P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.

OBJECTIVE

The purpose of this study was to determine whether digoxin use is associated with increased flow cytometric markers of endothelial cell and platelet activation in patients with nonvalvular atrial fibrillation (AF).

BACKGROUND

Increased intracellular calcium is a key event in platelet activation, and several studies have demonstrated that digitalis activates platelets in vitro. Intracellular calcium also is a key regulator of endothelial cell function, and endogenous digitalis-like substances have been shown to affect biologic processes in endothelial cells.

METHODS

We studied 30 patients with nonvalvular AF. We measured the levels of (1) platelet expression of P-selectin (CD62P), (2) platelet microparticles (PMP); and (3) endothelial microparticles (EMP) identified by anti-CD31 (EMP31) and by anti-E-selectin antibodies (EMP62E).

RESULTS

Patients who were taking digoxin (n = 16; mean digoxin level = 0.93 ng/dL) did not demonstrate any significant differences in clinical or echocardiographic characteristics compared with patients not taking digoxin (n = 14). Patients taking digoxin had significantly increased levels of CD62P expression in platelets and platelet-leukocyte conjugates and markedly increased markers of endothelial activation: EMP62E and EMP31. After adjusting for potential confounders (including age, congestive heart failure, coronary artery disease, ejection fraction, antiplatelet, beta-blocker, and calcium channel blocker use), the differences persisted.

CONCLUSIONS

Digoxin use in patients with AF is associated with increased levels of endothelial and platelet activation. If digitalis activates endothelial cells and platelets at pharmacologic doses, use of digitalis in conditions such as AF could predispose to thrombosis and vascular events.

BACKGROUND

In previous studies we have consistently shown a significant increase of platelet reactivity after exercise in patients with obstructive coronary artery disease (CAD). We also observed a significant individual variability in the response to exercise of platelet reactivity in these patients. Whether exercise-induced changes in platelet reactivity correlate with changes in platelet membrane receptors in patients with CAD is unknown.

METHODS

We studied 26 patients with stable CAD and 10 matched healthy controls who underwent a symptom-limited treadmill exercise stress test. Venous blood samples were collected at rest and within 5 min of peak exercise. Platelet reactivity was measured by the PFA-100 method as time to occlude (closure time, CT) a ring coated with collagen/adenosine diphosphate (C/ADP). Platelet expression of glycoprotein (GP) IIb/IIIa, in both global (CD41) and active form (PAC-1), and P-selectin (CD62P) and formation of leukocyte-platelet aggregates were assessed by flow cytometry.

RESULTS

After exercise CT did not change in controls (85.4+/-12 to 84.0+/-9 s, p=0.37), whereas it decreased in CAD patients (98.8+/-24 to 91.4+/-25 s, p<0.001). After exercise, CD41 and PAC-1 platelet expression increased significantly in CAD patients (p=0.04 for both), but not in controls (p=0.39 and p=0.98, respectively). To evaluate the relationship between the response to exercise of platelet reactivity and of platelet receptor expression, CAD patients were divided into two groups: CAD group 1 (16 patients, decrease in CT >5 s after exercise) and CAD group 2 (10 patients no increase in platelet reactivity after exercise). CD41 and PAC-1 expression increased in CAD group 1 (p=0.008 and p=0.026, respectively) but not in CAD group 2 (p=0.39 and p=0.50, respectively). No significant differences were observed between the 2 groups for changes in CD62P and leukocyte-platelet aggregates.

CONCLUSIONS

Our data show that, in patients with stable CAD, an increased platelet reactivity to C/ADP stimulation after exercise, as assessed by the PFA-100 method, is specifically associated with an increased expression of platelet GP IIb/IIIa receptor.

RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.

Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

OBJECTIVE

Endothelial microparticles (EMPs) are vesicles that are released from activated endothelial cells and serve as a surrogate for endothelial dysfunction (ED). ED may be involved in migraine pathophysiology and contribute to the increased risk of ischemic stroke, particularly in female migraineurs with aura (MA). We sought to determine whether EMPs are elevated in women with MA.

METHODS

In this case-control study, EMPs were detected by analysing surface markers using fluorescence-activated cell sorting (FACS). Surface markers were measured covering the main cell lines relevant in cardiovascular disease like endothelial cells, platelets, monocytes and leucocytes. Microparticles (MPs) were identified in correlation to calibration by 1 -µm calibrator beads (Beckman Coulter). Arterial stiffness was assessed using fingertip tonometry and the heart rate-adjusted augmentation index (AI).

RESULTS

We included 29 patients with MA and 29 matched controls. MA patients had significantly higher EMPs (CD62E(+)AnnexinV(+): 5142/µl vs 1535/µl; p < 0.001; CD144(+)AnnexinV(+): 6683/µl vs 3107/µl; p < 0.001), monocytic (CD14(+)AnnexinV(+) 6378 vs 3161; p < 0.001), and platelet MPs (CD62P(+)CD42b(+)AnnexinV(+) 5450 vs 3204; p < 0.001). Activated EMPs (CD62E(+)AnnexinV(+)) correlated with heart-rate adjusted AI (r = 0.46; p < 001).

CONCLUSIONS

EMP levels are significantly elevated in women with MA and correlated with increased AI. Our findings suggest that endothelial activation is present in women with MA. This might contribute to higher stroke risk in MA.

P-selectin (CD62P) is an adhesion molecule that mediates the initial attachment of leukocytes to activated platelets and endothelial cells in damaged tissues. We evaluated the role of P-selectin in concanavalin A (Con A)-induced hepatitis, a model characterized by CD4(+) T cell activation and infiltration of the liver. Con A injection induced transient P-selectin expression on hepatic venules and platelets. Mice lacking P-selectin showed impaired lymphocyte adhesion to liver venules and sinusoids, a striking reduction in intrasinusoidal occlusion, and decreased lymphocyte infiltration of liver parenchyma. The reduction in transaminase levels and the almost complete abolition of necrotic injury demonstrated that liver damage was lower in P-selectin-deficient mice. In wild-type mice, pretreatment with the P-selectin-blocking monoclonal antibody attenuated the sinusoidal occlusion and reduced the rise in transaminases after Con A treatment. These results implicate P-selectin in the development of Con A-induced liver injury and reveal the protective effect of blocking P-selectin in this hepatitis.

BACKGROUND

Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives.

METHODS

Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR.

RESULTS

We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement.

CONCLUSIONS

We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability.

BACKGROUND

Glatiramer acetate (GA, Copaxone, Copolymer-1) is an FDA approved drug for the treatment of MS and it is very effective in suppressing neuroinflammation in experimental autoimmune encephalitis (EAE), an animal model of MS. Although this drug was designed to inhibit pathogenic T cells, the exact mechanism of EAE/MS suppression by GA is still not well understood. Previously we presented evidence that platelets become activated and promote neuroinflammation in EAE, suggesting a possible pathogenic role of platelets in MS and EAE. We hypothesized that GA could inhibit neuroinflammation by affecting not only immune cells but also platelets.

RESULTS

We investigated the effect of GA on the activation of human platelets in vitro: calcium influx, platelet aggregation and expression of activation markers. Our results in human platelets were confirmed by in-vitro and in-vivo studies of modulation of functions of platelets in mouse model. We found that GA inhibited thrombin-induced calcium influx in human and mouse platelets. GA also decreased thrombin-induced CD31, CD62P, CD63, and active form of αIIbβ3 integrin surface expression and formation of platelet aggregates for both mouse and human platelets, and prolonged the bleeding time in mice by 2.7-fold. In addition, we found that GA decreased the extent of macrophage activation induced by co-culture of macrophages with platelets.

CONCLUSIONS

GA inhibited the activation of platelets, which suggests a new mechanism of GA action in suppression of EAE/MS by targeting platelets and possibly preventing their interaction with immune cells such as macrophages. Furthermore, the reduction in platelet activation by GA may have additional cardiovascular benefits to prevent thrombosis.

The white blood cell count and mean platelet volume determined shortly after the symptom onset are known as independent predictors for clinical outcome after stroke. In the present study we sought to evaluate the prognostic value of platelet-derived inflammatory biomarkers measured prospectively after an ischaemic event. Using five-colour flow cytometry, the platelet surface expression of CD40L, CD62P and subpopulations of leukocyte-platelet aggregates were assessed in 93 stroke patients on the first (V(0)), 10th (V(1)) and 90th (V(2)) day after stroke, and once in 65 disease controls. The clinical outcome was evaluated using the Scandinavian Stroke Scale (SSS) and modified Rankin Scale (mRS) at the same time points as blood sampling and 24 months after the event. Patients with either CD40L surface expression or the percentage of monocyte-platelet aggregates (M-plt) in the third tertile (T3) at V0 had a significantly lower score on the SSS at V(1). Patients with the percentage M-plt at V(0) higher than the median value of M-plt in controls were at increased risk of SSS < 40 at V(1) (odds ratio: 2.6; 95% confidence interval [CI]: 1.4 - 8.7; p=0.006). Patients with the percentage of M-plt in T3 at V(0) showed progressive decline in survival (hazard ratio [HR]: 1.6; 95% CI: 1.1-1.9; p=0.02) and a significantly higher number of recurrent vascular events (HR: 2.64; 95% CI: 1.3-3.2; p=0.02) when compared to the first tertile. In conclusion, increased levels of M-plt could be a predictive marker for both early outcome and long-term prognosis while increased CD40L was correlated with worse clinical outcome.

BACKGROUND

The aim of this study was to further characterize the effect of the antiplatelet agents, aspirin and eptifibatide, on the surface expression of CD40L and CD62P on platelets from patients with stable coronary artery disease.

METHODS

Platelet function was evaluated using standard light transmission aggregometry. Measurements of CD62P and CD40L were carried out by flow cytometry and ELISA assays.

RESULTS

All patients had the expected level of platelet aggregation inhibition in response to 20 muM ADP in the presence of increasing eptifibatide concentrations. Platelet activation by adenosine diphosphate (ADP) or thrombin agonist peptide (TRAP) increased CD62P and CD40L surface density in the presence of aspirin by 1.9 - 2.8 -fold. Aspirin treatment did not prevent either CD62P or CD40L expression. Eptifibatide pretreatment at pharmacologically relevant concentrations blocked agonist-induced increases in CD62P platelet surface density. A marked percentage of platelets still expressed low levels of surface CD62P suggesting slight platelet activation even with potent platelet inhibition. Eptifibatide also blocked agonist-induced increases in CD40L surface expression and decreased the percent of platelets positive for surface CD40L. Decreased expression of CD40L was due to an inhibition of CD40L translocation and not caused by enhanced shedding from the surface, as soluble CD40L (sCD40L). Eptifibatide concentrations that effectively blocked platelet aggregation correlated with total inhibition of increased CD62P and CD40L surface density.

CONCLUSIONS

Blockade of the GPIIb-IIIa receptor on platelets from coronary artery disease patients may have significant bearing on reducing proinflammatory and procoagulant events mediated by CD62P and sCD40L.

Platelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events. Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation in ACS. We consecutively investigated both the platelet count and the platelet surface expression of GPVI in 843 patients with a symptomatic coronary artery disease verified by coronary angiography. Four hundred fourteen patients presented with stable angina pectoris and 429 patients with ACS. Platelet surface expression of GPVI and CD62P was determined by flow cytometry and platelet count with a coulter counter, plasmatic soluble GPVI was measured by ELISA. Platelet GPVI expression in patients with ACS was compared to platelet count. Patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count [mean fluorescence intensity (MFI) +/- standard deviation): 1(st) vs. 4(th): 20.44 +/- 6.1 vs. 18.62 +/- 3.7; p=0.012; 2(nd)vs.3(rd):21.2+/-8.5vs.18.76+/-3.7;P=0.03; 2(nd)vs.4(th): 21.2+/-8.5vs.18.62+/-3.7;P=0.004], which was paralleled in trend for the CD62P expression [MFI: 1(st) vs. 4(th): 11.2 +/- 6.8 vs. 12.3 +/- 9; p=0.057; 2(nd) vs. 3(rd): 16.3 +/- 16 vs.12.7 +/- 5.3; p=0.138; 2(nd) vs. 4(th): 16.3 +/- 16 vs.11 +/- 4.4; p=0.043]. In a subgroup of 48 patients with ACS, determination of soluble GPVI showed similar results [plasma GPVI (ng/ml): 1(st)vs.4(th): 1.6 +/- 0.6 vs. 1.2 +/- 0.4; p=0.046; 1(st) vs. 3(rd): 1.6 +/- 0.6 vs. 1.1 +/- 0.5; p=0.038; 2(nd) vs. 3(rd): 1.9 +/- 0.8 vs. 1.1 +/- 0.5; p=0.04; 2(nd) vs. 4(th): 1.9 +/- 0.8 vs. 1.2 +/- 0.4; p=0.056]. Thus, a lower platelet count comes along with a higher GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased activation and enhanced recruitment of platelets to the site of vascular injury.

Microparticles (MPs) are vesicular structures shed from endothelial or circulating blood cells, after activation or apoptosis, and can be considered markers of vascular damage. We aimed to determine the levels of circulating MPs, their content of miRNA-126-3p and 5p, and their relationship with early endothelial activation/damage, in patients with different degree of glucose tolerance.

CD62E+, CD62P+, CD142+, CD45+ circulating MPs, their apoptotic (AnnexinV+) fractions, and miRNA-126 expression were determined in 39 prediabetic (PreDM), 68 type 2 diabetic (T2DM), and 53 control (NGT) subjects, along with main anthropometric and biochemical measurements. MPs were analysed by flow cytometry. miRNA-126 was measured by quantitative real-time PCR. Plasma antioxidant capacity was determined by electronic spin resonance; ICAM-1, and VCAM-1 by ELISA.

Activated endothelial cell-derived MPs (CD62E+) were significantly increased in PreDM and T2DM in comparison to NGT (p < 0.0001). AnnexinV+/CD62E+ MPs and Annexin V+ MPs were significantly increased in T2DM compared to PreDM and NGT (p < 0.001); other MPs were not significantly different among groups. Plasma antioxidant capacity was significantly decreased in PreDM and T2DM compared to NGT (p = 0.001); VCAM-1 significantly increased in PreDM and T2DM in comparison to NGT (p = 0.001). miR-126-3p expression, but not miR-126-5p, in MPs, decreased significantly and progressively from NGT, to PreDM, and T2DM (p < 0.001). In PreDM and T2DM, CD62E+ MPs level was significantly and negatively associated with plasma glucose (p = 0.004).

We show for the first time that circulating CD62E+ MPs level and miR-126-3p content in MPs are abnormal in subjects with pre-diabetes; the content of miR-126-3p correlates with markers of endothelial inflammation, such as VCAM-1, plasma antioxidant capacity, and microparticles, well-accepted markers of endothelial dysfunction.

OBJECTIVE

Type 2 diabetes is a potent cardiovascular risk factor, associated with proinflammatory and prothrombotic processes. The purpose of this study was to investigate whether platelet-bound CD40-CD40L signalling, P-selectin expression and soluble CD40L were increased in patients with diabetes mellitus and can be normalized by improving glycaemic control.

METHODS

Soluble (s) CD40L, platelet surface expression of CD40L, CD40 and P-selectin (CD62P) on platelets were measured by flow cytometry in 71 patients with Type 2 diabetes mellitus and 37 healthy volunteers. In addition, the relationship of HbA1c to CD40-CD40L and P-selectin expression was determined in a longitudinal follow-up.

RESULTS

In patients with Type 2 diabetes, platelet membrane CD40 expression (Type 2 diabetes 3.1+/-0.61 vs. controls 2.5+/-0.85 mean fluorescence intensity; P=0.001), platelet-bound CD40L (1.2+/-0.32 vs. 1.1+/-0.14; P=0.034) as well as surface expression of CD62P (0.66+/-0.19 vs. 0.57+/-0.12; P=0.007) were higher than in control subjects. Plasma sCD40L values (3.2+/-1.70 vs. 1.8+/-0.50 ng/ml; P<0.001) were also significantly increased in Type 2 diabetes. After improving glycaemic control in patients with initial HbA1c>8.5% (n=15), platelet P-selectin and CD40-CD40L expression decreased significantly by 54.0%, 36.22% and 16.26%, respectively 1 year later.

CONCLUSIONS

Type 2 diabetes is associated with up-regulation of the platelet-bound CD40-CD40L system, platelet hyperactivity (enhanced P-selectin expression) and increased sCD40L levels. Improved glycaemic control, however, does help to correct abnormal platelet activation via down-regulation of CD40-CD40L system and P-selectin expression.

Effect of in-water oxygen prebreathing at different depths on decompression-induced bubble formation and platelet activation in scuba divers was evaluated. Six volunteers participated in four diving protocols, with 2 wk of recovery between dives. On dive 1, before diving, all divers breathed normally for 20 min at the surface of the sea (Air). On dive 2, before diving, all divers breathed 100% oxygen for 20 min at the surface of the sea [normobaric oxygenation (NBO)]. On dive 3, before diving, all divers breathed 100% O2 for 20 min at 6 m of seawater [msw; hyperbaric oxygenation (HBO) 1.6 atmospheres absolute (ATA)]. On dive 4, before diving, all divers breathed 100% O2 for 20 min at 12 msw (HBO 2.2 ATA). Then they dove to 30 msw (4 ATA) for 20 min breathing air from scuba. After each dive, blood samples were collected as soon as the divers surfaced. Bubbles were measured at 20 and 50 min after decompression and converted to bubble count estimate (BCE) and numeric bubble grade (NBG). BCE and NBG were significantly lower in NBO than in Air [0.142+/-0.034 vs. 0.191+/-0.066 (P<0.05) and 1.61+/-0.25 vs. 1.89+/-0.31 (P<0.05), respectively] at 20 min, but not at 50 min. HBO at 1.6 ATA and 2.2 ATA has a similar significant effect of reducing BCE and NBG. BCE was 0.067+/-0.026 and 0.040+/-0.018 at 20 min and 0.030+/-0.022 and 0.020+/-0.020 at 50 min. NBG was 1.11+/-0.17 and 0.92+/-0.16 at 20 min and 0.83+/-0.18 and 0.75+/-0.16 at 50 min. Prebreathing NBO and HBO significantly alleviated decompression-induced platelet activation. Activation of CD62p was 3.0+/-0.4, 13.5+/-1.3, 10.7+/-0.9, 4.5+/-0.7, and 7.6+/-0.8% for baseline, Air, NBO, HBO at 1.6 ATA, and HBO at 2.2 ATA, respectively. The data show that prebreathing oxygen, more effective with HBO than NBO, decreases air bubbles and platelet activation and, therefore, may be beneficial in reducing the development of decompression sickness.

BACKGROUND

Circulating microparticles (MP) have been described in sickle cell anaemia (SCA); however, their interaction with endothelial markers remains unclear. We investigated the relationship between MP, protein C (PC), free protein S (PS), nitric oxide (NO), endothelin-1 (ET-1) and adrenomedullin (ADM) in a large cohort of paediatric patients.

METHODS

A total of 111 children of African ethnicity with SCA: 51 in steady state; 15 in crises; 30 on hydroxyurea (HU) therapy; 15 on transfusion; 17 controls (HbAA) of similar age/ethnicity. MP were analysed by flow cytometry using: Annexin V (AV), CD61, CD42a, CD62P, CD235a, CD14, CD142 (tissue factor), CD201 (endothelial PC receptor), CD62E, CD36 (TSP-1), CD47 (TSP-1 receptor), CD31 (PECAM), CD144 (VE-cadherin). Protein C, free PS, NO, pro-ADM and C-terminal ET-1 were also measured.

RESULTS

Total MP AV was lower in crisis (1.26×10(6) ml(-1); 0.56-2.44×10(6)) and steady state (1.35×10(6) ml(-1); 0.71-3.0×10(6)) compared to transfusion (4.33×10(6) ml(-1); 1.6-9.2×10(6), p<0.01). Protein C levels were significantly lower in crisis (median 0.52 IU ml(-1); interquartile range 0.43-0.62) compared with all other groups: HbAA (0.72 IU ml(-1); 0.66-0.82, p<0.001); HU (0.67 IU ml(-1); 0.58-0.77, p<0.001); steady state (0.63 IU ml(-1); 0.54-0.70, p<0.05) and transfusion (0.60 IU ml(-1); 0.54-0.70, p<0.05). In addition, levels were significantly reduced in steady state (0.63 IU ml(-1); 0.54-0.70) compared with HbAA (0.72 IU ml(-1); 0.66-0.80, p<0.01). PS levels were significantly higher in HbAA (0.85 IU ml(-1); 0.72-0.97) compared with crisis (0.49 IU ml(-1); 0.42-0.64, p<0.001), HU (0.65 IU ml(-1); 0.56-0.74, p<0.01) and transfusion (0.59 IU ml(-1); 0.47-0.71, p<0.01). There was also a significant difference in crisis patients compared with steady state (0.49 IU ml(-1); 0.42-0.64 vs. 0.68 IU ml(-1); 0.58-0.79, p<0.05). There was high correlation (R>0.9, p<0.05) between total numbers of AV-positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p<0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p<0.001). %MP CD201 was higher in crisis (11.6%) compared with HbAA (3.2%, p<0.05); %MP CD144 was higher in crisis (7.6%) compared with transfusion (2.1%, p<0.05); %CD14 (0.77%) was higher in crisis compared with transfusion (0.0%, p<0.05) and steady state (0.0%, p<0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared with the rest (40%); %MP CD235a was higher in crisis (17.9%) compared with transfusion (8.9%), HU (8.7%) and steady state (9.9%, p<0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. Pro-ADM levels were raised in chest crisis: 0.38 nmol L(-1) (0.31-0.49) versus steady state: 0.27 nmol L(-1) (0.25-0.32; p<0.01) and control: 0.28 nmol L(-1) (0.27-0.31; p<0.01). CT-proET-1 levels were reduced in patients on HU therapy: 43.6 pmol L(-1) (12.6-49.6) versus control: 55.1 pmol L(-1) (45.2-63.9; p<0.05). NO levels were significantly lower in chest crisis (19.3 mmol L(-1) plasma; 10.7-19.9) compared with HU (22.2 mmol L(-1) plasma; 18.3-28.4; p<0.05), and HbSC (30.6 mmol L(-1) plasma; 20.8-39.5; p<0.05) and approach significance when compared with steady state (22.5mmol L(-1) plasma; 16.9-28.2; p=0.07).

CONCLUSIONS

Protein C and free PS are reduced in crisis with lower numbers of platelet MP and higher percentage of markers of endothelial damage and of red cell origin. During chest crisis, ADM and ET-1 were elevated suggesting a role for therapy inhibiting ET-1 in chest crisis.

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli-LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli-LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli-LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP.

CONCLUSIONS

Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.

BACKGROUND

Nonvalvular atrial fibrillation (AF) confers a five-fold increased risk of stroke. Whether catheter ablation (CA) subsequently decreases prothrombotic risk is unknown.

OBJECTIVE

The purpose of this study was to assess the long-term effects of CA for AF on prothrombotic risk.

METHODS

Fifty-seven patients undergoing CA for AF were prospectively studied. Platelet activation (CD62P [platelet P-selectin] and PAC-1 [glycoprotein IIb/IIIa] expression) and endothelial function (asymmetric dimethylarginine [ADMA] levels) were measured at baseline and 6-months postablation.

RESULTS

Thirty-seven (65%) patients remained in sinus rhythm (SR group) and 20 (35%) sustained AF recurrence (AF recurrence group) at 6-months. Patients with AF-recurrence were older, had a higher proportion of hypertension and long-standing persistent AF. There were no significant differences in CD62P (P = .3), PAC-1 (P = .1) and ADMA (P = .7) levels at baseline between the two groups. In the SR group, markers of platelet activation decreased significantly at 6-month follow-up compared to baseline; log CD62P % 0.79 ± 0.28 vs 1.03 ± 0.27 (P <.05) and log PAC-1 % 0.22 ± 0.58 vs 0.89 ± 0.31 (P <.01). This was not significant in the AF-recurrence group (P = .8, log CD62P; P = .1, log PAC-1). For endothelial function, ADMA levels decreased significantly at 6-months compared to baseline in the SR group (log ADMA μM/L 0.15 ± 0.02 vs 0.17 ± 0.04; P <.05), but did not alter significantly in the AF-recurrence group (P = .4, log ADMA).

CONCLUSIONS

Catheter ablation and successful maintenance of SR leads to a decrease in platelet activation and improvement in endothelial function in patients with AF. These findings suggest that AF is an important determinant of the prothrombotic state and that this may be reduced by successful catheter ablation.

OBJECTIVE

Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation.

METHODS

In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation.

RESULTS

Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit.

CONCLUSIONS

The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

In this study, megakaryocytopoiesis was investigated in the recessive mutant mouse, jumonji, obtained by a gene-trap strategy. We investigated the number of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygous embryos by in vitro colony forming assay and monitored colony formation from single megakaryocyte progenitors. We also investigated the differentiation of jumonji-deficient megakaryocytes in terms of the expression of megakaryocyte differentiation markers PF4, CD62P, and GATA-1, proplatelet formation, cytoplasmic maturation, and endomitosis. We found that the population of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygotes increased. A fraction of megakaryocyte progenitors derived from the fetal liver of jumonji homozygotes formed larger colonies in vitro when compared with controls. This abnormality is caused by delayed growth arrest in the progeny. Immature megakaryocyte progenitors showed this abnormality. The megakaryocytes of jumonji homozygotes expressed PF4, CD62P, and GATA-1, obtained cytoplasmic maturation, extended proplatelet-like processes, and underwent endomitosis. The loss of the jumonji gene causes an increase in the number of megakaryocyte lineage cells. Our data suggest that the jumonji gene regulates proliferation but not differentiation of megakaryocyte lineage cells.

When lungs of experimental animals are repeatedly challenged with Ag, pulmonary inflammation wanes via unknown mechanisms. We hypothesized that changes in the balance of lung cytokines are responsible for immune down-regulation to repeated Ag challenge. We used intratracheal (IT) challenge of primed C57BL/6 mice with SRBC and on various days after single (1IT) or triple (3IT) challenge counted lung inflammatory cells and measured whole-lung cytokine mRNA and protein concentrations using RT-PCR and ELISA. We found that lung lymphocyte numbers and parenchymal lung inflammation decreased significantly at days 6 and 9 after final Ag challenge in 3IT mice compared with 1IT mice. Lungs of 3IT mice showed the following changes in relative mRNA expression: an earlier peak in IL-10, decreased IL-1beta, and a change from a Th2 response in 1IT mice to a Th1 response in 3IT mice (with pronounced increases in IL-12, IL-18, and IFN-gamma and decreased IL-4, IL-13, and IL-5). Similar types of changes were seen in whole-lung protein concentrations for TNF-alpha, IL-10, IL-12 p40, IFN-gamma, and IL-4. Additionally, mRNA expression of the endothelial selectins CD62E and CD62P decreased and lung lymphocyte apoptosis increased in the 3IT group. Thus, physiologic down-regulation of the pulmonary immune response to repeated Ag exposure is characterized by increased anti- and decreased proinflammatory cytokines that accompanies Th1 polarization. Similar mechanisms may act to minimize chronic lung inflammation in the majority of normal humans who do not develop progressive lung pathology when repeatedly exposed to inhaled or aspirated environmental Ags.

OBJECTIVE

Enhanced platelet reactivity may play a significant role in the genesis of the hypercoagulable state of nephrotic syndrome. However, the role of platelet function testing in nephrosis is controversial, partly because the methods used to assess platelet function (platelet aggregation and immunoassays of plasma beta-thromboglobulin and platelet factor 4) have such marked methodological problems. In the present study, we evaluated several tests assessing platelet function in 18 adult patients with idiopathic nephrotic syndrome and normal renal function.

METHODS

Platelet function was assessed by measurement of plasma beta-thromboglobulin (enzyme-linked immunosorbent assay, ELISA), plasma P-selectin (ELISA), circulating platelets exposing the activation-dependent antigens P-selectin (CD62P) and lysosomal GP53 (CD63) (flow cytometry), and by aggregation response to agonists such as ADP and collagen. Results were compared to those obtained in a group of 16 age- and gender-matched healthy subjects.

RESULTS

Levels of plasma beta-thromboglobulin (p = 0.001), plasma P-selectin (p < 0.001), and CD62P/CD63-positive platelets (p < 0.001 for both) were increased in nephrotic patients as compared to healthy controls. Platelet hyperaggregability in vitro was found in 13/18 patients. The reproducibility of platelet activation markers, as assessed by blood sample collection a week later from all patients, was found to be higher for plasma P-selectin (Spearman correlation coefficient, R = 0.99) and circulating activated platelets (CD62P: R = 0.97; CD63: R = 0.96) than for plasma beta-thromboglobulin (R = 0.78).

CONCLUSIONS

Pronounced platelet activation takes place in nephrotic syndrome and may contribute to the hypercoagulability of nephrosis. Whole blood flow cytometry assay of platelet activation and plasma P-selectin assay may represent useful tests to assess the hypercoagulable state in nephrotic patients.

One hundred and twenty-two samples from normal volunteers were processed for full blood count analysis through the Bayer ADVIA120. The samples were simultaneously tested using the marker CD62P to establish the absence of platelet activation. Samples were tested at two time intervals, 20 min and 3.5 h, to simulate the testing practice for routine and urgent samples in our laboratory. From these data the mean and reference ranges, with 95% confidence limits, for platelet parameters were calculated for the ADVIA120. The time difference for some platelet parameters was significant. Hence two ranges have been reported. The age or smoking habit of the volunteer did not significantly affect platelet results but there was a statistically significant sex difference in some platelet parameters.

Both the complement system and platelet-leukocyte aggregates are involved in chronic and acute stages of atherosclerosis. Properdin, a positive regulator of the complement system, is secreted by leukocytes and endothelial cells. In the present study, the role of properdin in the formation of platelet-leukocyte aggregates was investigated. Incubation of human whole blood with properdin (25-200 microg/ml) resulted in a dose-dependent formation of platelet-leukocyte aggregates, with an increase of up to 2.2-fold compared to controls (p < 0.05), as analysed by flow cytometry. In addition, properdin significantly amplified ADP-induced aggregation of platelets with leukocytes by 53% (p < 0.05), while it had no effect on ADP-induced aggregation of platelets alone. Consistent with these results, properdin did not activate platelets as shown by the expression of activated GPIIb/IIIa (PAC-1 epitope) and P-selectin (CD62P) on the platelet surface. However, properdin significantly induced expression of CD11b (MAC-1) on leukocytes by 12-fold (p < 0.05) as a measure of leukocyte activation. In conclusion, the complement system component properdin induces the formation of platelet-leukocyte aggregates via leukocyte activation. The data establish a link between the complement system and platelet-leukocyte aggregates with potential significance in atherosclerotic vascular disease.