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Whole Blood Flow Cytometry (whole + blood_flow_cytometry)
Selected AbstractsEndothelin attenuates endothelium-dependent platelet inhibition in manACTA PHYSIOLOGICA, Issue 4 2010R. E. Malmström Abstract Aim:, The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. Methods:, In 25 healthy male subjects (25 ± 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l -NMMA. Results:, Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 ,m ADP, fibrinogen binding decreased from 41 ± 4% to 31 ± 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l -NMMA. ET-1 did not affect platelet activity per se, whereas l -NMMA increased platelet P-selectin expression. Both ET-1 and l -NMMA attenuated the acetylcholine-induced inhibition of platelet activity. Conclusions:, Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications. [source] Increased circulating platelet,leukocyte aggregates in myeloproliferative disorders is correlated to previous thrombosis, platelet activation and platelet countEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2001Morten Krogh Jensen Abstract: Platelet,leukocyte adhesion may occur as a consequence of platelet activation and possibly plays a key role in the deposition of activated platelets and fibrin in the thrombotic plug. The aim of the present study was to assess by whole blood flow cytometry the presence of circulating platelet,leukocyte aggregates (PLA) and the platelet,leukocyte response to platelet agonist stimulation (ADP and TRAP) in 50 patients with chronic myeloproliferative disorders (MPD) and 30 controls. PLA were identified as platelet,granulocyte/monocyte aggregates (PGMA), platelet,monocyte aggregates (PMA) and defined as the percentage of leukocytes coexpressing the platelet-specific marker glycoprotein Ib. Compared to controls the mean percentage of PGMA and PMA was increased in unstimulated whole blood from patients with MPD (7.98 vs. 1.76%; p<0.001 and 12.34 vs. 3.2%; p<0.001, respectively). The percentage of PGMA was correlated to the platelet count (r=0.46; p<0.001), percentage of P-selectin (r=0.69; p<0.001) and thrombospondin (r=0.58; p<0.001) positive platelets and platelet expression of GPIV (r=0.33; p=0.02). The mean percentage of PGMA and PMA was significantly increased in ADP-stimulated whole blood of patients (57.14 vs. 47.92%; p=0.009 and 54.91 vs. 45.89%; p<0.001, respectively). Compared to patients without a history of thrombosis, patients having experienced microvascular disturbances or a thrombotic event had a higher mean percentage of PGMA and PMA in non-stimulated whole blood (10.07 vs. 6.34%; p=0.025 and 14.81 vs. 10.48%; p=0.021, respectively) and a higher percentage of PGMA in ADP stimulated whole blood (64.32 vs. 51.50%; p<0.01). These data document an increased frequency of PLA in non-stimulated whole blood in MPD associated with a previous history of thrombosis or microvascular disturbances. [source] Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibitionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2008M. RAZMARA Summary.,Background:,Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non-diabetic subjects. Objective:,We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Methods:,Patients with T2DM and gender/age/body mass index-matched non-diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet-derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet-dependent thrombin generation and plasma clotting time were monitored in recalcified platelet-rich plasma. Results:,Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Conclusions:,Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade. [source] The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activitiesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2008A. L. FRELINGER III Summary.,Background:,Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y12 adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities.Methods and results:,As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte,platelet aggregates. R-138727 reduced monocyte,platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 ± 0.1 ,m). Conclusions:,In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte,platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development. [source] High glucose levels enhance platelet activation: involvement of multiple mechanismsBRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2006Dzana Sudic Summary Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of d -glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l l -glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity). [source] Circulating CD10,/CD16low neutrophils provide a quantitative index of active bone marrow neutrophil releaseBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2005Y. Orr Summary Circulating neutrophil phenotype and function are altered during neutrophilia associated with acute inflammatory states, however, the contribution of bone marrow neutrophil release to these changes has been difficult to quantify in humans. Accelerated release of neutrophils, with potentially distinct attributes, from the bone marrow and their dilution within the circulating pool may produce these apparent changes. Unfortunately selective analysis of these newly emergent neutrophils is difficult given their morphologic similarity to those already in the circulation and the coincident effect of soluble inflammatory mediators on circulating neutrophil phenotype and function. Using whole blood flow cytometry and cardiac surgery as an inflammatory stimulus, we demonstrate the emergence of a unique subpopulation of circulating neutrophils characterised as CD10,/CD16low, indicative of active bone marrow neutrophil release peri-operatively. CD10,/CD16low neutrophils emerge at the same operative stages as band forms and a left shift, yet represent over 40% of circulating neutrophils postoperatively, and generate a greater stimulus-induced [Ca2+]i flux than their CD10+ counterparts. We conclude that CD10,/CD16low neutrophils represent a significant proportion of the circulating pool after cardiac surgery and that bone marrow release, a major contributor to neutrophilia, influences the phenotype and functional activity of circulating neutrophils following this acute inflammatory stimulus. [source] Thromboembolic events after carotid endarterectomy are not prevented by aspirin, but are due to the platelet response to adenosine 5,-diphosphateBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 4 2000P. D. Hayes Background: Aspirin therapy fails to prevent a number of postprocedural thrombotic events, yet it still remains the standard antiplatelet regimen in most vascular surgical centres. After carotid endarterectomy (CEA), thrombosis of the endarterectomized vessel is preceded by increasing numbers of microemboli that can be detected with transcranial Doppler (TCD). The number and rate of emboli is highly predictive of thrombotic stroke. It was hypothesized that a preoperative test of platelet function might identify the mechanism(s) underlying post-CEA thrombosis. Methods: Blood was taken from 120 patients using a standardized phlebotomy technique. Platelet fibrinogen binding was measured by whole blood flow cytometry, in unstimulated samples, and in response to adenosine 5,-diphosphate (ADP) (10,5,10,7 mol l,1) and thrombin (0·02,0·16 units ml,1). Platelet aggregation was measured using ADP (4,20 × 10,7 mol l,1). The ability of aspirin to inhibit platelets was assessed by the aggregation induced by arachidonic acid. For the first 3 h after operation, the number of emboli occurring was quantified using TCD. Results: Of the 120 patients studied, 110 were monitored by TCD. These were divided into patients with more than 25 postoperative emboli (n = 22) and those with fewer than 25 emboli (n = 88). The degree of platelet inhibition induced by aspirin was not significantly different between the two groups (P = 0·89). However, platelets from the group with high rates of embolization bound 58 per cent more fibrinogen on flow cytometry in response to stimulation with a physiological dose of ADP (10,7 mol l,1) (P = 0·006). Aggregation of platelets from this group was also increased in response to ADP (35 per cent) relative to the group with few emboli (P = 0·001). ADP also induced more rapid aggregation in the patients with more than 25 emboli (P = 0·04). There was no difference in the activity of resting platelets (P = 0·4) or platelets stimulated by thrombin (P = 0·43), between the two groups of patients. Conclusion: These data suggest that it is the platelet response to ADP which is important in arterial thrombotic complications rather than products of the cyclo-oxygenase pathway. This observation could have significant therapeutic implications for other vascular or interventional procedures in which the endothelium is disrupted. © 2000 British Journal of Surgery Society Ltd [source] | ||||||||||