Reduced sensitivity of platelets from type 2 diabetic patients to acetylsalicylic acid (aspirin)-its relation to metabolic control

Aspirin (acetylsalicylic acid, ASA), which is recommended for primary and secondary prevention in diabetes mellitus (DM), has been shown to have a lower antiplatelet activity in diabetic patients. We conducted a crossover designed observational study to evaluate whether there is an association between the parameters relevant to metabolic control of diabetes and platelet sensitivity to aspirin in type 2 diabetic patients. Platelets' ability to adhere and aggregate was monitored with the use of platelet function analyser (PFA-100 collagen/epinephrine closure time, CT(CEPI) or collagen/ADP closure time, CT(CADP)), classical turbidimetric aggregometry and whole blood electrical aggregometry (WBEA), using collagen (WBEA(coll)), ADP (WBEA(ADP)) and arachidonic acid (WBEA(AA)) as platelet agonists, in 48 control healthy volunteers (mean age+/-S.D., 49+/-9 years) and 31 type 2 DM patients (50+/-9 years; HbA(1c) 9.4+/-1.6%). In majority of control subjects (69%) and minority of diabetic patients (29%, p=0.0006), the use of 150 mg aspirin daily for 1 week significantly reduced platelet adhesiveness and reactivity (by 14.1% in diabetes vs. 78.6% in control, p(np)=0.0035, as expressed by the relative changes in CT(CEPI)). Aspirin reduced WBEA(coll) and WBEA(AA) to a lesser extent in diabetic patients (by 2.1% vs. 8.3% in controls, p(np)=0.0397, and by 97.3+/-12.8% vs. 100% in controls, p(np)=0.0383, respectively), which corresponded to ASA-mediated decreased aggregation in platelet-rich plasma (PRP, r(S)=0.45 and r(S)=0.78 for collagen- or arachidonate-agonized platelets, p<0.01 or lower). The maximal inhibition of platelet aggregation was lower and IC(50) higher in diabetic compared to control subjects, both in the presence of arachidonic acid (71% vs. 39%, p(np)0.0001; 0.5 microg/ml vs. 1.3 microg/ml, p<0.0001) and collagen (52% vs. 35%, p<0.0004; 1.6 microg/ml vs. 2.1 microg/ml, p<0.01). The reduced response of platelets from diabetic subjects to aspirin was associated with a higher level of HbA(1c), lower concentration of HDL-cholesterol and a higher total cholesterol concentration. Overall, there is evidence that reduced platelets response to aspirin may occur more often in diabetic patients. Poor metabolic control may play a role in the reduced platelet sensitivity to aspirin in DM patients. Thus, our findings strongly support the requirements for an excellent near-normal metabolic control and may suggest a need for alternative ASA dosing schedules in DM patients.     

Mechanism of ethanol-induced aggregation in whole blood.

Effects of ethanol on blood clotting and platelet aggregation have been reported in many models, but its in vitro actions in whole blood, impedance aggregometry have not been reported. We investigated the effect of ethanol in vitro in whole blood and platelet rich plasma of humans and rats, as measured by impedance aggregometry. Ethanol (34 to 170 mM) induced concentration-dependent aggregation in whole blood but not platelet rich plasma. In further studies in rats, aggregation was inhibited by pretreatment of whole blood with the prostacyclin analog iloprost or the enzyme apyrase, which degrades ADP to AMP. Levels of ethanol which produced aggregation in whole blood were also associated with concentration-dependent hemolysis. Based on the requirement for whole blood for ethanol-induced aggregation, the inhibitory effect of apyrase and our observation of hemolysis, and previous studies which have demonstrated the potential contribution of ADP from lysed red blood cells to platelet aggregation, we conclude that ethanol-induced aggregation in whole blood is mediated by erythrocyte lysis and the ADP released from these cells.     

Increased platelet aggregability and prostacyclin biosynthesis induced by intense physical exercise

Changes in platelet aggregability during maximal bicycle ergometry were studied in healthy untrained subjects. Ex vivo platelet aggregation in response to ADP and collagen was measured in whole blood by impedance aggregometry or by direct electronic counting in an Ultra-Flo 100 platelet counter. This last method revealed that the platelet aggregation induced by low concentration of ADP (0.5 - 1.0 microM) was significantly enhanced during exercise. The plasma level of beta-thromboglobulin and the urinary excretion of 2,3--dinor-6-keto prostaglandin F1 alpha were also increased. These data indicate that an intense physical exercise enhances the aggregability of human platelets and induces a compensatory increase in prostacyclin biosynthesis.     

Is platelet aggregation a more important contributor than platelet adhesion to the overall platelet-related primary haemostasis measured by PFA-100?

Background: Platelet-related primary haemostasis (PRPH), measured in PFA-100™ as a closure time (CT), reflects platelets' combined ability to adhere and aggregate under higher shear stress. The inputs of platelet aggregation and platelet adhesion into the real values of CT remain unknown, and this poor discrimination results in the complexity of the PFA-100™ measurement. Objective: To estimate the particular contributions of two physiological phenomena, platelet aggregation and adhesion, and the importance of various membrane receptors underlying platelets' capability of the plug formation in PFA-100™ cartridges. Materials and methods: Effects of various blockers antagonizing ligands binding to platelet surface membrane receptors (antagonists of GPIIb-IIIa complex, collagen receptors and purinoreceptors), and aurintricarboxylic acid (ATA), the antagonist of GPIb–von Willebrand factor (vWF) interaction, were monitored in 47 healthy donors with the use of PFA-100™ and whole blood electrical aggregometry (WBEA). Results: PFA-100™ collagen/ADP CT was the most sensitive in probing the effect of platelet membrane receptor antagonists acting via the blockade of GPIIb-IIIa complex and those antagonizing GPIb–vWF interaction (GR144053F, Integrilin, ATA), whereas the other blockers, acting on collagen receptors or purinoreceptors, remained much less efficient. For the examined GPIIb-IIIa and GPIb antagonists, the overall variability in WBEA explained a very significant part (30–60%) of the overall variability in PFA-100™ CT. Conclusions: GPIIb-IIIa-mediated platelet aggregation and von Willebrand factor interactions with GPIb and/or GPIIb-IIIa seem to be the major determinants of PFA-100™ CT. On the contrary, other platelet receptors participating in platelet aggregation and/or platelet adhesion are of secondary importance and minor significance in blood flow at higher shear stress monitored in PFA-100™.     

Influence of hypercapnia and hypoxia on rabbit platelet aggregation

The influence of changes in pCO₂, pH and pO₂ on the aggregation of rabbit blood platelets was studied , with emphasis on hypercapnia, acidocis and hypoxia. Hypercapnia combined with acidosis caused a reduction in rabbit platelet aggregation, as induced by collagen, thrombin and ADP; the effect being most pronounced with collagen and smallest with ADP. Hypoxia reduced thrombin induced platelet aggregation, but had no effect on ADP and collagen induced aggregation. Synergistic activation of rabbit platelets, as induced by the addition of serotonin to platelet rich plasma together with collagen or ADP, seemed to be equally sensitive to changes in pCO₂ and pH as activation by the individual agents, and insensitive to changes in pO₂.     

The influence of sample age on collagen-induced platelet aggregation in whole blood

Whole blood electrical aggregometry (WBEA) has become an accepted method to gain quick information on platelet disorders. Compared to the optical method WBEA is closer to physiology and less complicated, but on the other hand more difficult to standardize. Different approaches have been attempted in the past to improve the reliability and practicability of this technique. The influence of sample age has not been defined so far. In a first step a mathematical modelling program was established, which is able to characterize the aggregation curves obtained after collagen stimulation. In a mathematical analysis various characteristics of the curve function were calculated and their sensitivity for aging investigated. Regression was performed for each characteristic, and correction factors defined.

Our results indicate, that whole blood specimen for collagen induced aggregation can be used without correction factor up to 30 minutes. Data obtained with an age exeeding half an hour have to be corrected following a quadratic regression.     

Enhanced platelet aggregation with TRAP-6 and collagen in platelet aggregometry in patients with venous thromboembolism

The role of platelet hyperaggregability as a possible risk factor for venous thromboembolism is not well defined. Some authors described enhanced maximal platelet aggregation in platelet aggregometry as a contributing factor for arterial and venous thrombosis. This syndrome has been termed “sticky-platelet syndrome” (SPS). The diagnosis of SPS is based on the demonstration of platelet hyperaggregability in aggregometry after stimulation with epinephrine (EPI) and/or adenosine diphosphate (ADP).

We investigated platelet hyperaggregability in platelet-rich plasma (PRP) of patients (n=34) with unexplained venous thromboembolism in comparison to healthy individuals (n=53). For analysis, platelet aggregometry was performed and the influence of epinephrine, adenosine diphosphate, collagen (Coll) and thrombin receptor-activated peptide (TRAP-6) as agonist were determined. Compared to the control group, patients with venous thromboembolism showed an enhanced maximal platelet aggregation with low concentrations of TRAP-6 (2 μM) and collagen (0.05 μM). In contrast, we could not detect an increased platelet aggregation with EPI or ADP.

Our results indicate that platelet hyperaggregability may represent an independent risk factor in patients with otherwise unexplained venous thromboembolism. In our study, low concentrations of TRAP-6 and collagen are superior to EPI and ADP to define platelet hyperreactivity in platelet aggregometry.     

Enhancement of collagen-induced aggregation of platelets in whole blood

In order to elucidate the features of platelet aggregation in whole blood, studies were carried out on human blood. The platelet aggregation reaction was monitored by counting the residual free platelet number with an electronic particle counter (Coulter). Platelets in citrated whole blood were aggregated by a very small amount of collagen which did not aggregate platelets in citrated plasma. Such enhancement was not observed if ADP or epinephrine was used. By addition of isolated erythrocytes to platelet rich plasma, enhancement of the platelet response to collagen was obtained. The erythrocyte membrane stabilizer, Dilazep, abolished the enhancement effect of erythrocytes. Those results indicated that erythrocytes enhanced the platelet response to collagen. Although participation of ADP from the erythrocytes in the enhancement was suggested, the results of ADP determinations on suspensions of erythrocytes indicated that other factors of the erythrocytes might be involved in the enhancement.     

A new platelet function test

A method for screening EDTA(K3) blood samples for platelet function is described. The general availability of whole blood platelet counters in the clinical laboratory suggested their use to measure platelet aggregation. To simplify the collection and storage of blood samples, a method utilizing EDTA collected blood is described. Addition of calcium and citrate restores platelet function to blood samples even when stored at room temperature for several hours. Thus, the blood sample used to assay RBC, WBC and platelet parameters may also be employed in platelet aggregation. A comparison of this method with aggregometry on 120 subjects indicated a similar response to arachidonate, collagen, ristocetin and ADP.     

Effect of diet-induced hyperlipidemia on in vitro-function of rabbit platelets

Hyperlipidemia was induced in rabbits by feeding them a diet enriched with egg yolk for a minimum period of sixteen weeks. Platelets isolated from the blood of these rabbits and resuspended in Tyrode-albumin solution showed a significant increase in collagen- or thrombin-induced aggregation and serotonin release but not in aggregation induced by ADP. Collagen-induced platelet factor 3-availability was also greater with platelets from hyperlipidemic rabbits compared with platelets from control rabbits. There were no significant differences between the diet and control groups in platelet aggregation induced by ADP, collagen, or thrombin or serotonin release induced by collagen or thrombin in citrated platelet-rich plasma. With washed platelets there was no correlation between the percentage increase in aggregation induced by collagen or thrombin and the plasma cholesterol or triglyceride levels in hyperlipidemic or control rabbits. When washed normal rabbit platelets were resuspended in citrated plasma from a hyperlipidemic rabbit their response to aggregating or release-inducing stimuli was not significantly different from that observed when platelets from normal rabbits were resuspended in normal plasma. When washed normal platelets were incubated for 30 min in hyperlipidemic plasma at 37°C, isolated from the plasma and resuspended in Tyrode-albumin solution, their response to aggregating or release-inducing stimuli was not significantly different from that observed with normal platelets incubated in normolipidemic plasma. The findings indicate that diet-induced hyperlipidemia in rabbits may induce an intrinsic functional alteration of the platelets which cannot be readily reproduced by short-term in vitro exposure of platelets to hyperlipidemic plasma.     

Alloxan-induced hyperglycemia in rabbits and the response of platelets to aggregating agents in vitro and to exposed subendothelium in vivo

Many patients with diabetes mellitus show increased platelet aggregation and prostaglandin synthesis in response to physiological agents such as ADP and collagen when their platelets are tested in platelet-rich plasma or washed platelet suspensions. However, the relationship between increased platelet aggregation in vitro and increased thrombosis in vivo is difficult to establish with certainty. We have developed an in vivo model system in rabbits which tests the response of platelets in circulating native blood to an arterial vessel wall with limited damage such as might occur in arteries of patients with diabetes mellitus. We have used this model system to investigate whether 5 to 9 weeks of alloxan-induced hyperglycemia increases platelet adhesion and aggregation on a damaged vessel wall in vivo as well as platelet aggregation in vitro. Our results show that rabbit platelet function is not affected by extreme hyperglycemia and suggest that alloxan-induced diabetes in the rabbit may not be a good model for human diabetes mellitus.     

Influence of Platelet Aggregate Formation in Blood Samples on Light Transmission Aggregometry Results

Background: Light transmission aggregometry is a standard method used to evaluate platelet function. However, in clinical settings, light transmission aggregometry results sometimes fail to reflect actual platelet hyperactivity. In patients with suspected platelet hyperactivity such as thrombosis, platelet aggregates are frequently detected in citrated blood samples using a scattergram of a hematology analyzer. This study aimed to evaluate the effects of platelet aggregate formation on light transmission aggregometry results. Methods: We used 19 citrated blood samples in which platelet aggregate formation was intentionally induced by a hematology analysis process. Employing fully automated light transmission aggregometry and agonists including adenosine diphosphate or collagen, light transmission aggregometry maximum aggregation percentage, platelet count, and mean platelet volume of platelet-rich plasma before and after platelet aggregate formation were evaluated. Results: Light transmission aggregometry maximum aggregation percentage with adenosine diphosphate or collagen was significantly lower in the samples after than before platelet aggregate formation. Platelet count and mean platelet volume were both decreased by platelet aggregate formation (P < .01), suggesting that maximum aggregation percentage reduction was caused by the decrease in activated large platelets in the platelet-rich plasma. Conclusion: This study clarified that platelet aggregate formation in blood samples interfered with an accurate assessment of platelet hyperactivity. To ensure reliability of light transmission aggregometry results, we must confirm that platelet aggregates have not formed in the sample, especially in those of patients with platelet hyperactivity.     

Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood

Several methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p < 0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.     

Pharmacology of a potent, new antithrombotic agent, 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844)

The effects of 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844) on platelet function and experimental thrombosis were evaluated in a series of in vitro, ex vivo and in vivo experiments. The compound inhibited platelet aggregation in vitro in platelet rich plasma obtained from humans, rats and rabbits with EC50s of less than 1 microgram/ml when aggregation was induced by ADP, collagen or thrombin. Supra-additive interaction against ADP aggregation was also observed when BMY-20844 was combined with prostacyclin. BMY-20844 was orally active with an ex vivo ED50 in the rat of 3.2 mg/kg vs ADP. Significant antithrombotic activity was observed in two animal models (laser induced thrombosis in the microcirculation of the rabbit ear and coronary artery thrombosis in the dog). Inhibitions of 52% at 3 mg/kg p.o. in the laser model and 100% at 1 mg/kg i.d. in the coronary artery thrombosis model were obtained. Modest inotropic and hemodynamic effects were observed in ferrets and dogs. BMY-20844 was found to be a potent, specific inhibitor of platelet low Km cyclic AMP phosphodiesterase.     

Effect of iron therapy on the whole blood platelet aggregation in infants with iron deficiency anemia

This study was performed to investigate the platelet aggregation alterations in whole blood samples of infants with iron deficiency anemia. Platelet aggregation induced by various concentrations of adenosine diphosphate (ADP) and collagen was studied with impedance aggregometry in 25 patients before and after oral iron therapy and in 12 children of the control group. The posttreatment mean maximum aggregation values were significantly higher (p<0.01) and the posttreatment mean aggregation times were significantly lower (p<0.01) in the study group at all concentrations of ADP and collagen. The aggregation time and maximum aggregation values revealed no significant difference except for the maximum aggregation value at 5 microM ADP (p<0.05) between the study group after therapy and the control group. The differences between the pretreatment and posttreatment mean platelet counts and mean platelet volume values in the study group were statistically significant (p<0.01), whereas those values in the study group after therapy and in the control group were not significantly different. We conclude that iron deficiency anemia in infants, even without clinically meaningful platelet abnormality, may cause dysfunction of the ex vivo whole blood platelet aggregation, and can be reversed by iron therapy. Further studies should be carried out at the enzymatic level to determine whether this platelet aggregation dysfunction in iron deficiency anemia is due to a deficiency in the activation of iron-containing enzymes.     

Early detection of preeclampsia by determination of platelet aggregability

Preeclampsia is still a leading cause of maternal and fetal morbidity and mortality. There is evidence for the involvement of platelets. Therefore, we investigated the suitability of corrected whole blood impedance aggregometry as an early predictor of preeclampsia in 71 consecutive, high-risk pregnancies. According to the occurrence of preeclampsia, defined postpartum by an independent investigator, and the stage of pregnancy (early and late, cutoff: 25 weeks of gestation), four study groups were defined. Platelet aggregation data were corrected for the influence of hematocrit and platelet count by a special purpose software package. Women developing preeclampsia showed significantly higher platelet aggregation response compared to controls in early and late pregnancy. In early pregnancy, all women developing preeclampsia had aggregation responses to collagen higher than the highest responses among the controls. Hence, this test had a 100% positive predictive value of subsequent preeclampsia. Despite being significantly increased, platelet aggregability was of minor predictive value in late pregnancy. We conclude that preeclampsia is accompanied by exaggerated platelet aggregability, particularly perceptible early in the course of pregnancy. We propose collagen-induced whole blood platelet aggregation with correction for the influence of hematocrit and platelet count for early detection of preeclampsia.     

Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment

The level of platelet aggregation, measured with light transmission aggregometry (LTA) in platelet rich plasma (PRP), has been shown to predict outcomes after percutaneous coronary intervention (PCI). However, measuring parameters of platelet function with LTA is time consuming and weakly standardized. Thus, a fast and standardized method to assess platelet function after clopidogrel treatment would be of great value for clinical practice. A new method, multiple electrode platelet aggregometry (MEA), to rapidly measure platelet aggregation in whole blood has recently been developed. The aim of this study was to assess parameters of platelet function with MEA and LTA before and after administration of 600 mg clopidogrel. Blood samples from 149 patients scheduled for coronary angiography were taken after clopidogrel treatment; in addition, in 60 of the patients samples were available before clopidogrel treatment. ADP-induced platelet aggregation was measured with LTA and simultaneously in whole blood with MEA on the Multiplate analyzer. Platelet aggregation measured with MEA decreased significantly after clopidogrel treatment (P < 0.0001). ADP-induced platelet aggregation assessed with MEA and LTA correlated significantly (Spearman rank correlation coefficient = 0.71; P < 0.0001). The results of MEA, a fast and standardized method to assess the platelet response to ADP prior to and after clopidogrel treatment, correlate well with LTA.     

Exercise-induced changes in platelet aggregation; a comparison of whole blood and platelet rich plasma techniques

Studies have been performed to assess the effect of exercise on spontaneous platelet aggregation in shaken whole blood, and on agonist-induced platelet aggregation in whole blood and platelet rich plasma (PRP). Spontaneous platelet aggregation in shaken whole blood was increased following exercise compared to pre-exercise values. The increase in spontaneous aggregation after exercise correlated inversely with the increase in white cell count in whole blood. Platelet sensitivity in whole blood to adrenaline, collagen and adenosine diphosphate (ADP) was increased following exercise. Changes in platelet sensitivity to adrenaline following exercise correlated with increases in plasma noradrenaline levels but not with changes in blood cell counts. In PRP, platelet sensitivity to ADP and to collagen was increased following exercise when the pre and post-exercise PRP platelet counts were not corrected to allow for the increase in platelet count which occurred with exercise. When the PRP platelet counts were corrected, no changes in platelet sensitivity to any agonist after exercise were observed.     

Dipyridamole inhibits platelet aggregation in whole blood

Dipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine-5'-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p less than 0.001). A statistically significant inhibition of both collagen (p less than 0.0025) and ADP-induced (p less than 0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37 degrees C with dipyridamole (3.9 microM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood. Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.     

Dose-response aggregometry--contribution to the precise platelet function evaluation.

A method for the determination of blood platelet aggregability as the function of ADP concentration is described. Individual platelet aggregability was characterised by means of parameters of the sigmoidal relationship between the dose of the aggregating reagent and the aggregation curve amplitude. The method was applied to establish ADP-induced aggregability of various mammalian species. The aggregation curves of rabbit and rat platelets reached significantly lower maximum amplitudes than those of human and dog platelets. The eman concentration of ADP was significantly higher in dog platelets and the slope of the dose-response curve was significantly steeper in rat platelets compared to the other species studied. Some of the causes of individual intra-species ADP-induced aggregation variability revealed by means of dose-response aggregometry are discussed.