The P2Y1 receptor plays an essential role in the platelet shape change induced by collagen when TxA2 formation is prevented.

ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.     

Enhanced antiplatelet effect of clopidogrel in patients whose platelets are least inhibited by aspirin: a randomized crossover trial

Summary. Objective: We aimed to determine whether adding clopidogrel to aspirin in patients at high risk of future cardiovascular events would suppress laboratory measures of the antiplatelet effects of aspirin; and have greater platelet inhibitory effects in patients with the least inhibition of platelets by aspirin. Methods: We performed a randomized, double‐blind, placebo‐controlled, crossover trial, comparing clopidogrel 75 mg day^?1 versus placebo, in 36 aspirin‐treated patients with symptomatic objectively confirmed peripheral arterial disease. Results: The addition of clopidogrel to aspirin did not suppress platelet aggregation induced by arachidonic acid, urinary 11 dehydro thromboxane B₂ concentrations, or soluble markers of platelet activation markers (P‐selectin, CD40‐ligand) and inflammation (high sensitivity serum C‐reactive protein, interleukin‐6). Clopidogrel significantly inhibited platelet aggregation induced by ADP (reduction 26.2%; 95% CI: 21.3–31.1%, P < 0.0001) and collagen (reduction 6.2%; 95% CI: 3.2–9.3%, P = 0.0003). The greatest inhibition of collagen‐induced platelet aggregation by clopidogrel was seen in patients with the least inhibition of arachidonic acid induced aggregation by aspirin [lower tertile of arachidonic acid‐induced platelet aggregation: 2.8% (95% CI: ?0.8 to 6.3%) reduction in mean collagen‐induced aggregation by clopidogrel; middle tertile: 4.0% (95% CI: 0.4–7.6%); upper tertile 12.6% (95% CI: 4.5–20.8%); P‐value for interaction 0.01]. Conclusions: The greatest platelet inhibitory effect of clopidogrel occurs in patients with the least inhibition of arachidonic acid‐induced platelet aggregation by aspirin. This raises the possibility that the clinical benefits of adding clopidogrel to aspirin may be greatest in patients whose platelets are least inhibited by aspirin. Confirmation in clinical outcome studies may allow these patients to be targeted with antiplatelet drugs that inhibit the ADP receptor, thereby overcoming the problem of laboratory aspirin resistance.     

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin

BACKGROUND: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)-1 and COX-2. Objectives: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. METHODS: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325-mg dose of aspirin. Platelet studies included light transmission aggregometry; P-selectin and integrin alpha(IIb)beta(3) expression, and serum thromboxane B(2) (TxB(2)) levels. Reticulated platelets and platelet COX-2 expression were measured using flow cytometry. RESULTS: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 microg mL(-1) collagen, and postaspirin aggregations to 5 microm and 20 microm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P-selectin and integrin alpha(IIb)beta(3) expression were also higher in the upper tertile both before and after aspirin. Platelet COX-2 expression was detected in 12 +/- 7% (n = 10) of platelets in the upper tertile, and in 7 +/- 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB(2) levels were higher in the upper (5.5 +/- 4 ng mL(-1)) than in the lower tertile (3.2 +/- 2.5 ng mL(-1), P = 0.03), and decreased even further with ex vivo additional COX-1 and COX-2 inhibition. The incidence of aspirin resistance (>or= 70% platelet aggregation to 5 microm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). CONCLUSIONS: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX-1 and COX-2 activity.     

Aspirin resistance detected with aggregometry cannot be explained by cyclooxygenase activity: involvement of other signaling pathway(s) in cardiovascular events of aspirin-treated patients

OBJECTIVES: Although the concept of aspirin resistance is extensively reported in medical literature, its precise mechanisms and clinical outcomes are largely unknown. In this study, we examined individual thromboxane biosynthesis and platelet aggregation in aspirin-treated patients, and whether the results of a platelet aggregation test influenced clinical outcomes. RESULTS: Subjects taking 81 mg of aspirin (n = 50) and controls (n = 38) were evaluated for platelet aggregation and platelet cyclooxygenase-1 (COX-1) activity by measuring collagen-induced thromboxane B2 production. For aggregometry, both light transmission (LT) and laser-light scattering methods were employed to quantitatively evaluate aggregate sizes and numbers. Aspirin treatment resulted in the inhibition of collagen-induced platelet aggregation, particularly the transition from small to large platelet aggregates. Although platelet COX-1 activity seemed to be uniformly inhibited in all patients, platelet aggregation studies showed great inter-individual differences; variation in platelet COX-1 activity only accounted for 6-20% of the individual aggregations. Factor analysis revealed the existence of a common factor (other than platelet COX-1) that explained 48.4% of the variations in platelet aggregation induced by collagen, adenosine diphosphate (ADP), and collagen-related peptide. We then prospectively enrolled 136 aspirin-treated patients in our study, and we found that being in the upper quartile level of LT, or with large aggregate formation induced by collagen, was an independent risk factor for developing cardiovascular events within 12 months [hazard ratio (HR) = 7.98, P = 0.008 for LT; HR = 7.76, P = 0.007 for large aggregates]. On the other hand, the existence of diabetes mellitus was an independent risk factor for overall outcomes (HR 1.30-11.9, P = 0.015-0.033). CONCLUSIONS: Aspirin resistance expressed as unsuppressed platelet COX-1 activity is a rare condition in an out-patient population. Other factor(s) affecting collagen-induced platelet aggregation may influence early outcomes in aspirin-treated patients.     

Persistent platelet activation in patients with type 2 diabetes treated with low doses of aspirin

BACKGROUND: The percentage of diabetic patients who do not benefit from the protective effect of aspirin is larger than in other populations at cardiovascular risk. OBJECTIVE: We compared the ability of aspirin to suppress TxA2 and platelet activation in vivo, in type-2 diabetics vs. high-risk non-diabetic patients. METHODS: Urinary 11-dehydro-TXB2, plasma sCD40 L, and sP-selectin were measured, together with indices of low-grade inflammation, glycemic control, and lipid profile, in 82 patients with type-2 diabetes and 39 without diabetes, treated with low doses of aspirin. RESULTS: Urinary 11-dehydro-TxB2, plasma sCD40L and sP-selectin were significantly higher in diabetics than in controls: [38.9 (27.8-63.3) vs. 28.5 (22.5-43.9) ng mmol(-1) of creatinine, P = 0.02], [1.06 (0.42-3.06) vs. 0.35 (0.22-0.95) ng mL(-1); P = 0.0001], [37.0 (16.8-85.6) vs. 20.0 (11.2-35.6) ng mL(-1), P = 0.0001], respectively. The proportion of individuals with diabetes increased across quartiles of 11-dehydro-TxB2, sCD40L, and sP-selectin, with the highest quartiles of 11-dehydro-TxB2, sCD40L and sP-selectin, including 66%, 93.3%, and 93.3% of individuals with diabetes. Markers of platelet activation positively correlated with indices of glycemic control but not with markers of low-grade inflammation. CONCLUSIONS: Platelet dysfunction associated with insufficient glycemic control, may mediate persistent platelet activation under aspirin treatment.     

Pathways responsible for platelet hypersensitivity in rats with diabetes. I. Streptozocin-induced diabetes

Several pathways are activated when platelets aggregate and undergo the release reaction. We have examined the relative importance of these pathways in the responses to adenosine diphosphate (ADP), thrombin, or collagen of washed platelets from rats with diabetes induced by streptozocin. ADP-induced aggregation was enhanced without the release reaction with platelets from diabetic rats. Collagen-induced aggregation and release, and the adherence of platelets to collagen-coated glass were similar with platelets from diabetic and control rats. Thrombin (1 U/ml) induced more extensive loss of tritium from 3H-arachidonic acid-labeled platelets from diabetic rats than from control rats. Platelet aggregation and the release of 14C-serotonin from prelabeled platelets was greater in response to low concentrations of thrombin (0.04 U/ml). Creatine phosphate-creatine phosphokinase (CP/CPK) and aspirin completely blocked aggregation and partially blocked the release of granule contents from platelets from control and diabetic rats exposed to this low concentration of thrombin. Thus, the enhanced platelet aggregation in response to low concentrations of thrombin was likely mediated in part by released ADP and products formed from arachidonate. In contrast, with a higher concentration of thrombin (0.0625 U/ml), CP/CPK and aspirin did not inhibit the increased sensitivity of diabetic platelets to thrombin-induced aggregation and release; the concentrations of CP/CPK completely blocked aggregation induced by ADP (10 mumol/L), and the aspirin inhibited thromboxane B2 production in response to thrombin (1 U/ml) by 99%. Thus, a thrombin-induced pathway(s) of aggregation and release independent of released ADP and the products of arachidonate metabolism is enhanced in platelets from diabetic rats.     

Near-normal glycaemic control does not correct abnormal platelet reactivity in diabetes mellitus

The effect of improved glycaemic control, effected by 16 weeks continuous subcutaneous insulin infusion (CSII), on platelet aggregation and platelet prostaglandin biosynthesis has been assessed in a group of 11 diabetic patients with painful peripheral neuropathy. Before CSII and compared with results obtained on samples from age- and sex-matched control subjects, there was enhanced reactivity of the platelets from diabetic patients to ADP, collagen and sodium arachidonate (NaAA). There was also increased thromboxane B2 (TXB2) production after platelet stimulation by NaAA. In contrast, collagen-induced thromboxane production by platelets from diabetic patients was significantly less than that of platelets from controls. Treatment by CSII resulted in a statistically significant improvement in glycaemic control and this was maintained for the 16 week period of the study. At 16 weeks and in the presence of near-normal glycaemic control, the enhanced platelet reactivity in response to collagen and NaAA persisted and that to ADP was further increased. Collagen-induced thromboxane production was, however, corrected by CSII.     

Evidence for a role in thrombus stabilization for thromboxane A2 in human platelet deposition on collagen.

The role of thromboxane A2 (TxA2) in platelet deposition onto collagen was studied in flowing whole heparinized human blood in vitro by using a cyclooxygenase inhibitor, aspirin, and a TxA2 receptor antagonist, GR32191B. Previous studies have demonstrated a role for TxA2 in platelet aggregation in citrated plasma, and for platelet deposition in flowing citrated human and rabbit blood, but not in flowing heparinized rabbit blood. In contrast with the literature regarding rabbit blood, aspirin was demonstrated to be effective in reducing platelet accumulation in heparinized human blood, as was GR32191B. The temporal pattern of the platelet deposition, which reached an asymptote with TxA2 inhibition at 2.0 minutes but did not do so without inhibition, suggested that TxA2 plays a role in thrombus stabilization. The reduction in platelet deposition (which included some aggregation) seen with aspirin and GR32191B in the first 1.5 minutes of perfusion indicated that some inhibition of platelet recruitment occurred. Scanning electron microscopy revealed that less fibrin was present in thrombi derived from GR32191B-treated heparinized blood than in thrombi derived from control heparinized blood. No fibrin formation was observed in citrated blood with or without TxA2 inhibition. It is proposed that, in addition to its role as a mediator of platelet recruitment, TxA2 is involved in the stabilization of platelet-platelet interactions in the thrombus, perhaps by enhancing local fibrin formation or binding.     

Action of platelet-activating factor on type 1 diabetic human platelets

Platelets from patients with type 1 diabetes have exhibited more sensitivity to aggregation when compared with platelets from controls without diabetes after challenge with platelet-activating factor (PAF). The production of thromboxane B2 (TxB2) and 12-hydroxyeicosatetraenoic acid (12-HETE) and the release of 5-hydroxytryptamine (5HT) were increased when the platelets were challenged by PAF (5.0 X 10(-6) mol/L and 1.0 X 10(-6) mol/L). The production of TxB2 and 12-HETE and the release of 5HT were related to the irreversible biphasic aggregation profiles observed in the patients with diabetes. Inhibition of thromboxane A2 (TxA2) production by acetylsalicylic acid abolished the secondary wave of aggregation of platelets from patients with diabetes, changing an irreversible aggregation to a reversible one. Inhibition of both TxA2 and 12-HETE production by eicosatetraynoic acid did not contribute further to the inhibition caused by acetylsalicylic acid alone, indicating that 12-HETE was not involved in the secondary wave of aggregation. These data show that the increased aggregation observed in the platelets from the group with diabetes in response to PAF results in part from their higher production of TxA2 and release of 5HT.     

Role of thromboxane A2 as a mediator of platelet-activating-factor-induced aggregation of human platelets

1. At present it is unclear whether platelet-activating-factor (PAF)-induced aggregation is mediated by thromboxane. To obtain further information about this event we have compared the affects of aspirin on platelet aggregation and secretion induced by PAF and collagen. 2. Collagen and PAF induced aggregation and secretion in human platelets in a dose-related manner. 3. Aspirin inhibited the magnitude of both platelet aggregation and secretion induced by PAF and collagen, but the degree of inhibition was much greater for collagen. 4. Aspirin strongly inhibited the aggregation rate of collagen-induced platelet aggregation, but had no measurable effect on the rate of PAF-induced aggregation. 5. Inconsistencies reported in previous studies of the effect of aspirin on PAF-induced platelet aggregation may be explained, in part, by the doses of PAF used and the method of inactivating cyclo-oxygenase (in vitro compared with in vivo). 6. Our results suggest that the initial events of PAF-induced aggregation are independent of thromboxane A2 formation and that thromboxane A2 plays only a minor role in the later phase of PAF-induced aggregation.     

Nitric oxide pathway, Ca2+, and serotonin content in platelets from patients suffering from chronic daily headache

An alteration in serotonin concentration has been found in patients with chronic headache caused by abuse of analgesic substances as well as an up-regulation of 5HT2 platelet receptors, which has been correlated with chronicization of the headache. In a previous study we demonstrated an increase in L-arginine/nitric oxide (NO) pathway activity in platelets from patients affected by migraine with or without aura, particularly during attacks. In the present research we assessed the variations in platelet L-arginine/NO pathway and cyclic guanosine monophosphate (cGMP) levels in 32 patients affected by chronic daily headache (CDH) (8 M, 24 F, age range 34-50 years) both during and between attacks. In these same patients, the platelet aggregation to different collagen concentrations (0.3, 1, 3 micrograms/ml) was determined as well as the intracellular platelet calcium concentration using fluorescence polarization spectrometry. These parameters were compared with those of an age- and sex-matched control group (n = 25; n = 10, n = 15, age range 35-51 years). A reduction found in platelet aggregation response to each collagen concentration used (p < 0.001) was coupled with an increased NO and cGMP production (NO: p < 0.0001; cGMP: p < 0.001). This was accompanied by a significant increase in intracytosolic Ca2+ (p < 0.0001) concentration and a reduced platelet serotonin content compared to those in control individuals (p < 0.0002). Changes in the above platelet parameters were accentuated more in patients with analgesic abuse than in CDH patients with no drug abuse. These findings suggest the occurrence of an activation of cGMP-Ca2+ mediated events in CDH patients with analgesic abuse. This physiologic compensatory mechanism, which intervenes in overcoming the increase in cytosolic Ca2+ levels, is not as efficient at limiting serotonin depletion by platelet dense bodies. A similar depletion in the central serotoninergic pathway can be assumed in the same patients.     

Clinical and preclinical pharmacology of KC-764, a novel antiplatelet agent]

Preclinical pharmacological studies showed that KC-764 was more potent and more selective in inhibiting platelet aggregation than aspirin. The concentration of KC-764 for inhibiting PGI2 production in the aorta was 70 times higher than that for inhibiting TXA2 in platelets. Antiplatelet action of KC-764 was augmented by plasma components. This augmentation by plasma may lead to selective antiplatelet activity. KC-764 has been investigated for platelet function in patients with chronic cerebral infarction. KC-764 at 10, 20 and 40 mg b i d, inhibited platelet aggregation induced by arachidonic acid, collagen, and ADP, and its potency was almost equal to aspirin at 100-330 mg daily. Plasma TXB2 levels were markedly depressed by KC-764 but plasma 6-keto-PGF1 alpha levels were not influenced. On the contrary, aspirin depressed both plasma prostanoids. These findings suggest that KC-764 can overcome the 'aspirin dilemma'.     

The binding of fibrinogen to platelets in diabetes mellitus

Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125I-fibrinogen than platelets from normal controls Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thromboxane A2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from non-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinopathic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from non-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis.     

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’.     

Measurements of the in vivo synthesis of thromboxane and prostacyclin in humans

Our studies on the urinary excretion of 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1 alpha in humans strongly indicate that these metabolites are good indicators of the in vivo synthesis of TxA2 and PGI2. Our finding that physical exercise increases PGI2 synthesis was of particular importance for the design of adequate studies on the effects of various drugs on the in vivo formation of PGI2. The rapid recovery (within 3-4 h) of PGI2 formation found following administration of 1.0 g of aspirin together with the long-lasting inhibition of TxA2, suggests that in the prophylaxis of thromboembolic events an intermittent dosage of 0.5 g of aspirin every third day should be a better alternative than daily low or high doses of aspirin. The increased TxA2 formation found in patients with acute myocardial infarction, deep vein thrombosis and in patients following insertion of synthetic surfaces into the circulation, is very likely a reflection of an increased activation of platelets. The increased TxA2 synthesis may cause further platelet activation, vasoconstriction and activation of the coagulation system. Thus, theoretically, inhibition of TxA2 could diminish platelet activation and reduce the risk of thrombotic complications. It is well known that the interaction between platelets and the vessel wall plays an important role in haemostasis and in the development of thrombosis. On the basis of its biological properties, PGI2 may play a local haemostatic role in the regulation of this interaction. Our studies of myocardial infarction and deep vein thrombosis clearly demonstrate the involvement of PGI2 in those diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of platelet aggregation and thromboxane production by low concentrations of aspirin in vitro

1. The aspirin concentrations previously reported to inhibit platelet aggregation in vitro (40-500 mumol/l) are much greater than those required in vivo in man (5 mumol/l). 2. Human platelet-rich plasma was incubated with buffer or various aspirin concentrations at 37 degrees C for up to 4.5 h. Platelet aggregation and thromboxane generation were measured in response to collagen (0.4-6.3 micrograms/ml) and adenosine 5'-pyrophosphate (0.5-4 mumol/l). 3. The concentration of aspirin needed to inhibit platelet aggregation in response to a critical concentration of aggregating agent (lowest concentration to cause greater than 50% aggregation) was lower than that required for higher concentrations of aggregating agent. 4. With more prolonged incubation times with aspirin, lower concentrations of aspirin inhibited platelet aggregation. 5. Inhibition of platelet aggregation and thromboxane formation by 10 mumol/l aspirin was maximal by 90 min. There was progressive inhibition by 3 mumol/l aspirin during incubation for 270 min. By the end of this time there was also significant inhibition by 1 mumol/l aspirin. 6. The apparent discrepancy between inhibitory aspirin concentrations in vivo and those observed in vitro in previous studies appears to have been resolved by extending the incubation time of platelets with low aspirin concentrations, thus mimicking the conditions in vivo.     

Presystemic acetylation of platelets by aspirin: reduction in rate of drug delivery to improve biochemical selectivity for thromboxane A2.

The utility of aspirin in the treatment of vascular occlusive disease has been ascribed to its blockade of platelet thromboxane A2 (TxA2), a potent inhibitor of vascular smooth muscle contraction and platelet aggregation. Coincident with the inhibition of TxA2 by aspirin at currently used dose regimens is a decrease in formation of prostacyclin (PGI2), an eicosanoid with opposing effects to TxA2 in vivo. The coincident inhibition of PGI2 could potentially limit the therapeutic efficacy of aspirin. This study addressed the issue as to whether a reduction in the rate of drug delivery could enhance the biochemical selectivity of aspirin toward inhibition of TxA2. Intubation studies were performed in parallel groups of healthy volunteers in which a 50-mg aspirin dose was administered as either a bolus or a 5 or 10 mg/hr infusion via a nasogastric tube. A control group received buffer solution. Systemic plasma levels of aspirin, major urinary metabolites of TxA2 and PGI2 and serum thromboxane B2 levels ex vivo were evaluated. Relative to the group receiving bolus aspirin, the slower rates of aspirin delivery increased the effective selectivity of the administered aspirin for TxA2. To investigate further this apparent selectivity, controlled release (CR) dose forms of aspirin were prepared and evaluated in a preliminary dose-ranging study in healthy subjects. The doses studied were 50 and 75 mg CR and a 75-mg aspirin solution. The CR aspirin dose forms decreased the maximal plasma concentration of aspirin relative to a bolus dose. The pharmacodynamic effects of the CR formulations were assessed via measurement of ex vivo serum thromboxane B2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet aggregation profiles in cluster headache

BACKGROUND: Platelets are activated in patients with cluster headache, during both the remission period and the active cycles. OBJECTIVE: To delineate more clearly the origin of platelet activation in cluster headache. Methods.-Platelet aggregation induced by collagen (0.5 micro g/mL and 2 micro g/mL), adenosine diphosphate (10-5 M and 10-6 M), and platelet-activating factor (10-6 M and 10-7 M) was determined by the Born's method in 26 patients with cluster headache and 24 sex- and age-matched controls. RESULTS: The platelets of patients with cluster headache aggregated significantly less to collagen at a concentration of 0.5 micro g/mL compared to those of controls (P =.04). The extent of platelet aggregation obtained with a higher dose of collagen (2 micro g/mL) was in the same range in both groups. Platelet aggregation obtained via adenosine diphosphate at a concentration of 10-6 M was significantly reduced in patients with cluster headache in comparison to controls (P =.002), but no differences were found at a concentration of 10-5 M. In contrast, the platelets of patients with cluster headache aggregated significantly more to platelet-activating factor at both the concentrations of 10-6 M (P =.001) and 10-7 M (P =.00001) compared to those of controls. CONCLUSIONS: This study suggests that platelet aggregation is impaired in patients with cluster headache during the active phase of the disease. We found hypoaggregation in response to low doses of collagen and adenosine diphosphate, and hyperaggregation when platelets were stimulated with platelet-activating factor. Any interpretation of these results can only be speculative. It may be that impairment of platelet aggregation with collagen and adenosine diphosphate may indicate a derangement of nitric oxide function, while the hypersensitivity to platelet-activating factor may be due to fluctuations in its plasma levels.     

Insulin inhibition of platelet-endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets

Summary.  Objectives:  Platelet hyperreactivity contributes to adverse vascular events in diabetes mellitus. It is unclear whether platelet hyperreactivity is due to impaired insulin effects directly on platelets and /or originates from endothelial cells. Here, acute effects of insulin on platelet activation and platelet-endothelial cell interactions were analyzed. Methods and results:  Washed human platelets were treated with insulin alone or in combinations with thrombin, collagen and ADP. Insulin signaling was analyzed by intracellular phosphorylation markers of platelet activation (ERK, p38 MAPK, PKB) or inhibition (VASP), platelet aggregation, intracellular Ca²⁺ levels, and platelet adhesion to collagen coated surfaces and endothelial cells under flow. Insulin up to 100 n m for 5 min did not change phosphorylation status of VASP, p38, ERK or PKB in platelets. Integrin α_IIbβ₃ activation, P-selectin expression, aggregation, and platelet adhesion to collagen coated surfaces and endothelial cells under flow were not altered by insulin. An insulin receptor was detected on endothelial cells but not on human platelets. Insulin treatment decreased platelet adhesion to endothelial cells through insulin stimulation of endothelial NO production and NOS inhibition interfered with this process. Conclusions:  Insulin exerts no direct acute effects on platelet function but inhibits platelet-endothelial interaction by insulin stimulation of endothelial NO production.     

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.