Inhibition Of ADP-induced Aggregation Research Articles

Effects of the direct thrombin inhibitor dabigatran etexilate vs warfarin on platelet function in patients with atrial fibrillation

Purpose: The impact of novel anticoagulants on platelet function is largely unknown. We characterized the effects of dabigatran etexilate (DE) compared with warfarin (W). Methods: We evaluated 22 atrial fibrillation (AF) patients treated with DE enrolled in the RELY-ABLE study, 26 AF patients treated with W, and 22 control age- and sex-matched non-anticoagulated non-AF patients. We assessed light transmittance platelet aggregation with 2 and 5 μM ADP, 5 and 10 μM thrombin-activated peptide (TRAP), and 1.5 and 6 ng/mL human γ-thrombin. We quantified platelet membrane thrombin protease-activated receptor (PAR)-1 expression by flow-cytometry. Results: We found no inhibition of ADP-induced aggregation in the DE or W groups vs control. γ-thrombin-induced aggregation (1.5 ng/mL) was significantly reduced by 90% in DE and 18% in W (P<0.001 and P<0.01) vs control, and by 88% in DE vs W (P<0.001). TRAP-induced aggregation (5 μM) was reduced in the W (by 60%, P<0.01) and DE (by 36%, P=NS) groups vs control (Figure, left). PAR-1 expression (mean fluorescence intensity, MFI ratio) was higher in the DE and W groups vs control (Figure, right). ![Figure][1] Conclusions: Both DE and W inhibit γ-thrombin-induced aggregation. W also reduces TRAP-induced aggregation. These findings were unexpected, since W is not known to affect γ-thrombin-induced aggregation, and TRAP-induced aggregation should bypass any indirect effect of W on thrombin generation (and direct effects of DE on thrombin activity). The higher platelet PAR-1 expression in platelets of anticoagulated patients suggests that reduced PAR-1 expression on the platelet surface does not explain these reduced platelet responses, which likely involve post-receptor changes. Such effects may contribute to the differential in vivo antithrombotic profile of DE vs W. [1]: pending:yes

Left Ventricular Assist Device Induced Coagulation and Platelet Activation and the Effect of Current Anticoagulant Therapy Regimen

Abstract 48530 Background:Left ventricular assist devices (LVAD) are used to improve circulation in heart failure patients until an available transplant. Despite the benefits of these pumps, they have also been shown to cause a higher rate of adverse effects than the customary medical treatment. Specifically, these implants have been shown to heighten the incidence of both bleeding events (such as intestinal bleeds) and thromboembolic events. Previous studies have attributed these episodes to shear stress caused by blood flow through the pump and to contact of the blood with the artificial surfaces of the device. Contact of blood with artificial surfaces can activate the clotting cascade, while shear stress can directly activate platelets and can impact the functionality of von Willebrand factor. Therefore, a testing regimen that can predict the occurrence of adverse events could lead to enhanced clinical benefit of ventricular assist devices. Methods:Patients implanted with the HeartMate II LVAD at Loyola University Medical Center were enrolled in the study. Blood samples are drawn on a monthly basis and at time of an adverse event. These blood samples were analyzed using flow cytometry to assess P-selectin expression and PAC-1 binding (activated GPIIb/IIIa), the ICHOR PlateletWorks assay (Helena Laboratories; Beaumont, TX) and thrombelastography using the Platelet Mapping assay (Haemoscope; Niles, IL). Patient demographic and clinical data was obtained from medical records. Additionally, blood drawn from 5 individuals per group of warfarin alone, aspirin alone, and normal individuals served as controls. Results:Patients included 15 males and 5 females with a mean age of 53.3 ±13.5 years (range: 23.5 to 70 years) at the time of enrollment. Of the patients 35% were diabetic, 36.7 % were hypertensive, and 45% were hyperlipidemic. Of the 20 patients 18 were on warfarin (mean starting dose of 4.6±2.5 mg) and 19 of 20 patients were on aspirin (81–325 mg/day). Additionally, 60% of patients were treated with an ACE inhibitor, 80% with a β-blocker and 80% with a diuretic. Of the 20 patients, 2 patients had experienced adverse events during the study period. One had a small bowel bleed requiring multiple transfusions. The second had a 3-day GI bleed requiring multi-unit transfusion. By flow cytometry, both patients with bleeding episodes exhibited less of an activation response to platelet agonists ADP, arachidonic acid and TRAP in terms of P-selectin expression and PAC-1 binding than the mean of the stable LVAD group or the normal individuals. In the Platelet Mapping assay, the stable LVAD population exhibited a 31.5 ± 18.4% (mean ± SD) inhibition of ADP-induced activation (p<0.05 vs normals at 15.1 ± 4.8%); the patient with the small bowel bleed exhibited a 65.6% inhibition. In the ICHOR PlateletWorks assay, the stable LVAD population exhibited a 37.3 ± 19.7% inhibition of collagen-induced aggregation (p=0.004 vs normals at 7.9 ± 1.9%); the patients experiencing bleeding events had inhibition levels of 63.7% and 74.2%, respectively. In the PlateletWorks assay, while the stable LVAD population exhibited a 21.2 ± 18.2% inhibition of ADP-induced aggregation (p<0.004 vs normals at 6.1 ± 2.6%), the patient with the GI bleed exhibited an inhibition level of 72.6%. In the PlateletWorks and PlateletMapping assays, several patients exhibited results indicative of low platelet reactivity, but had not experienced a hemorrhagic event to date. These patients will be followed with periodic monitoring of platelet function to determine the predictive nature of each assay for any coagulation disorder. Discussion:Through these studies a baseline of normal platelet function has been established for our LVAD patient population. Further, while early in our evaluation the selected assays of PlateletWorks, PlateletMapping, and flow cytometry suggest that it may be possible to identify patients at risk of LVAD-associated bleeding complications. Disclosures:No relevant conflicts of interest to declare.

Left Ventricular Assist Device Induced Coagulation and Platelet Activation and the Effect of Current Anticoagulant Therapy Regimen

Abstract 5201 Background:Left ventricular assist devices (LVAD) are used to improve circulation in heart failure patients until an available transplant. Despite the benefits of these pumps, they have also been shown to cause a higher rate of adverse effects than the customary medical treatment. Specifically, these implants have been shown to heighten the incidence of both bleeding events (such as intestinal bleeds) and thromboembolic events. Previous studies have attributed these episodes to shear stress caused by blood flow through the pump and to contact of the blood with the artificial surfaces of the device. Contact of blood with artificial surfaces can activate the clotting cascade, while shear stress can directly activate platelets and can impact the functionality of von Willebrand factor. Therefore, a testing regimen that can predict the occurrence of adverse events could lead to enhanced clinical benefit of ventricular assist devices. Methods:Patients implanted with the HeartMate II LVAD at Loyola University Medical Center were enrolled in the study. Blood samples are drawn on a monthly basis and at time of an adverse event. These blood samples were analyzed using flow cytometry to assess P-selectin expression and PAC-1 binding (activated GPIIb/IIIa), the ICHOR PlateletWorks assay (Helena Laboratories; Beaumont, TX) and thrombelastography using the Platelet Mapping assay (Haemoscope; Niles, IL). Patient demographic and clinical data was obtained from medical records. Additionally, blood drawn from 5 individuals per group of warfarin alone, aspirin alone, and normal individuals served as controls. Results:Patients included 15 males and 5 females with a mean age of 53.3 ±13.5 years (range: 23.5 to 70 years) at the time of enrollment. Of the patients 35% were diabetic, 36.7 % were hypertensive, and 45% were hyperlipidemic. Of the 20 patients 18 were on warfarin (mean starting dose of 4.6±2.5 mg) and 19 of 20 patients were on aspirin (81–325 mg/day). Additionally, 60% of patients were treated with an ACE inhibitor, 80% with a β-blocker and 80% with a diuretic. Of the 20 patients, 2 patients had experienced adverse events during the study period. One had a small bowel bleed requiring multiple transfusions. The second had a 3-day GI bleed requiring multi-unit transfusion. By flow cytometry, both patients with bleeding episodes exhibited less of an activation response to platelet agonists ADP, arachidonic acid and TRAP in terms of P-selectin expression and PAC-1 binding than the mean of the stable LVAD group or the normal individuals. In the Platelet Mapping assay, the stable LVAD population exhibited a 31.5 ± 18.4% (mean ± SD) inhibition of ADP-induced activation (p<0.05 vs normals at 15.1 ± 4.8%); the patient with the small bowel bleed exhibited a 65.6% inhibition. In the ICHOR PlateletWorks assay, the stable LVAD population exhibited a 37.3 ± 19.7% inhibition of collagen-induced aggregation (p=0.004 vs normals at 7.9 ± 1.9%); the patients experiencing bleeding events had inhibition levels of 63.7% and 74.2%, respectively. In the PlateletWorks assay, while the stable LVAD population exhibited a 21.2 ± 18.2% inhibition of ADP-induced aggregation (p<0.004 vs normals at 6.1 ± 2.6%), the patient with the GI bleed exhibited an inhibition level of 72.6%. In the PlateletWorks and PlateletMapping assays, several patients exhibited results indicative of low platelet reactivity, but had not experienced a hemorrhagic event to date. These patients will be followed with periodic monitoring of platelet function to determine the predictive nature of each assay for any coagulation disorder. Discussion:Through these studies a baseline of normal platelet function has been established for our LVAD patient population. Further, while early in our evaluation the selected assays of PlateletWorks, PlateletMapping, and flow cytometry suggest that it may be possible to identify patients at risk of LVAD-associated bleeding complications. Disclosures:No relevant conflicts of interest to declare.

Safety and Feasibility of Inhaled Iloprost on Exercise Capacity in Patients with Pulmonary Arterial Hypertension

Introduction: Endothelial dysfunction (EDF), defined on the basis of impaired eNOS-mediated vasodilatation, is a marker of atherogenic and coronary risk. Vascular nitric oxide (NO) resistance shares these prognostic implications, despite independence from NO generation. Furthermore,NOresistanceatplatelet level carries similar prognostic information. Nevertheless, previous data are limited as regards correlations between these parameters. Methods: In a population of randomly selected aging subjects (n= 177) (mean age 67± 6 years), we tested the hypotheses that (a) EDF predicts vascular NO resistance; (b) vascular NO resistance is correlated with platelet NO resistance. Vasomotor responsiveness was assessed utilizing applanation tonometry, with salbutamol and GTN as eNOS-dependent and independent dilators respectively, and platelet responsiveness to NO determined via inhibition of ADP-induced aggregation. Results: There was a significant (R= 0.3; p< 0.001) correlation between vascular eNOS-dependent and independent responses (Figure), but no significant correlation existed between vascular and platelet NO resistance (R= 0.1; p= 0.1).

Antiaggregant Effect of Taurine Chloramines in the Presence of Serum Albumin

The effects of taurine chloramine derivatives on initial aggregation of isolated platelets suspended in buffered saline were studied. Inhibition of ADP-induced aggregation in pure cell suspension depended on the structure of chloramine antiaggregants. The most effective of them was N,N-dichlorotaurine; its concentration needed for 50% inhibition of aggregation was about 0.1 mM. Weaker antiaggregants N-chloro-N-methyltaurine and N-chlorotaurine in a final concentration of 0.5 mM reduced platelet aggregation by only 10%. The studied chloramines considerably differed by their characteristics (velocity of the reaction with sulfur-containing groups of atoms). N,N-dichlorotaurine exhibited the weakest reactivity with methionine thioester group. In turn, the velocity constant with reduced glutathione was by 2-3 orders of magnitude higher than that of other chloramines. Antiaggregant effect of taurine chloramine derivatives was 2-fold higher in the presence of serum albumin, presumably due to special interactions of taurine chloramines in complex with albumin with platelets.

Another view on prasugrel

Thromb Haemost 2009; 101: 12–13 The combination of aspirin with clopidogrel is currently the mainstay of antiplatelet therapy for patients with acute coronary syndromes (ACS) (1). While aspirin inhibits platelet thromboxane A2 production and platelet activation, clopidogrel inhibits adenosine diphosphate (ADP)-induced platelet activation by blocking the P2Y12 platelet receptor. When added to aspirin therapy in patients with ACS, clopidogrel significantly reduces the risk of recurrent ischaemic events (1). Clopidogrel s onset of action is delayed and variable between individuals. A therapeutic level of inhibition is reached 4–6 hours (h) after a 300 mg loading dose, and 2 h after a 600 mg loading dose. Laboratory analysis reveals that a substantial proportion of patients do not show adequate inhibition of ADP-induced aggregation. Low responsiveness to clopidogrel has been shown to be significantly associated with adverse outcomes after percutaneous coronary intervention (PCI) (2). Prasugrel is a novel thienopyridine pro-drug with a greater rate, magnitude, and consistency of platelet ADP inhibition, as compared to clopidogrel. The therapeutic level of the inhibition of platelet aggregation is reached within 1 h after a 60 mg loading dose (3). TRITON-TIMI 38 (4) validated the hypothesis that more intensive antiplatelet blockade via the ADP receptor decreases the incidence of ischaemic events. Patients (13,608) with acute coronary syndromes scheduled for PCI and receiving aspirin were randomly assigned to receive either prasugrel or clopidogrel. After a median duration of 14.5 months, the primary efficacy outcome of cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke occurred in 12.1% of patients taking clopidogrel and in 9.9% of those taking prasugrel. Stent thrombosis was also reduced in the prasugrel group (2.4% clopidogrel vs. 1.1% prasugrel). However, major bleeding was increased (1.7% vs. 2.5%), as was fatal bleeding (0.1% vs. 0.4%) in the prasugrel group. Overall mortality did not differ significantly between treatment groups. In their viewpoint article, Serebruany et al. (5) discuss different aspects of the clinical development of prasugrel and, in particular, the pivotal trial TRITON-TIMI-38. Although their interpretations and evaluations are definitely debatable, this article highlights several points which are of outstanding interest for clinical and research approaches to platelet function inhibition: – Standardization: Serebruany et al. (5) highlight two aspects of standardization. First, the standardization of definitions of endpoints for clinical trials; in order to be able to compare event rates in different studies, a standardized definition of endpoints is desirable. Second, standardization of monitoring techniques for the assessment of anti-platelet therapy is crucial. The authors comment on discrepancies that occurred in the early phase of clinical development of prasugrel, which may be attributed to the use of different equipment for aggregation analysis in participating centers. Today, there is still a wide variation in the literature and clinical practice with respect to instruments, reagents, sample preparation, sample anticoagulants, as well as the analysis algorithms and clinical cut-offs applied for anti-platelet drug monitoring. – Patient compliance: An intensified anti-platelet treatment is usually equalized with a decreased rate of low patient responsiveness to anti-platelet treatment. Serebruany et al. (5) remind us that in a real-life scenario, patient compliance is crucial. Intensifying anti-platelet therapy might generate an increased rate of annoying minor bleeding complications during everyday activities, such as shaving or brushing teeth, which may again lead to patient non-compliance. For the physician, this includes the comforting fact that even with more modern and intensified anti-platelet regimens, the role of personal communication with the patient is still important for the appropriate adherence of the patient to his life-saving treatment. – Focus on safety: With the growing efficacy of antithrombotic treatments in ACS and in other areas such as prophylaxis of deep venous thrombosis, there is an increased focus on drug safety. Melagatran and ticlopidine are examples of antithrombotic drugs that were either withdrawn from the market, or experienced a sharp drop in use due to safety concerns. Referring to prasugrel, Bhatt (6) states that in a “realworld” setting, such as an elderly patient with multiple coexisting conditions, the risk of major bleeding and even fatal bleeding may increase to an even greater degree than was seen in TRITON–TIMI 38. This expectation is also expressed by Serebruany et al. in their viewpoint article (5). – Transformation of clinical trials into routine patient management: Clinical trials have inclusion and exclusion © 2009 Schattauer GmbH, Stuttgart

Clopidogrel Attenuates Coated-platelet Production in Patients Undergoing Elective Coronary Catheterization

Coated-platelets are a subclass of highly thrombotic activated platelets with an enhanced ability to generate thrombin. Excessive numbers of coated-platelets are believed to increase thrombotic risk. A previous report demonstrated that P2Y12 inhibition in vitro attenuates coated-platelet formation. The aim of this study was to determine the effect clopidogrel has on coated-platelet formation. We enrolled 27 patients undergoing elective coronary angiography. A total of 3 blood samples were taken from eligible patients: baseline, 24-hour postclopidogrel (preangiography), and 6-hour postangiography. Coated-platelet levels, expressed as percentage of total platelets, were determined with convulxin and thrombin with or without 1.5 or 6 microM adenosine diphosphate (ADP). Baseline levels of coated-platelets were 40.0% +/- 14.3% (mean +/- 1 SD). After clopidogrel exposure, the coated-platelet level was 32.8% +/- 13.6%, representing a significant 7.2% absolute reduction (AR) (17.8% relative reduction (RR); P < 0.0001). Clopidogrel significantly lowered the convulxin, thrombin plus ADP coated-platelet production (11.0% AR; 20.1% RR for 1.5 microM and 11.2% AR; 19.1% RR for 6 microM). This is the first report on the impact of in vivo administration of a P2Y12 antagonist on coated-platelet formation. The significance of a partial attenuation in coated-platelet potential has yet to be determined, but this could represent a new antithrombotic mechanism of clopidogrel beyond inhibition of ADP-induced aggregation.

Clopidogrel anti-platelet effect: An evaluation by optical aggregometry, impedance aggregometry, and the Platelet Function Analyzer (PFA-100™)

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4–30% of patients. Traditionally, optical aggregometry is used to assess clopidogrel's anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100™). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 µM) and collagen (1 and 2 µg/mL). For PFA-100™ analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71 ± 34% vs. 34.2 ± 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100™ ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100™ was unable to detect clopidogrel effect in the majority of the subjects.

Platelet Nitric Oxide Responsiveness

Nitric oxide (NO) is critically important in the regulation of vascular tone and the inhibition of platelet aggregation. We have shown previously that patients with acute coronary syndromes (ACS) or stable angina pectoris have impaired platelet responses to NO donors when compared with normal subjects. We tested the hypotheses that platelet hyporesponsiveness to NO is a predictor of (1) cardiovascular readmission and/or death and (2) all-cause mortality in patients with ACS (unstable angina pectoris or non-Q-wave myocardial infarction). Patients (n=51) with ACS had evaluation of platelet aggregation within 24 hours of coronary care unit admission using impedance aggregometry. Patients were categorized as having "normal" (> or =32% inhibition of ADP-induced aggregation with the NO donor sodium nitroprusside; 10 micromol/L; n=18) or "impaired" (<32% inhibition of ADP-induced aggregation; n=33) NO responses. We then compared the incidence of cardiovascular readmission and death during a median of 7 years of follow-up in these 2 groups. Using a Cox proportional hazards model adjusting for age, sex, index event, postdischarge medical treatment, revascularization status, left ventricular systolic dysfunction, concurrent disease states, and cardiac risk factors, impaired NO responsiveness was associated with an increased risk of the combination of cardiovascular readmission and/or death (relative risk, 2.7; 95% CI, 1.03 to 7.10; P=0.041) and all-cause mortality (relative risk, 6.3; 95% CI, 1.09 to 36.7; P=0.033). Impaired platelet NO responsiveness is a novel, independent predictor of increased mortality and cardiovascular morbidity in patients with high-risk ACS.

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2 × 103 leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10 µM) and ATP (10–100 µM) was markedly reduced, but to ATP (1000 µM) was enhanced (all p < 0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2 = 4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.

Comparison of turbidimetric aggregation and in vitro bleeding time (PFA-100) for monitoring the platelet inhibitory profile of antiplatelet agents in patients undergoing stent implantation

The present study compared classical ADP-induced platelet aggregation vs. PFA-100 closure times using collagen/ADP membrane cartridges to monitor the degree of platelet-inhibiting effect of three drug regimens: ticlopidin, abciximab/ticlopidin and loading dose clopidogrel, each on top of aspirin (acetylsalicylic acid, ASA) during and after elective stent placement (intervention) in a total of 31 patients with acute coronary syndrome. Ticlopidin was started directly after stent implantation, abciximab was started before coronary intervention and given intravenously for 12 h, and a clopidogrel loading dose was given before intervention. The 10 patients treated with ticlopidin (500 mg daily) showed no significant prolongation of PFA closure times and a slight increase of ADP-induced platelet aggregation shortly after intervention. In 11 patients treated with abciximab/ticlopidin, the PFA closure times were significantly prolonged, and ADP-induced platelet aggregation was reduced by more than 80% during the 12-h abciximab infusion after intervention. The 10 patients pretreated with loading dose clopidogrel (450 mg followed by 75 mg daily) showed an intermediate but significant prolongation of PFA closure times and reduction of ADP-induced platelet aggregation at levels between the ticlopidin/aspirin- and the abciximab/ticlopidin/aspirin-treated groups. At 20 h after intervention, a similar degree of PFA closure time prolongation and inhibition of ADP-induced aggregation was observed in the abciximab/ticlopidin/aspirin- and the clopidogrel/aspirin-treated patient groups. Both measurement of PFA-100 closure times and inhibition of ADP-induced platelet aggregation showed a similar degree of platelet inhibition, but had rather broad SD ranges, which limit their precision for the follow-up of individual patients. In conclusion, abciximab on top of ticlopidin/aspirin showed a stronger antiplatelet effect for only less than 20 h, as compared to loading dose clopidogrel/aspirin in acute coronary syndrome patients undergoing stent implantation. Whether such a short-term superiority of abciximab, as compared to loading dose clopidogrel, translates into an overall clinical benefit of thombotic and bleeding complications remains to be established in a randomized clinical trial.

P-Aminobenzoic Acid and Its Metabolite p-Acetamidobenzoic Acid Inhibit Agonist-Induced Aggregation and Arachidonic Acid-Induced [Ca 2+] i Transients in Human Platelets

We have previously found that the naturally occurring amine p-aminobenzoic acid (PABA) inhibits the thrombin-induced thromboxane B 2 production in human platelets. In this report we show that PABA and its acetylated metabolite p-acetamidobenzoic acid (PACBA) inhibit platelet aggregation induced by agonists such as adenosine diphosphate (ADP) and arachidonic acid (AA). Both substances were equipotent to acetylsalicylic acid regarding inhibition of ADP-induced aggregation and approximately 50% as potent as acetylsalicylic acid regarding arachidonic acid-induced aggregation. Although not significantly inhibiting collagen aggregation, PABA and PACBA reduced the concomitant adenosine triphosphate (ATP) secretion by approximately 30 and 20%, respectively. The antiaggregatory effect does not seem to be mediated through cyclic adenosine monophosphate (cAMP) increase because in our experiments PABA and PACBA did not significantly affect cAMP levels. However, we have found that PABA and PACBA inhibit the intracellular aequorin indicated Ca 2+ transient upon arachidonic acid stimulation. Our results describe a hitherto unknown effect of PABA and PACBA on platelet aggregation.

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro.

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.

The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T AC receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptorselective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T AC , in mouse platelets with similar function as in human platelets.

Influence of fibrinolysis system on ADP- and serotonin-induced aggregation of human platelets

Urokinase, a component of the fibrinolytic system, induces a time-dependent decrease in platelet aggregation activated by ADP and serotonin. Significant inhibition of ADP-induced aggregation was observed on the 30th–60th min and serotonin-induced on the 3th–10th min of preincubation with urokinase and depended on urokinase concentration. The plasmin inhibitor aprotinin partially abolished urokinase-induced reduction of the amplitude and rate of ADP-induced aggregation and had no effect on serotonin-induced aggregation. Our results favor multiple mechanisms of urokinase influence on platelet activity.

Effects of 2-methylthioadenosine 5?-?,?-methylenetriphosphonate and 2-ethylthioadenosine 5?-monophosphate on human platelet activation induced by adenosine 5?-diphosphate

Adenosine 5′-diphosphate (ADP) induces human platelet aggregation, increases intracellular levels of free calcium, and inhibits stimulated adenylate cyclase. These effects of ADP are mediated by P2T-purinoceptors that are inhibited specifically and competitively by adenosine 5′-triphosphate (ATP). Inhibition of ADP-induced aggregation and increases in calcium by 2-alkylthio analogs of ATP and of adenosine 5′-monophosphate (AMP) are also specific, but the inhibition is non-surmountable. To examine further the nature of inhibition of ADP-induced platelet activation by 2-alkylthio analogs, the effects of 2-methylthioadenosine 5′-β,γ-methylenetriphosphonate (2-MeS-AMP-PCP) and 2-ethylthioadenosine 5′-monophosphate (2-EtS-AMP) were tested on ADP-induced platelet aggregation and inhibition of adenylate cyclase. 2-MeS-AMP-PCP inhibited platelet aggregation induced by ADP but not by epinephrine, arachidonic acid, 5-hydroxytryptamine (5-HT), platelet activating factor (PAF), or 11α,9α-epoxymethano-prostaglandin H2 (U46619). Inhibition of ADP-induced platelet aggregation by 2-MeS-AMP-PCP was non-surmountable, and it achieved only 50% inhibition of ADP (5 μM)-induced aggregation. 2-MeS-AMP-PCP achieved 100% inhibition of ADP (5 μM)-induced inhibition of prostaglandin E1-stimulated adenylate cyclase, and Schild analysis showed the inhibition to be potent (pA2 7.3) and competitive (slope 1.12). 2-MeS-AMP-PCP inhibited platelet aggregation induced by adenosine 5′-O-2-thiodiphosphate (ADP-β-S), which inhibited stimulated adenylate cyclase activity, but did not inhibit aggregation induced by adenosine 5′-O-1-thiodiphosphate (ADP-α-S), which does not inhibit stimulated adenylate cyclase. 2-EtS-AMP behaved similarly to 2-MeS-AMP-PCP. These results suggest that ADP may induce aggregation by interacting with two forms of the calcium-mobilizing P2T-purinoceptor, only one of which is coupled to inhibition of adenylate cyclase and at which 2-alkylthio analogs of ATP and AMP are specific competitive antagonists. © 1996 Wiley-Liss, Inc.

Antiplatelet Effects of Ticlopidine Are Reduced in Experimental Hypercholesterolemia

This study determines the antiplatelet effects of oral ticlopidine (100 mg/kg x day) in experimental hypercholesterolemia. Rabbits were fed either a standard diet or a cholesterol-enriched diet (0.5% for 3 months, 1% for 1 month). In normocholesterolemic controls ADP-, but not collagen-induced platelet aggregation was inhibited by ticlopidine treatment. This was accompanied by a significantly enhanced inhibition of ADP-induced platelet aggregation and stimulation of cyclic AMP accumulation by iloprost. Hypercholesterolemia considerably attenuated the inhibition of ADP-induced aggregation by ticlopidine but did not change its effect on the iloprost-induced inhibition of platelet function and cyclic AMP formation. ADP-induced platelet-derived thromboxane formation was considerably greater in hypercholesterolemic rabbits and not reduced by ticlopidine. Ticlopidine did also not significantly influence the extent and severity of atherosclerotic plaque formation although a tendency for improvement was observed in a subgroup of animals. The data suggest that hypercholesterolemia attenuates the inhibitory effect of ticlopidine on ADP-induced platelet aggregation. This might be related to the stimulation of thromboxane formation by ADP in hypercholesterolemia. The maintained protection from ADP-induced inhibition of cAMP accumulation suggests a minor role of this mechanism in the progression of hypercholesterolemia-induced vessel disease in this model.

Pharmacodynamics of ticlopidine in man in relation to plasma and blood cell concentration

In 6 normal volunteers given single oral doses of 250,500 and 1000 mg ticlopidine (T), the peak plasma level of unchanged drug was reached after about 2 h. There was no correlation between the plasma T level and its inhibitory effect on platelet function, expressed as % inhibition of ADP-induced aggregation. By means of HPLC and GC/MS significant concentrations of T were demonstrated in washed red cells, platelets and neutrophils, with a marked difference in the time course of the appearance of cell-associated drug. The time course of platelet-associated T very accurately fitted that of the antiaggregatory activity. After subacute oral administration (250 mg b.d. for 7 days), the maximum effect on platelet function was observed after 3 to 4 days, when a significant concentration of platelet-associated T had been reached. The pharmacological effect persisted as long as drug was detectable in platelet. An in vitro study strongly suggested that the antiaggregating effect was retained by treated washed platelets but not by treated plasma. It is suggested that the platelet compartment represents the pharmacological target of T via a specific uptake system.

Low molecular weight heparin as an anticoagulant for in vitro platelet funtion studies

We evaluated the suitability of low molecular weight (LMW) heparin as an anticoagulant for in vitro platelet function tests in 11 normal volunteers. Results with citrated platelets were considered as the standard. Spontaneous platelet aggregation and the aggregation responses to ADP, epinephrine, collagen, ristocetin and thrombin were measured turbid imetrically in an aggregometer. Dose-response and dose-rate curves were constructed for ADP- and epinephrine-induced aggregation. The maximum aggregation response (ED max) and rate (EDR max), and the estimated dose of agonist to induce 50% of the maximum response (ED 50) and rate (EDR 50) were calculated from these curves. The inhibition of ADP-induced aggregation with PGI 2 was expressed as per cent inhibition. The release of ATP and TxA 2 from platelets aggregated with collagen was measured. No spontaneous aggregation occured with either anticoagulant. The ED 50 and the EDR 50 for heparinized platelets were significantly lower for ADP induced aggregation (0.8±0.3μM vs 2.1±1.0μM [p=0.001] and 0.4±0.1μM vs 0.8±0.3μM [p=0.003]). The EDR max with ADP was significantly higher (p=0.004) for heparinized platelets (64±17.0 units/ml vs 50.4±7.6 units/ml). The heparinized platelets aggregated slighty, but significantly, less in response to ristocetin than the citrated platelets. The response of washed heparinized and citrated platelets to thrombin was not significantly different. The per cent inhibition of ADP aggregation with PGI 2, was significantly lower with heparinized platelets. The release of TxA 2 and ATP was similar for both anticoagulants. These results indicate that LMW heparin is a satisfactory anticoagulant for platelet aggregation tests.

Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic amp and reducing cytoplasmic free calcium mobilization

Cyclic nucleotide phosphodiesterase inhibitors (HL-725, RO 15-2041, cilostamide, quercetin and MY-5445) potently inhibit human platelet aggregation induced by ADP. In parallel, PDE inhibitors inhibit the increase in cytoplasmic free Ca 2+ evoked by ADP, as measured with the fluorescent probe quin 2. The inhibition of ADP-induced aggregation and rise in [Ca 2+] i is potentiated by PGE 1 which stimulates adenylate cyclase and is inhibited by adrenaline which inhibits adenylate cyclase. PDE inhibitors increase human platelet cAMP levels in the presence of low concentrations of PGE 1. It is suggested that PDE inhibitors prevent platelet aggregation by raising cAMP levels and by subsequent inhibition of cytoplasmic free Ca 2+ mobilization.