Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Leukocyte recruitment to sites of inflammation requires the functions of selectins and integrins. P-selectin null (CD62P-/-) mice show a mild and CD18 null (CD18-/-) mice a more severe neutrophil recruitment defect in some inflammatory models. To investigate the possible cooperative interactions between CD18 integrins and P-selectin in mediating neutrophil recruitment, we generated CD18-/-CD62P-/- double null mice. CD18-/-CD62P-/- mice were apparently normal at weaning and fertile but later failed to gain weight, showed increased susceptibility to infection by fecal and commensal bacteria, and survived only 5-6 months. Some CD18-/-CD62P-/- mice showed severe spontaneous skin lesions; most showed neutrophil infiltration in the lungs and liver, and positive bacterial cultures from internal organs. The number and velocity of rolling leukocytes in tumor necrosis factor alpha treated venules of CD18-/-CD62P-/- mice was similar to those in wild-type mice, but neutrophil adhesion was severely reduced. Only 25% of adhered leukocytes were neutrophils in CD18-/-CD62P-/- mice vs. >90% in wild-type, CD62P-/-, and CD18-/- single mutants. Our data show that removing both P-selectin and CD18 integrins from mice leads to severe neutrophil recruitment defects and spontaneous pathology.

BACKGROUND

Atherosclerotic cardiovascular disease is a major cause of morbidity and mortality in patients with end-stage renal disease. This excess morbidity cannot be entirely explained by well-recognized conventional and novel risk factors alone, and occurs irrespective of dialysis modality. Recent evidence suggests that the activation of platelets and their interaction with circulating cells are important independent risk factors for atherosclerosis in non-uraemic patients. We therefore studied platelet activation and circulating platelet-leucocyte aggregates in stable patients without evidence of cardiovascular disease on continuous ambulatory peritoneal dialysis (CAPD) and haemodialysis and investigated an association with cardiovascular events.

METHODS

Immunofluorescent flow cytometry was used to measure the percentage of P-selectin- (CD62P) positive platelets, the percentage of platelet-neutrophil and platelet-monocyte aggregates, and the expression of the P-selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1, CD162) on leucocytes in haemodialysis and CAPD patients and normal controls. The platelet count and the mean platelet component (MPC, a measure of platelet activation) were determined on the ADVIATM 120 Haematology System (Bayer, NY).

RESULTS

Platelet activation as assessed by MPC or CD62P expression was significantly increased in haemodialysis but not CAPD patients compared with controls. Circulating platelet-monocyte aggregates were significantly increased in parallel with a significant reduction in PSGL-1 expression on monocytes in both patient groups compared with normal controls. The presence of higher platelet-monocyte aggregates in dialysis patients was associated with increased cardiovascular events.

CONCLUSIONS

We describe increased platelet-monocyte aggregates with reduced leucocyte PSGL-1 expression in patients with end-stage renal disease irrespective of dialysis modality, associated with an increased risk of cardiovascular disease. These findings may suggest a novel mechanism by which accelerated atherosclerosis occurs in uraemic patients.

We compared the levels of microparticles, platelet activation markers, soluble cell adhesion molecules, soluble selectins, and antioxidized low-density lipoprotein (anti-Ox LDL) antibody between patients with hyperlipidemia and control subjects. Binding of anti-glycoprotein (GP) IIb/IIIa and anti-GPIb monoclonal antibodies to platelets did not differ significantly between the hyperlipidemic patients and controls. However, expression of activation markers (CD62P, CD63, PAC-1, and annexin V) by platelets was higher in the hyperlipidemic patients with Type 2 diabetes. The levels of platelet-derived microparticles (PDMPs) and monocyte-derived microparticles (MDMPs) were significantly different in hyperlipidemic patients with Type 2 diabetes and controls. Soluble P-selectin (sP-selectin), soluble E-selectin (sE-selectin), and anti-Ox LDL antibody also showed higher levels in the hyperlipidemic patients with Type 2 diabetes. After treatment with eicosapentaenoic acid (EPA), the levels of CD62P, CD63, annexin V, PDMPs, and MDMPs, sE-selectin, and oxidized LDL antibody were reduced significantly. Triglyceride (TG) and total cholesterol levels were also decreased. Anti-Ox LDL antibodies and MDMPs were correlated positively with platelet CD62P (plt-CD62P) levels. These findings suggest that in hyperlipidemic patients with Type 2 diabetes, EPA may prevent complications caused by oxidized LDL, E-selectin, and activated platelets or monocytes.

As microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS-PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.

BACKGROUND

Renal injury remains a persistent complication of cardiopulmonary bypass (CPB) that, when sufficient to require dialysis, increases mortality eight-fold. The high prevalence of renal failure in sepsis and adult respiratory distress syndrome has been linked to the systemic inflammatory response associated with those disorders. We hypothesized that components of the inflammatory response to CPB may similarly contribute to post-CPB acute renal injury.

METHODS

Markers of leukocyte and platelet activation peri-CPB were measured in 75 patients undergoing cardiac operation with CPB and were correlated with acute renal injury, defined as an increase >> or = 50%) in peak serum creatinine post-CPB.

RESULTS

Eleven patients sustained post-CPB acute renal injury. This subset of patients demonstrated significantly greater increases in neutrophil CD11b density (p = 0.01), as well as higher total neutrophil counts (p = 0.045), compared with patients with preserved renal function. Hemodynamic instability sufficient to require postoperative hemodynamic support also predicted an increased risk of acute renal injury. However, neutrophil CD11b upregulation did not correlate with this or any other clinical variables associated with renal risk, suggesting that this marker of the neutrophil inflammatory response may independently predict renal injury. By contrast other inflammatory markers, neutrophil myeloperoxidase levels, monocyte CD11b, base line C-reactive protein, and platelet CD62P expression did not differ between the two patient groups.

CONCLUSIONS

Upregulation of the neutrophil adhesion receptor CD11b and high circulating neutrophil numbers are associated with acute renal injury after CPB, suggesting a contribution by activated neutrophils to the pathophysiology of this complication.

BACKGROUND

Increased platelet activation occurs in ischemic heart disease (IHD), but increased platelet activation is also seen in cerebrovascular atherosclerosis and peripheral artery disease. It is not clear therefore whether platelet activation is an indicator of IHD or a marker of generalized atherosclerosis and inflammation. South Asian subjects are at high risk of IHD, but little is known regarding differences in platelet and leukocyte function between European and South Asian subjects.

METHODS

Fifty-four male subjects (age 49-79 years) had coronary artery calcification measured by multislice computed tomography (CT), aortic atherosclerosis assessed by measurement of carotid-femoral pulse wave velocity (aortic PWV), and femoral and carotid atherosclerosis measured by B-mode ultrasound. Platelet and leukocyte activation was assessed by flow cytometry of platelet-monocyte complexes (PMC), platelet expression of PAC-1 binding site and CD62P, and expression of L-selectin on leukocytes.

RESULTS

Elevated circulating PMC correlated significantly with elevated aortic PWV and PMC were higher in subjects with femoral plaques. In contrast PMC did not differ by increasing coronary artery calcification category or presence of carotid plaques. Higher numbers of PMC were independently related to elevated levels of C-reactive protein (CRP), higher aortic PWV, hypertension and smoking in a multivariate model. Markers of platelet and leukocyte activation did not differ significantly by ethnicity.

CONCLUSIONS

Increased PMC are related to the extent of aortic and femoral atherosclerosis rather than coronary or carotid atherosclerosis. The association between elevated CRP and increased PMC suggests that inflammation in relation to generalized atherosclerosis may play an important role in PMC activation.

BACKGROUND

Depression is associated with an increased risk of cardiovascular disease (CVD). Although the mechanism is uncertain, prothrombotic and inflammatory factors may play a role.

OBJECTIVE

As platelets play a key role in CVD, we determined first, whether depressed individuals had more activated platelets than non-depressed individuals and second, whether treatment of depression reduced platelet activation levels.

METHODS

We recruited 108 depressed outpatients and 45 control subjects all without a history of CVD. After psychological assessment, the depressed patients were offered treatment with medication and/or psychotherapy. Flow cytometric markers of platelet activation and level of depression were assessed at baseline and at 4 weeks and 6 months after treatment.

RESULTS

Depression was associated with increased platelet activation with a higher number of circulating CD62p (0.76x10(9) L(-1) vs. 0.46, P=0.019) and CD63 (P=0.05) positive platelets compared with controls. Patients with depression also had more circulating platelet-leukocyte aggregates than controls (P<0.001). There was a positive correlation between the severity of depression and the level of platelet activation. Platelets from depressed patients were also hyperreactive to adenosine 5 -diphosphate (ADP) stimulation with increased CD62p and CD63 exposure (P=0.003 and 0.019, respectively). Six months of treatment resulted in a reduced number of circulating CD62p and CD63 positive platelets (29.84% and 53.38% decrease) and a 20.9% reduction in CD63 exposure after ADP activation.

CONCLUSIONS

Depression is associated with increased in vivo platelet activation and resolution of depression using psychotherapy and/or medication reduces platelet activation. These findings provide insights into the link between depression and cardiovascular risk.

Severe sepsis is associated with high morbidity and mortality rates. Inflammation and coagulation play pivotal roles in the pathogenesis of sepsis leading to multiple organ failure, especially in the liver. The aim of the present study was to assess the mechanism from sepsis to liver damage in a mouse model.

We created a sepsis model by injecting lipopolysaccharide (LPS) intraperitoneally in mice. At 0, 6, 12, and 24 h following intraperitoneal injection of LPS, mice were euthanised and analyzed. Primary antibodies against myeloperoxidase (MPO), hepatic sinusoidal endothelial cells (SE-1), and P-selectin (CD62p) were used. Expression and localization in neutrophil, sinusoidal endothelial, and platelet cells were assessed by immunohistochemistry.

Immunohistochemical analyses revealed a positive staining for MPO, most abundantly in neutrophil granulocytes, within the hepatic sinusoids immediately after injection. Neutrophil extracellular trap (NET)-like structures stained for MPO, indicating the presence of neutrophils undergoing NETosis, were confirmed at 6 h after LPS administration. SE-1 staining for liver sinusoidal endothelial cells was significantly reduced at 12 h post-LPS administration through sinusoidal endothelial injury or detachment. Furthermore, the presence of extravasated platelets was confirmed in the space of Disse at 24 h after LPS administration. Blood sample analyses showed that white blood cell counts and platelet counts decreased gradually, while MPO amounts increased until 12 h after LPS administration.

We conclude that NET formation and intravasated platelet aggregation are the first steps from sepsis to liver damage, and that extravasated platelet aggregation promoted by NET-facilitated detachment of sinusoidal endothelial cells is the origin of sepsis-induced liver dysfunction.

OBJECTIVE

Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke.

METHODS

We consecutively evaluated 205 patients, who admitted the stroke unit with symptoms for stroke. Surface expression of the platelet activation markers (GPVI, CD62P, GPIb) was determined by two-color whole blood flow cytometry.

RESULTS

Patients with transient ischemic attack (TIA) (n = 18; 8.8%) as well as with stroke (n = 133; 64.9%) showed a significantly enhanced GPVI expression (mean fluorescence intensity +/- SD) on admission compared to patients with non-ischemic (NI) events (n = 54; 26.3%) (TIA: 20.9 +/- 7.1 vs. NI: 16.2 +/- 3.9; P = 0.002; stroke: 20.4 +/- 5.7 vs. NI; P = 0.002). Neither CD62P nor GPIb surface expression showed a significant difference. Logistic regression analysis revealed that on admission GPVI was associated with stroke independent of conventional laboratory markers such as C-reactive protein, blood glucose, and creatine kinase. Using a receiver operating characteristic curve on GPVI, we have determined the cut off value of 18.2 for stroke. Thus, patients with enhanced GPVI expression levels >>or=18.2) had a 2.4-fold relative risk for stroke. Patients with elevated platelet GPVI expression level had a poorer clinical outcome in cumulative event-free survival for stroke, myocardial infarction, and cerebro-/cardiovascular death at 3-month follow-up (log rank; P = 0.045).

CONCLUSIONS

These findings indicate that platelet GPVI surface expression is significantly enhanced in patients with TIA and stroke compared to patients with NI events. Determination of platelet-specific GPVI may be useful as an early biomarker for cerebral ischemia.

We evaluated the plasma concentrations of platelet activation markers, microparticles and soluble adhesion molecules in patients with arteriosclerosis obliterans (ASO) and compared the beneficial effects of cilostazol alone and combination therapy of cilostazol and dipyridamole in these patients. There was a significant elevation of CD62P, CD63, PAC-1, annexin V, platelet-derived microparticles (PDMPs), sP-selectin, sE-selectin, sICAM-1 and sVCAM-1 in the ASO patients compared with the controls. Platelet aggregation was decreased by 2 weeks of cilostazol monotherapy in the ASO patients. Adding dipyridamole to the cilostazol therapy for 2 weeks further reduced platelet aggregation. While treatment with cilostazol alone reduced levels of CD62P, CD63, PAC-1, annexin V, PDMP, and sP-selectin, the combination therapy reduced these parameters further. While sE-selectin and cell adhesion molecules did not change significantly after 2 weeks of combination therapy, they exhibited a remarkable decrease after 16 weeks of combination treatment. These findings suggest that platelets are activated in ASO patients, and cilostazol is effective to reduce platelet activation. Furthermore, dipyridamole may potentiate the beneficial effect of cilostazol in ASO patients. Combination use of both drugs may help to prevent the onset of cardiovascular complications in patients with ASO by activated platelets and PDMP.

OBJECTIVE

Inflammatory conditions contribute to increased expression of various activity markers in platelets and endothelial cells, leading to atherosclerotic changes in the vascular wall. The objective of this study was to investigate possible protective effects of 1α,25-dihydroxyvitamin D3 in an endothelial cell model.

METHODS

After a 24-hour incubation with 1α,25-dihydroxyvitamin D3, human umbilical vein endothelial cells were stimulated with lipopolysaccharide (LPS) and incubated in direct contact with platelets. The expression of CD40L and CD62P in platelets, the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), the urokinase receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP) in endothelial cells and endothelial cell reactive oxygen species generation were measured by flow cytometry. Endothelial nitric oxide synthase was analyzed by Western blot.

RESULTS

The increased expression of VCAM-1 and MT1-MMP in endothelial cells by proinflammatory stimulation with LPS and by direct contact with activated platelets was significantly reduced through preincubation with 1α,25-dihydroxyvitamin D3. Platelets in direct contact with preincubated endothelial cells showed significantly reduced CD62P expression when compared to platelets incubated with untreated endothelial cells.

CONCLUSIONS

1α,25-Dihydroxyvitamin D3 attenuates platelet activation and the expression of VCAM-1 and MT1-MMP in human endothelial cells and could have early therapeutic relevance in atherosclerotic diseases.

The use of electronic cigarettes is increasing dramatically on a global scale and its effects on human health remain uncertain. In the present study, we measured endothelial progenitor cells (EPCs) and microvesicles (MVs) in healthy young volunteers following short-term exposure to inhalation of e-cigarette vapor (ECV) to determine vascular changes.

Sixteen healthy seldom smokers were randomized into two groups either exposed or not exposed to 10 puffs of ECV for 10 min, in a crossover design. Blood samples were obtained at baseline and 1, 4 and 24 h following exposure. EPCs (CD34 + CD309) and MVs were analyzed by flow cytometry. MVs were phenotyped according to origin (platelet (CD41), endothelial (CD144), leukocytes (CD45), monocytes (CD14)) and nuclear content (SYTO 13 dye). In addition, expression of inflammation markers such P-selectin (CD62P), E-selectin (CD62E), CD40-ligand (CD154) and HMGB1 was investigated. Fractional exhaled nitric oxide (FeNO) was also measured at baseline and after 24 h.

EPC levels in blood were significantly increased 1 h following exposure to ECV and returned to baseline values after 24 h. Only E-selectin positive MVs (endothelial origin) were slightly elevated (p < 0.038). FeNO was unaffected by exposure to ECV.

In healthy volunteers, ten puffs of e-cigarette vapor inhalation caused an increase in EPCs. This increase was of the same magnitude as following smoking of one traditional cigarette, as we previously demonstrated. Taken together, these results may represent signs of possible vascular changes after short e-cigarette inhalation. Further studies analyzing potential cardiovascular health effects are critical as the e-cigarette market continues to burgeon.

BACKGROUND

Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis.

RESULTS

We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis.

CONCLUSIONS

The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention.

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets' physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald's criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

OBJECTIVE

The aim of this study was to confirm clinically a hypothesis that cilostazol inhibits leukocyte Mac-1, leading to prevention of post-stent restenosis.

BACKGROUND

The platelet phosphodiesterase III inhibitor called cilostazol also inhibits alpha-granule release of P-selectin in platelets. The P-selectin-mediated platelet-leukocyte interaction promotes activation and upregulation of leukocyte Mac-1 after coronary stenting, which plays a key role on the mechanism of restenosis. Thus, cilostazol's potential inhibition of this process may lead to prevention of restenosis.

METHODS

Using flow cytometric analysis of whole blood obtained from the coronary sinus, the expression of platelet membrane glycoproteins and neutrophil adhesion molecules was observed in 70 consecutive patients undergoing coronary stenting. The patients were randomly assigned to either a cilostazol or ticlopidine group before stent placement.

RESULTS

The restenosis rate was lower (15% vs. 31%, p < 0.05) in the cilostazol group (n = 34) than in the ticlopidine group (n = 32). A stent-induced increase in platelet P-selectin (CD62P) expression and an increase in neutrophil Mac-1 (CD11b) expression were suppressed in the cilostazol group compared with the ticlopidine group. Angiographic late lumen loss was correlated with the relative changes in platelet P-selectin and neutrophil Mac-1 at 48 h after coronary stenting.

CONCLUSIONS

Cilostazol may have effects on suppression of P-selectin-mediated platelet activation, platelet-leukocyte interaction, and subsequent Mac-1-mediated leukocyte activation, which might lead to a reduced restenosis rate after coronary stent implantation.

OBJECTIVE

To comparatively study the expressive conditions of platelet activation related factors (GP I b, GP II b- III a and GMP-140) in healthy subjects and patients with coronary heart disease (CHD) of blood-stasis (BS) or non-blood-stasis (non-BS) syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes.

METHODS

With case control design adopted, patients with the CHD (40 of BS, 37 of non-BS) and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity (MFI) of GP I b, GP II b- III a, and GMP-140 (CD42b, CD61, CD62p) in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing.

RESULTS

MFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome (P<0.05); MFI of CD42b was lower in the CHD patients than in the healthy control (P<0.05), but showing insignificant difference between BS and non-BS syndrome (P>0.05); at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GP II b HPA-3 and GP I b HPA-2 polymorphism loci (P>0.05).

CONCLUSIONS

(1) The activities of GP II b- III a and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. (2) The level of GP I b was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. (3) Levels of GP II b- III a, GP I b and GMP-140 were not related with the number of affected coronary branches in CHD patients. (4) The changes in amino-acids expression induced by the two loci brought no significant influence on GP I b and GP II b- III a activities.

We investigated the effects of probucol and ticlopidine on circulating levels of platelet activation markers, microparticles, soluble selectins, and malondialdehyde-low density lipoprotein (MDA-LDL) in hyperlipidemic patients with or without type 2 diabetes. There were significant differences in the levels of CD62P, PAC-1, annexin V, PDMP, MDMP, sP-selectin, sE-selectin and MDA-LDL between the hyperlipidemic patients and the controls. In particular, these markers were significantly increased in hyperlipidemic patients who had type 2 diabetes. In the hyperlipidemic patients with diabetes, MDA-LDL was decreased by both monotherapy with probucol and combination therapy (probucol and ticlopidine). In these patients, CD62P, PAC-1, annexin V, MDMP, PDMP, sP-selectin, and sE-selectin were also significantly decreased after treatment. The decreases of CD62P, PAC-1, annexin V, PDMP and sP-selectin were greater combination therapy than with monotherapy. These findings suggest that administration of probucol and ticlopidine to hyperlipidemic patients with type 2 diabetes may help to prevent the development of cardiovascular complications caused by modified LDL, selectins, or activated platelets and monocytes.

BACKGROUND

Recent studies have demonstrated that infused platelets (PLTs) can promote inflammation. The objective of this study was to evaluate the impact of storage of transfusion-grade PLTs on the peripheral blood mononuclear cells (PBMNCs) of the recipient.

METHODS

An in vitro cell model system was established to measure the degree of activation of donor PLTs during 5 days of their storage and then to measure immune cell activation by detecting marker expression in coculture experiments.

RESULTS

The level of soluble CD62p increased significantly by Day 3, and membrane expression of CD62p increased significantly from Day 2, indicating some degree of PLT activation over time during storage (p < 0.05). Donor PLTs and PBMNC subsets (monocytes, B cells, and T cells) from recipients were cocultured for 48 hours. The number of PLT-PBMNC subset doublets detected by flow cytometry was correlated with the PLT storage time after Day 3 (p < 0.05), indicating consistent binding of PLTs to PBMNCs. The results of these experiments showed that there was a consistent and significant increase in expression of conventional activation markers of T cells, B cells, and monocytes compared with appropriate controls (p < 0.05 to <0.01).

CONCLUSIONS

The results of this study indicate that, from Day 3 onward, activation markers are consistently expressed on PLTs. From these results, we conclude that activated PLTs may affect PBMNC interactions in recipients.

We evaluated the significance of platelet activation in patients with rheumatoid arthritis (RA). The expression of CD62P and CD63 by platelets was determined using flow cytometry in 18 active RA patients, 10 remission RA and 15 normal controls. Meanwhile, the erythrocyte sedimentation rate (ESR) and C-reactive protein was also determined in all groups. The expression of CD62P in active RA patients (11.88 +/- 2.47%) was significantly higher than that in remission RA group (2.85 +/- 1.60%; P < 0.01) and control group (2.78 +/- 1.04%; P < 0.01). The expression of CD63 in active RA patients (9.90 +/- 3.02%) was significantly higher than that in remission RA group (4.11 +/- 2.00%; P < 0.01) and control group (4.13 +/- 1.85%; P < 0.01). The level of CRP (54.33 +/- 23.35 mg/l) and ESR (86.06 +/- 33.67 mm/h) in active RA patients was higher than that in remission RA group (2.55 +/- 1.01 mg/l, 14.70 +/- 4.57 mm/h; P < 0.01 for both) and normal control group (3.21 +/- 2.18 mg/l, 12.25 +/- 5.05 mm/h; P < 0.01 for both). There was a positive correlation between CD62P and ESR (r = 0.5224, P < 0.01) and also a positive correlation between CD62P and CRP (r = 0.7048, P < 0.01) as well as between CD63 and ESR (r = 0.4476, P < 0.05) but no correlation between CD63 and CRP. Platelet activation may be a sign of RA exacerbation.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Some malignant tumors induce a cellular immune response that results in the formation of an inflammatory infiltrate and subsequent tumor regression. The infiltrating leukocytes extravasate from the bloodstream after binding to adhesion receptors on the surface of the endothelium. One of these receptors is the P-selectin molecule (CD62P) that is constitutively present on normal capillaries. We observed that P-selectin expression is absent from the microvasculature in advanced primary melanoma and in melanoma metastasis in contrast to benign melanocytic lesions where P-selectin expression was identical to that in normal skin. We suggest that one of the mechanisms by which advanced melanoma lesions evade inflammatory regression operates via a decrease of endothelial P-selectin expression.

We evaluated the plasma concentrations of soluble adhesion molecules and platelet-derived microparticles (PMP) in patients with non-insulin dependent diabetes mellitus (NIDDM) and studied the effect of cilostazol on PMP generation. There were differences in the levels of soluble adhesion molecules between NIDDM patients (N = 43) and the control subjects (N = 30) (soluble thrombomodulin: 11.5+/-5.3 vs. 7.0+/-1.2 TU/ml, p<0.0001; soluble vascular cell adhesion molecule-1: 708+/-203 vs. 492+/-113 ng/dl, p<0.0001; soluble intercellular cell adhesion molecules- 1: 274+/-65 vs. 206+/-48 ng/dl, p<0.0001; soluble P-selectin: 194+/-85 vs. 125+/-43 ng/dl, p<0.0001). There were also differences in the levels of PMP and platelet activation markers between NIDDM patients and the controls (PMP: 943+/-504 vs. 488+/-219/10(4) plt, p<0.0001; platelet CD62P: 9.2+/-4.6 vs. 4.4+/-4.3%, p<0.001; platelet CD63: 10.2+/-4.5 vs. 4.5+/-3.3%, p<0.0001; platelet annexin V: 9.1+/-3.9 vs. 5.3+/-3.8%, p<0.001). To study the release of PMP into plasma, a modified cone-and-plate viscometer was used. Increased release of PMP from platelets was observed in diabetic plasma compared to normal plasma under high shear stress conditions (2,672+/-645 vs. 1,498+/-386/10(4) plt, p<0.05). Therefore, one cause of PMP elevation in NIDDM may be high shear stress. The levels of PMP, activated platelets, and soluble adhesion molecules all decreased significantly after treatment with cilostazol. These results suggest that cilostazol may be useful for the inhibition of both PMP-dependent and -independent vascular damage in NIDDM.