Do cardiac glycosides affect platelet function? A flow cytometric study in healthy volunteers.

OBJECTIVE: Cardiac glycosides exert their inotropic effect by increasing intracellular calcium. Increased intracellular calcium is a key event in platelet aggregation. In aggregometer studies, digitalis has been found to augment platelet agonist responses. A prothrombotic effect of digitalis might be concealed since heart failure and atrial fibrillation per se predispose to thromboembolism. The present study investigates the effects of digitoxin on platelet function in healthy volunteers. METHODS: Twenty healthy, non-smoking volunteers were randomised to receive digitoxin ( n = 10, 0.6 mg day 1, 0.4 mg day 2, then 0.1 mg daily) or placebo ( n = 10) for 10 days. Platelet function was then analysed ex vivo using three-colour whole-blood-flow cytometry, both in non-stimulated mode and after agonist stimulation with 0.1 micromol/l adenosine diphosphate (ADP), 10 micromol/l ADP and 5.0 micromol/l epinephrine (final concentrations). Expression of activated fibrinogen receptor, von Willebrand's factor receptor and P-selectin, formation of platelet-platelet and platelet-leukocyte aggregates and particle size were examined. RESULTS: No significant difference between the placebo and the digitoxin group (digitoxin levels 17-42 nmol/l) was found, neither on a global level nor for any isolated parameter. CONCLUSIONS: Theory and in vitro data suggest that digitoxin treatment could activate platelets. No evidence for this was found in healthy volunteers. This observation is strengthened by the unequivocal results for all parameters measured. However, thrombosis-prone patients with heart failure and/or atrial fibrillation may respond differently to digitalis therapy.

Evaluation of circulating platelet-leukocyte conjugates: a sensitive flow cytometric assay well suited for clinical studies.

Circulating platelet-leukocyte conjugates are found in patients with a variety of diseases. We have developed a flow cytometry-based assay for monitoring the presence of such conjugates. By preloading the sampling tubes with 1/8 volume of 4% paraformaldehyde, basal levels of platelet-leukocyte conjugates was stable for at least 48 h after blood sampling. The leukocytes were discriminated from the other cells in fixed whole blood by binding of anti-CD45, and no lysing, washing, or centrifugation steps were required. In whole blood from healthy individuals, platelets were found adherent to 3.2% of the lymphocytes, 4.1% of the monocytes and to 2.5% of the granulocytes. The corresponding values for mean number of platelets per leukocyte were 1.3 x 10(-2), 2.5 x 10(-2) and 1.6 x 10(-2), respectively. Upon in vitro agonist (ADP, TRAP) stimulation, the platelet adhesion to monocyte and granulocytes increased several-fold. Microscopy verified the flow cytometry results and revealed that platelet adhered to the leukocytes as platelet-platelet aggregates and not as single platelets as the agonist concentration increased. At high concentrations of TRAP, the strong agonist, large multiconjugates with several granulocytes per platelet-platelet aggregate evolved. Being an assay well suited for clinical studies, the recommended approach may be a valuable tool in establishing the clinical significance of circulating platelet-leukocyte conjugates.

Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies.

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1 x 10(3) (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet-platelet aggregates as well as for aggregate size.

Effects of short-term nitrogen monoxide inhalation on leukocyte adhesion molecules, generation of reactive oxygen species, and cytokine release in human blood.

Increased nitrogen monoxide (NO) concentrations change leukocyte function under a multitude of experimental conditions. NO inhalation is an experimental treatment for lung failure and exposes leukocytes to increased NO concentrations during passage through the lungs. To investigate whether short-term NO inhalation induces lasting changes in the function of circulating human leukocytes, venous blood samples were drawn from eight healthy male volunteers before and at the end of a 35-min period of breathing 40 ppm NO in 30% O(2). The leukocytes in the samples were subsequently analyzed for NO-induced changes in expression of cell surface molecules, generation of reactive oxygen species (ROS), and cytokine production by flow cytometry and ELISA techniques. The results were (1) NO inhalation changed neither the baseline nor the Escherichia coli lipopolysaccharide (LPS)-induced expression of the cell adhesion molecules CD11a, CD11b, CD11c, and CD62L (l-selectin) on neutrophilic granulocytes (PMN) or monocytes (Mo). The expression of CD14 and HLA-DR was also unchanged. (2) The generation of ROS in response to activation with phorbol myristate acetate increased in PMN after NO inhalation; an increase in Mo did not reach significance. (3) Baseline and LPS-stimulated production of IL-1beta decreased after NO inhalation, while the LPS-stimulated production of TNF-alpha increased. No changes in IL-6 production were detected.

Effect of calcium on reactive oxygen species in isolated rat cardiomyocytes during hypoxia and reoxygenation.

It has been suggested that calcium (Ca(2+)) overload and oxidative stress damage the myocardium during ischemia and reperfusion. We investigated the possible effect of varying extracellular Ca(2+)and total cell Ca(2+)on reactive oxygen species (ROS) levels in resting adult rat cardiomyocytes. Cardiomyocytes were isolated by trypsin/collagenase digestion and exposed to 1 h of hypoxia (H) (95% N(2)/5% CO(2), no glucose) and 2 h of reoxygenation (R) (95% air/5% CO(2), glucose 5.5 m M) in suspension. Cell Ca(2+)was measured by uptake of(45)Ca(2+). ROS was measured by flow cytometry of ethidium's red fluorescence formed by oxidation of dihydroethidium mostly by superoxide anion. Cell viability decreased during H and R, expressed as uptake of trypan blue, loss of rod shape morphology and release of lactate dehydrogenase. Rapidly exchangeable cell Ca(2+)was closely correlated with extracellular Ca(2+)concentration. Cell Ca(2+)was unchanged during H but increased three to four times after R. This increase was attenuated by adding 3,4-dichlorobenzamil, 10 microm at R, and amplified by adding ouabain 1 m M (from start), respectively. Levels of ROS in hypoxic cells were unchanged or slightly reduced at the end of H and increased significantly by 20% compared to control after R. Levels of ROS were significantly decreased by lowering total extracellular Ca(2+)from 1 m M to 0.1 m M or by decreasing free extracellular Ca(2+)with EGTA 0.9 m M at the onset of R. Keeping extracellular Ca(2+)constant, ROS levels were neither affected by attenuating the increase in cell Ca(2+)by DCB nor by amplifying the increase in cell Ca(2+)by ouabain. In conclusion, ROS (superoxide anion) levels increase rapidly after reoxygenation, are correlated with extracellular-free Ca(2+)and are reduced by lowering extracellular-free Ca(2+). Levels of ROS are apparently not consistently correlated with total cell Ca(2+).

Apheresis-induced platelet activation:comparison of three types of cell separators.

BACKGROUND: Platelet-harvesting technology differs in various cell separators. Alteration in shear stress and biocompatibility of surfaces may give variable platelet activation and thereby affect the quality of the component. STUDY DESIGN AND METHODS: Four groups (n = 10) of single-needle apheresis procedures using three cell separators, were compared: 1) Spectra LRS, 90-minute harvesting time; 2) MCS+, 90-minute harvest; 3) Amicus, 90-minute; and 4) Amicus, 45-minute. Whole-blood samples were collected from the donors as were samples from the final components at intervals during the first 4 hours after cessation of the apheresis. Platelet activation status and platelet activation capacity after agonist stimulation were assessed by flow cytometry. RESULTS: No activated platelets were found in preapheresis and postapheresis samples from the donors. The platelets in the components from the Amicus (90-min) were significantly more activated than those in the other groups of components: that is, there was increased size of platelet aggregates, increased fraction of microparticles, increased degranulation, increased fibrinogen receptor activation, and decreased von Willebrand factor receptor expression. Moreover, the response of these platelets to agonist stimulation was reduced for all activation variables. CONCLUSIONS: After 90 minutes' processing time, platelets obtained with the Amicus cell separator were significantly more activated than platelets harvested with the Spectra and the MCS+.

Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release.

To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear.

Adhesion of leukocytes to growing arterial thrombi.

Since the role of leukocytes found present in thrombi and haemostatic plugs is not clearly understood. we have investigated the interaction between leukocytes and growing thrombi in a human ex vivo model of arterial thrombogenesis. At a wall shear rate characteristic of moderately stenosed arteries (2600 s(-1)), granulocytes selectively accumulated at the luminal surface of platelet thrombi. The leukocyte adhesion seemed independent of fibrin formation and was clearly correlated to thrombus growth and platelet activation. In contrast, flow cytometry revealed that the expression of adhesion molecules (CD11a, CD11b, CD11, CD3, CD14, CD62L, HLA-DR and binding of fibrinogen) on the surface of circulating leukocytes passing the thrombi was, on short term conditions (15 min), independent of thrombus growth. The adhered granulocytes probably play a pivotal role in limiting the size of the evolving thrombi, as suggested by our electron micrographs of the arterial thrombi showing lysed and phagocytosed platelets. Thus, granulocytes might play an active role in the acute/semiacute phase of local thromboregulation.

Platelet activation in flowing blood passing growing arterial thrombi.

We investigated the combined effect of wall shear rate and immobilized collagen on platelet activation in flowing nonanticoagulated human blood. By combining an ex vivo model of thrombogenesis with flow cytometry, we showed that activated platelets can be detected in the bloodstream passing growing thrombi at a wall shear rate characteristic of moderately stenosed arteries (2600 s-1). The activation of the circulating platelets was clearly correlated with thrombus growth. Different antibodies against platelet activation-dependent surface markers had distinct sensitivity to the thrombotic process. alpha-Granule release detected by surface expression of CD62P seemed to be the most sensitive marker, as judged by both mean fluorescence intensity and fraction of platelets activated. The conformational change in glycoprotein IIb-IIIa, as detected by PAC-1, also seemed to be a sensitive marker and preceded binding of fibrinogen to activated glycoprotein IIb-IIIa, as detected by anti-fibrinogen. Large thrombi also elicited lysosome exocytosis, detected by surface expression of CD63. Finally, we observed a small decrease of glycoprotein Ib-IX expression, as detected by anti-CD42a. Thus, our study provides further information on the dynamics of platelet activation in relation to thrombus growth at arterial shear conditions in flowing nonanticoagulated human blood.