Evaluation of platelet surface glycoproteins in patients with Glanzmann thrombasthenia: Association with bleeding symptoms.

BACKGROUND & OBJECTIVES: Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbß3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. METHODS: Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. RESULTS: Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). INTERPRETATION & CONCLUSIONS: Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

Combined accurate platelet enumeration and reticulated platelet determination by flow cytometry.

BACKGROUND: Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. GOAL: To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method. METHODS: TO concentration, incubation, and fixation method were determined to be 10% of stock concentration, 30 min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control tube to set the limit of TO staining. RESULTS: Normal range (n = 51) was 9.9 ± 3.1%. Analysis of 40 patients with immune-thrombocytopenia-purpura (ITP) showed a RP range from 4.3% to 81.2%. Platelet enumeration was consistent with our previous studies in this area. CONCLUSIONS: Combining CD41/CD61 platelet enumeration with TO RP percentage is possible. Accurate RP percentage requires an effective gating strategy, as background fluorescence cursor placement is important. This method for enumeration of RP percentage combined with accurate platelet enumeration, particularly in the low range, should prove useful in differentiating production from consumption issues in thrombocytopenia and monitoring response to therapy.

Presurgical levels of circulating cell-derived microparticles discriminate between patients with and without transfusion in coronary artery bypass graft surgery.

OBJECTIVES: Improved understanding of presurgical risk factors for transfusions will lead to reduction in their number and related complications. The goal of this study is to identify these factors in coronary artery bypass graft (CABG) surgery. METHODS: Presented herein are results of analyses of data from an ongoing study of transfusion in CABG surgery. Of 122 patients, 81 received transfusion (Tx) and 41 did not (NoTx). In addition to routine tests, presurgical levels of microparticles from platelets (PMPs), red cells (RMPs), and other lineages were assayed. RESULTS: The Tx and NoTx groups were similar with respect to most presurgical variables but differed in distribution of gender, blood type, diabetes prevalence, activated partial thromboplastin time (aPTT), hemoglobin (HGB), and microparticle levels. Stepwise multiple logistic regression was used to evaluate presurgical variables and to develop a model to assess risk factors for transfusion. CD41(+) PMP and CD235(+) RMP levels were found to be the main risk factors for transfusion. The Model's discriminating ability was assessed using receiver operating characteristic curve analysis, which showed that the area under the model curve (± standard error) was 0.86 ± 0.04 (95% confidence interval, 0.77-0.94). According to the model, patients with higher presurgical levels of circulating CD41(+) PMP, CD235a(+) RMP, and HGB, as well as a shorter aPTT, are less likely to receive transfusion(s). CONCLUSIONS: Presurgical levels of CD41(+) PMPs and CD235a(+) RMPs are the main risk factors for transfusion in CABG, followed by HGB and aPTT.

Unexplained pregnancy loss: a marker of basal endothelial dysfunction?

OBJECTIVE: To compare the microparticle levels of women referred for unexplained pregnancy loss with those of parous controls. DESIGN: Incident case-control study. SETTING: University medical center. PATIENT(S): 124 women consecutively referred for unexplained pregnancy losses (two or more losses at or before 21 weeks of gestational age, or at least one later loss), and 273 parous women without pregnancy loss. INTERVENTION(S): Numeration of circulating microparticles by flow cytometry after differentiation of subpopulations according to the expression of membrane-specific antigens (CD51, CD144, or CD146 for endothelial, CD41 for platelet, CD45 and CD66b for leukocyte and neutrophil microparticles). MAIN OUTCOME MEASURE(S): Plasma levels of microparticles. RESULTS: A relative hypercoagulable state assessed by thrombin generation test had been previously reported in such cases, so we hypothesized that this could be explained by an excess of procoagulant microparticles. The study women displayed statistically significantly lower platelet and higher endothelial microparticle levels than the controls. The parameters of the thrombin generation test were only correlated with the level of endothelial microparticles, with a low coefficient of Speerman's correlation (r=0.15). CONCLUSION(S): The difference in microparticle levels between the patients and controls does not clearly explain the hypercoagulable state reported in the patients but could reflect chronic endothelium damage.

Preserved thrombin-inducible platelet activation in thienopyridine-treated patients.

BACKGROUND: Abundant thrombin generation may be a major reason for subsequent thromboembolic events in patients with cardiovascular disease receiving dual antiplatelet therapy. We therefore investigated the susceptibility of thienopyridine responders and nonresponders to thrombin receptor-activating peptide (TRAP)-6- and adenosine diphosphate (ADP)-inducible platelet activation. MATERIALS AND METHODS: Response to clopidogrel or prasugrel was determined by the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay and multiple electrode aggregometry (MEA) in 317 patients undergoing angioplasty and stenting for cardiovascular disease. Baseline, TRAP-6-, and ADP-inducible P-selectin expression, activated glycoprotein IIb/IIIa (GPIIb/IIIa) and monocyte-platelet aggregate (MPA) formation were measured as sensitive parameters of platelet activation. RESULTS: In patients with high on-treatment residual ADP-inducible platelet reactivity (HRPR), baseline P-selectin expression, GPIIb/IIIa and MPA formation were similar to those in patients without HRPR (all P > 0.05). After platelet activation with TRAP-6 or ADP, patients with HRPR by both assays exhibited significantly higher levels of P-selectin expression, GPIIb/IIIa and MPA formation than patients with an adequate thienopyridine-mediated platelet inhibition (all P ≤ 0.02). However, high levels of TRAP-6-inducible P-selectin, GPIIb/IIIa and MPA formation also occurred in 20.4%, 19.1% and 20.1% of the good responders by the VASP assay, and in 19.6%, 16.6% and 20.6% of the good responders by MEA, respectively. CONCLUSIONS: Thienopyridine nonresponders are more susceptible to thrombin- and ADP-inducible platelet activation than patients with good platelet inhibition. However, even patients with adequate thienopyridine-mediated platelet inhibition often show a preserved responsiveness to thrombin. These patients may benefit from additional thrombin receptor blockage or inhibition of thrombin generation.

Platelet reactivity and thrombogenicity in postmenopausal women.

OBJECTIVE: Age-adjusted incidence of cardiovascular disease, including myocardial infarction, is significantly lower in premenopausal women than in men, which is thought to be caused by the cardioprotective effects of estrogen. However, there is a consistent increase in the incidence of coronary artery disease in postmenopausal women in comparison with premenopausal women. The protective benefit of hormone therapy has not been observed in postmenopausal women. It is unknown whether measures of platelet reactivity and clot strength contribute to the disproportionate incidence of cardiovascular disease between premenopausal and postmenopausal women. METHODS: Fifty healthy volunteers, including 25 premenopausal women and 25 postmenopausal women, aged between 40 and 65 years were enrolled. Total estradiol and follicle-stimulating hormone levels were measured for confirmation of menopausal state and comparison testing. Platelet reactivity was assessed using light transmission aggregometry and P-selectin, and glycoprotein IIb/IIIa receptor expression was assessed using flow cytometry. Thrombelastography was used to measure clot strength, clotting time, and fibrinogen activity. Serum cholesterol, C-reactive protein, complete blood count, and comprehensive metabolic panel were also measured. RESULTS: Platelet reactivity did not differ among menopausal states or hormone levels. Clotting time was increased in postmenopausal women (6.6 ± 2.0 vs. 7.8 ± 1.2 min, P = 0.013) and significantly correlated with estradiol levels (r = 0.68, P < 0.001). A significantly higher low-density lipoprotein cholesterol level was observed in postmenopausal women (P = 0.05). Mean C-reactive protein levels were numerically higher in the postmenopausal group. CONCLUSIONS: The thrombotic risk profile between premenopausal and postmenopausal women is similar. However, improved management of cholesterol may be of clinical benefit. Large-scale studies are required to validate these findings.

Platelets express and release osteocalcin and co-localize in human calcified atherosclerotic plaques.

BACKGROUND: Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease. METHODS: Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues. RESULTS: Human platelets expressed OC and co-localized with CD63 in δ-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone. CONCLUSIONS: Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further.

Increased circulating endothelial microparticles in COPD patients: a potential biomarker for COPD exacerbation susceptibility.

RATIONALE: The influence of COPD exacerbation on the endothelium is not completely understood. Circulating endothelial microparticles (EMPs) are membrane vesicles in circulating blood that are shed by activated or apoptotic endothelial cells. OBJECTIVE: To compare EMP numbers in stable COPD patients with those during and after exacerbation. METHODS: We examined the EMP numbers in 80 stable COPD patients, 27 patients with exacerbated COPD, and 20 healthy non-COPD volunteers. EMPs were defined as CD144+ MPs (VE-cadherin EMPs), CD31+/CD41- MPs (PECAM EMPs), CD146 MPs (MCAM EMPs) and CD62E+ EMPs (E-selectin EMPs) as analysed by FACS. Von Willebrand factor (vWF) expression was utilised to identify the origins of the EMPs. RESULTS: VE-cadherin, PECAM and E-selectin EMP numbers were significantly higher in the stable COPD patients than in the non-COPD volunteers, and they were significantly higher in the patients with exacerbated COPD than in the stable COPD patients. The majority of these increased EMPs were vWF-negative, indicating a pulmonary capillary origin. Baseline E-selectin EMP levels were significantly higher in COPD patients who experienced frequent exacerbations than in those who did not have frequent exacerbations (p<0.001). Twenty-eight days after the onset of exacerbation, E-selectin EMP levels returned to those observed in stable COPD patients, whereas PECAM EMP levels remained high. MCAM EMP numbers were not elevated in stable or exacerbated-COPD patients. CONCLUSIONS: Endothelial damage, mainly in pulmonary capillaries, occurs during exacerbation and continues even after clinical symptoms disappear. Higher baseline E-selectin EMP levels may indicate COPD patients who are susceptible to exacerbation.

Inherited thrombophilia and IVF failure: the impact of coagulation disorders on implantation process.

In connection with the embryo acceptance process after IVF procedure, endometrial cells surface receptors, extracellular matrix (ECM) molecules, endothelium and blood circulation factors were involved in remodelling of endometrium. Plasminogen activator inhibitor type 1 plays a significant role during the early phases of placental vascular remodelling and regulates the trophoblast invasion through controlling plasmin activity. Endometrial cell surface protein integrin alphaV/beta3, responsible for the adhesion of the embryo, has had also the same subunit beta3, which is component of integrin alphaIIb/beta3 connected with platelet aggregability. Prothrombin, furthermore, has had a debatable effect upon endothelial and mesenchymal cells and possible contribution on embryo vascular development. Confoundable data have been present about the role of coagulation factor V and its role for implantation. These and other coagulation factors have relatively common gene polymorphisms that enhanced their activity. This review discusses the effect of increased coagulation activity on implantation process, which is not yet fully determined. The establishment of the positive or negative impact of mother hypercoagulability on the success of embryo implantation after assisted reproduction technology could determine the timing of preventing anticoagulant therapy in women with history of early embryo loss.

Circulating microparticles in children with sleep disordered breathing.

BACKGROUND: Endothelial dysfunction is a common complication of pediatric obstructive sleep apnea (OSA). Circulating cell-derived microparticles (MPs) have emerged as reliable biomarkers of endothelial dysfunction and atherosclerosis. METHODS: Children underwent blood drawing the morning after a sleep study. Endothelial function was assessed using a modified hyperemic test after cuff-induced occlusion of the brachial artery. Circulating MP levels in plasma, including levels of endothelial MPs, endothelial progenitor MPs, leukocyte MPs, and platelet MPs, were measured using flow cytometry after staining with cell-specific antibodies. RESULTS: The levels of endothelial MPs, endothelial progenitor MPs, leukocyte MPs, and platelet MPs were significantly different according to the severity of OSA in children. Leukocyte CD11b+ MPs and platelet CD41a+ MPs correlated with the apnea-hypopnea index (AHI) (r = 0.334, P < .001; and r = 0.301, P < .001, respectively), and associations emerged between leukocyte CD11b+ MPs and apolipoprotein B (r = 0.206, P < .05) and between endothelial MPs and low-density lipoprotein cholesterol (r = 0.240, P < .01). In a multivariate regression model, the BMI z score (ß ± SE, 0.045 ± 0.020; P = .020) and the CD41a MPs to leukocyte CD45 MPs ratio (ß ± SE, 0.074 ± 0.032; P = .021) were independently associated with peak hyperemic responses. After controlling for age, gender, race, BMI z score, and apolipoprotein B levels, endothelial MPs, endothelial progenitor MPs, and leukocyte MPs showed independent associations with the AHI. Complex significant associations emerged between endothelial function, the AHI, and CD41a MPs. CONCLUSIONS: Childhood OSA is associated with higher circulating MP levels that can promote cardiovascular risk. Platelet-derived MPs emerge as being significantly associated with the vascular dysfunction associated with OSA in children and could potentially account for increased risk for altered endothelial function. However, the clinical use of MPs as reliable biomarker indicators of vascular risk will have to await further studies.

The combined effects of sidestream smoke extracts and glycated serum albumin on endothelial cells and platelets.

BACKGROUND: The purpose of this study was to test the hypothesis that sidestream tobacco smoke extracts would inhibit the culture of endothelial cells and enhance platelet aggregation under diabetic vascular conditions. Sidestream tobacco smoke and advanced glycation end products are known cardiovascular risk factors and we aimed to determine the combined interaction between these two risk factors to promote cardiovascular diseases associated with diabetes. METHODS: Human umbilical vein endothelial cells were cultured in the presence of sidestream tobacco smoke extracts (SHS) or nicotine and glycated albumin (AGE) or non-glycated albumin. After 3 days, endothelial cell viability and density were investigated. Platelets were also incubated with these compounds for up to 6 hours. Platelet aggregation and the surface expression of CD41 and CD62P were examined. In some experiments, platelets were added to the endothelial cell culture to determine if an interaction between platelets and endothelial cells occurs that can alter the responses to SHS or AGE. RESULTS: In general, the endothelial cell culture conditions were reduced in the presence of AGE and SHS. Nicotine, did not play a role in this reduction. Platelet aggregation proceeded faster in the presence of AGE and SHS. Interestingly, with the combined culture of endothelial cells and platelets, the endothelial cell culture conditions were improved and the platelet functional changes were diminished in the presence of SHS and AGE, as compared with the individual incubations. CONCLUSIONS: Our data suggests that diabetics that are exposed to SHS may have a higher likelihood for cardiovascular disease development through a diminished endothelial cell viability and an increased platelet activity, which are partially mediated by CD41 and not CD62P. This study provides support for an increased cardiovascular risk for diabetic patients that are exposed to SHS. This study also provides a new experimental technique to monitor platelet-endothelial cell interactions.

Associations between dietary patterns and flow cytometry-measured biomarkers of inflammation and cellular activation in the Atherosclerosis Risk in Communities (ARIC) Carotid Artery MRI Study.

BACKGROUND: Specific foods and overall dietary patterns are associated with soluble biomarkers of systemic inflammation and endothelial activation. However, no large epidemiological studies have evaluated relationships between such dietary factors and cell-specific markers of activation and inflammation as measured by flow cytometry. METHODS: Cell aggregates and multiple platelet and leukocyte markers were quantified by flow cytometry in fresh whole blood from 1101 white adults participating in the Carotid Artery MRI Study, a subset of the larger Atherosclerosis Risk in Communities (ARIC) Study. Two dietary patterns ("Healthy" and "Western") were empirically derived via principal components analysis using data collected by food frequency questionnaire. Cross-sectional associations between dietary patterns and flow cytometry-measured biomarkers were evaluated, adjusting for demographics and lifestyle factors, including medications use. RESULTS: After multivariable adjustment, monocyte lipopolysaccharide receptor (CD14), monocyte toll-like receptor-2, and platelet glycoprotein IIb (CD41) showed inverse associations with the Healthy dietary pattern (p=0.01, 0.04, and 0.01, respectively). In contrast, the Western dietary pattern was positively associated with CD41 and platelet-granulocyte aggregates (p=0.01 and 0.04, respectively). Independent of other dietary factors, alcohol consumption was inversely associated with levels of pan-leukocyte marker (CD45), P-selectin (CD62P) on PLA1 and on PLA2 platelets, and platelet-monocyte, platelet-granulocyte, and platelet-lymphocyte aggregates. CONCLUSION: Dietary patterns and alcohol intake were each cross-sectionally associated with select markers of cellular activation and inflammation measured by flow cytometry. These data are consistent with the hypothesis that holistic measures of dietary intake are associated with inflammation.

Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles.

BACKGROUND: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets. OBJECTIVES: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. METHODS: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG1 isotype control was used as a negative control. RESULTS: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation=16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (dsph) ranged from 10 to 475 nm (mean: 67.5+/-26.5 nm) and from 5 to 204 nm (mean: 51.4+/-14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702x10(9) L(-1) plasma vs. 11-626x10(6) L(-1) plasma). After filtration of isolated MPs through a 0.22-microm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean dsph: 37.2+/-11.6 nm), but not by flow cytometry. CONCLUSIONS: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.

CD41 Western blotting: a new method to detect platelet adhesion to artificial surfaces used in extracorporeal circulation procedures.

Cardiopulmonary bypass (CPB) surgery is associated with platelet activation and reduced platelet counts due to artificial surface activation of blood elements and non-physiological flow-patterns. As shown in former studies, coating of medical devices can improve hemocompatibility in extracorporeal circulation systems. In this study, we demonstrate a new method to determine platelet adhesion on 18 coated and non-coated membrane oxygenators in a simulated CPB model with CD41 Western blot. Platelet loss and the release of beta-TG (platelet activation marker) were determined during a 120 min recirculation phase. At the end of the run the membrane oxygenators (with tubing system) were rinsed and the amount of adsorbed proteins on the surface was analyzed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. Uncoated devices showed significantly higher concentrations of CD41 and of fibrinogen adsorption compared to the coated membrane oxygenators. These results correspond with the release of beta-TG and platelet loss indicating less platelet adhesion on the coated oxygenators compared with the uncoated group. This new method may be useful in choosing less platelet activating materials for all kind of blood contacting devices to improve thrombogenicity including platelet functionality.

Mild-to-moderate renal impairment is associated with platelet activation: a cross-sectional study.

The high incidence of cardiovascular disease in patients with moderate renal impairment is not fully explained by traditional atherothrombotic risk factors. Independently from these factors, blood platelet activation may increase the cardiovascular disease risk of patients with mild-to-moderate renal impairment. Blood platelet activation has not been studied in nondiabetic patients with mild-to-moderate renal impairment. Therefore, we measured the extent of platelet activation by means of fluorescence cytometry in 93 nondiabetic patients with MDRD-estimated creatinine clearance ranging from 13 - 63 ml/min/1.73 m2. As platelet activation parameters we used the expression of CD62P (P-selectin), CD 63 (glycoprotein 53), PAC-1 (activated fibrinogen receptor), CD42b (von Willebrand factor receptor) and CD41 (fibrinogen receptor) on the platelet surface membrane. The expression of CD62p, CD63 and PAC-1 was statistically significantly inversely related to the estimated glomerular filtration rate in these patients (standardized b -0.28, -0.32 and -0.39, respectively). We conclude that nondiabetic mild-to-moderate renal impairment is associated with blood platelet activation. Whether this contributes to the increased cardiovascular risk in these patients needs further study.

Effects of enoxaparin and unfractionated heparin on platelet activity and reactivity during carotid endarterectomy.

The aim of this study was to determine platelet activity and reactivity and the effects of unfractionated heparin (UFH) and enoxaparin on platelet function during carotid eversion endarterectomy under local anesthesia. Twenty symptomatic patients undergoing carotid endarterectomy were randomly assigned to either 5,000 units of UFH or body weight-adjusted enoxaparin (0.5 mg/kg body weight) as an intraoperative intravenous bolus. The activity of platelets was assessed by measuring the expression of CD62p and CD41 with flow cytometry. Additionally, platelet-leukocyte aggregates (PLAs) were enumerated. The reactivity of platelets was evaluated by measuring the expression of the same antigens after stimulation. In addition, platelet reactivity was also analyzed using a PFA-100 analyzer. A significant increase in platelet activity was observed during surgery for CD41 and CD62p (p = .002 and < .001, respectively). The number of PLAs showed no significant changes during surgery. Yet there was a significant difference between patients treated with UFH and patients treated with enoxaparin. No difference for platelet activity or reactivity for patients receiving either UFH or enoxaparin prior to cross-clamping of the carotid arteries was seen. The formation of PLAs after endarterectomy was significantly higher in the UFH group; thus, PLAs are probably a useful surrogate parameter for measuring platelet activity.

Mechanisms of lumbrokinase in protection of cerebral ischemia.

The present study was designed to explore the mechanisms involved in the anti-ischemic action of lumbrokinase (LK) in brain. The enzyme immunoassay, spectrofluorimeter and flow cytometry were used to detect the level of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), the Ca(2+) mobilization, and human platelet surface antigen expression in order to elucidate the anti-platelet action involved in LK cerebroprotection. RT-PCR and western blot were used to identify the role of Intercellular adhesion molecule-1 (ICAM-1) and Janus Kinase1/Signal Transducers and Activators of Transcription1 (JAK1/STAT1) pathway in protecting brain against ischemic injury by anti-thrombosis and anti-apoptosis. Results showed that LK significantly potentiated the activity of adenylate cyclase (AC), increased the cAMP level in vivo, remarkably inhibited the rise of rat platelet intracellular Ca(2+) ([Ca(2+)](i)), and attenuated the expression of Glycoprotein IIB/IIIA (GPIIB/IIIA) and P-selectin in human platelet stimulated by thrombin in vitro. Furthermore, the expressions of ICAM-1 and JAK1/STAT1 were remarkably regulated by LK in Human Umbilical Vein Endothelial Cell (HUVEC) and ischemic cerebral tissues. These data indicated that the anti-ischemic activity of LK was due to its anti-platelet activity by elevating cAMP level and attenuating the calcium release from calcium stores, the anti-thrombosis action due to inhibiting of ICAM-1 expression, and the anti-apoptotic effect due to the activation of JAK1/STAT1 pathway.

Delayed inhibition of agonist-induced granulocyte-platelet aggregation after low-dose sevoflurane inhalation in humans.

BACKGROUND: Sevoflurane can be used as sedative-analgesic drug with endothelial protective properties. We tested whether low-dose sevoflurane inhalation provides sustained inhibition of detrimental granulocyte-platelet aggregation in humans. METHODS: Ten healthy male volunteers were enrolled in this crossover study. Each subject inhaled sevoflurane for 1 h at 0.5-1 vol % end-tidal concentration in oxygen (50 vol %). Inhaling oxygen (50 vol %) alone served as control. Venous blood samples were collected at baseline before inhalation, immediately after inhalation, and 24 h thereafter, and were used for flow cytometry to determine platelet surface marker (CD41, CD42b, CD62P/P-selectin, and PAC-1) on platelets and granulocytes and for kaolin-induced clot formation, as assessed by thromboelastography. In flow cytometry experiments, platelets were stimulated with arachidonic acid (AA, 30 microM), adenosine diphosphate (ADP, 1 microM), and thrombin receptor agonist peptide-6 (TRAP-6, 6 microM). RESULTS: AA, ADP, and TRAP-6 markedly increased the expression of CD62P on platelets, whereas CD42b (shedding) and PAC-1 (heterotypic conjugates) expression decreased. The amount of granulocyte-platelet aggregates increased upon agonist stimulation. Low-dose sevoflurane inhalation reduced ADP-induced CD62P expression on platelets 24 h after inhalation, and inhibited the formation of granulocyte-platelet aggregates under stimulation with AA and ADP after 1 and 24 h, and with TRAP-6 after 24 h compared with control. Inhibition of granulocyte-platelet aggregates was accompanied by reduced clot firmness 24 h after sevoflurane inhalation compared with control. CONCLUSIONS: We demonstrated for the first time that inhaling low-dose sevoflurane (<1 vol % end-tidal) inhibits agonist-induced granulocyte-platelet interactions 24 h after administration and thus counteracts thromboinflammatory processes.

Effects of selective COX-2 inhibition on prostanoids and platelet physiology in young healthy volunteers.

BACKGROUND: Selective inhibitors of cyclooxygenase-2 (COX-2) called coxibs, are effective anti-inflammatory and analgesic drugs. Recently, these drugs were associated with an increased risk for myocardial infarction and atherothrombotic events. The hypothesis of thromboxane-prostacyclin imbalance has been preferred to explain these unwanted effects. METHODS: We studied the effects of 14 days intake of rofecoxib (25 mg q.d.), celecoxib (200 mg b.i.d.), naproxen (500 mg b.i.d.) and placebo in a randomized, blinded, placebo-controlled study in young healthy volunteers (median age 25-30 years, each group n = 10). We assessed prostanoid metabolite excretion (PGE-M, TXB(2), 6-keto-PGF(1alpha), 11-dehydro-TXB(2), 2,3-dinor-TXB(2), and dinor-6-keto-PGF(1alpha)), the expression of platelet activation markers (CD62P, PAC-1, fibrinogen), platelet-leukocyte formation, the endogenous thrombin potential, platelet cAMP content and plasma thrombomodulin level. RESULTS: Naproxen suppressed biosynthesis of PGE-M, prostacyclin metabolites and thromboxane metabolites and thrombomodulin levels. In contrast, both coxibs had an inhibitory effect only on PGE-M, 6-keto-PGF(1alpha), and on dinor-6-keto-PGF(1alpha), whereas TXB(2), 2,3-dinor-TXB(2) and 11-dehydro-TXB(2) excretion were unaffected. None of the coxibs exerted significant effects on the expression of platelet activation markers, cAMP generation, platelet-leukocyte formation, or on thrombomodulin plasma levels. Interestingly, platelet TXB(2) release during aggregation was enhanced after coxib treatment following arachidonic acid or collagen stimulation. CONCLUSION: In young healthy volunteers coxibs inhibit systemic PGE(2) and PGI(2) synthesis. Platelet function and expression of platelet aggregation markers are not affected; however, coxibs can stimulate TXB(2) release from activated platelets. Combined decrease in vasodilatory PGE(2) and PGI(2) together with increased TXA(2) in proaggregatory conditions may contribute to coxib side effects.