted with autologous PPP or Tyrode's buffer. DHT (2 nM) replacement significantly inhibited the ADP-induced platelet adhesion. However, presence of flutamide (3 μM) increased ADP-induced platelet adhesion again. DHT replacement obviously reduced the ratio of TXB 2 to 6-keto-PGF 1α in castrated rats. However, administration of flutamide and DHT to castrated rats caused an increase in the ratio of TxB 2 to 6-keto-PGF1α. Conclusion Inhibition of experimental arterial thrombosis by androgen at physiological doses and its receptor is mediated via modulation of platelet activation.