Platelet CD62P Expression Research Articles

VacA, the vacuolating cytotoxin of Helicobacter pylori, binds to multimerin 1 on human platelets

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive experiments, as the binding protein for VacA. Plasmon resonance confirmed their binding, and dot blot analysis revealed that the peptide sequence AA 321-340 of multimerin 1 is the binding site for VacA. In conclusion, we propose a new interaction between multimerin1 and VacA , which may give another insight into H. pylori-induced platelet activations under H. pylori infection.

Evaluation of therapeutic efficacy on combined use of clopidogrel and ozagrel sodium in the treatment of acute ischemic stroke

Background Antiplatelet aggregation treatment has become a regular treatment of ischemic stroke. The affirmation of antiplatelet therapy is mainly derived from patients with clinical use, which can not provide the laboratory indexes for evaluation of a recognized accuracy. Studies have confirmed that the degree of platelet activation is associated with atherosclerosis and ischemic stroke, and recognized that both CD62p (α-platelet granule membrane glycoprotein) and CD63 (lysosomal membrane glycoprotein) were important indexes of platelet activation. This study aims to explore the differences of efficacy between combined use of clopidogrel and ozagrel sodium and monotherapy by aspirin in the treatment of acute ischemic stroke by investigating the expression of CD62p and CD63. Methods Flow cytometry was employed to detect CD62p and CD63 expression on circulating platelet in patients with ischemic stroke and normal control group. The positive rate of CD62p and CD63 was detected in patients with ischemic stroke who were treated with aspirin 0.15 g (single drug therapy) and clopidogrel 75 mg + ozagrel sodium 80 mg (combination therapy) before and after one and two weeks' treatment. National Institute of Health Stroke Scale (NIHSS) scores were measured in patients with ischemic stroke at the same time in three periods respectively to evaluate the improvement of neural function. Results Platelet CD62p and CD63 positive expression rate in ischemic stroke group were higher than normal control group before treatment (P = 0.001, 0.032). CD62p and CD63 positive expression rate and NIHSS score were measured at different times, and the differences were statistically significant (F = 56.693, P = 0.000; F = 21.544, P = 0.000; F = 216.271, P = 0.000, respectively). Compared with before treatment, CD62p and CD63 positive expression rate and NIHSS score decreased significantly after treatment (P = 0.000, for all), but the differences between aspirin group and combination group were not statistically significant (P > 0.05, for all). There was no interaction between the treatment groups and measuring time with CD62p and CD63 positive expression rate (F = 1.403, P = 0.250; F = 2.830, P = 0.063), while there was interaction between treatment groups and measuring time with NIHSS score (F = 4.518, P = 0.013). Conclusion Antiplatelet drug treatment of acute ischemic stroke is effective. The curative effect of combined treatment (clopidogrel and ozagrel sodium) is not superior to aspirin alone. CD62p positive expression rate in acute stage of ischemic stroke can measure the effect of antiplatelet therapy, while the determination of CD63 needs further research.

The apoptotic actions of platelets in acute ischemic stroke

Stroke is a disease that affects the blood vessels that supply blood to the brain. Although platelets are implicated in the pathophysiology of stroke the mechanism is still not clear and there antiplatelet agents available for the prevention and treatment of stroke. We herein examined the relationship between the potential cytokine, TNF-α platelet activation and apoptosis in acute ischemic stroke patients. We selected 60 patients (mean age 57.9±10.2years) who had not taken any antiplatelet drugs for 14days. A group of 45 participants (mean age 51.05±9.07years) were selected as the control group. For both the patients and for the control group, P-selectin (CD62p) and Annexin-V binding, cytochrome-c levels, caspase-3 gene expression and caspase-3 releasing and plasma TNF-α levels were measured in platelets. The results showed significant increase in plasma TNF-α and platelet Annexin-V, CD62p, cytochrome-c and caspase-3 gene expression in stroke patients compared to the control group. The data of this work suggests that inflammation may have a role in platelet apoptosis in stroke which may suggest a new aspect of the role of inflammation in the development of acute ischemic stroke.

Newly diagnosed depression is associated with increased beta-thromboglobulin levels and increased expression of platelet activation markers and platelet derived CD40-CD40L

BackgroundInflammation plays a key role in atherosclerotic disease. Up until now only limited evidence exists on the mechanism of cardiovascular complications in patients with depression. In addition depression was also linked to an increase in cardiovascular mortality. The present study was designed to evaluate the extent of platelet activation and platelet-derived markers of atherosclerotic disease in patients with newly diagnosed depression. MethodsThis study used whole blood aggregometry, flow cytometry and ELISA to investigate platelet CD62P (P-selectin) expression and atherosclerotic markers (CD40, CD40L) as well as serum platelet factor 4 (PF-4) and beta-thromboglobulin (β-TG) levels in 46 participants. Patients with newly diagnosed, but not yet medically treated depression (n = 21) were compared to healthy control patients. ResultsThe platelet activation marker CD62P was significantly higher in patients with depression (2.62% depression versus 1.27% controls; p = 0.006). Further we found basal CD40 (6.7% vs. 4.8%; p = 0.002) and basal CD40L (31.0% vs. 22.0%; p = 0.025) to be elevated in patients with depression as compared to control persons. In addition sCD40L (52.7 vs. 44.4 ng/ml; p = 0.023) and β-TG differed significantly in depressed patients (206.9 vs. 182.8 ng/ml; p = 0.001). However, basal CD41 (97.0% vs. 96.3%; p = 0.57), CD42b (96.7% vs. 94.7%; p = 0.28) and PF-4 (89.61 vs. 81.75 IU/ml; p = 0.10) and the aggregometry results did not differ significantly between the study groups. ConclusionsOur findings with elevated CD40 and CD40L as well as CD62P and β-TG in newly diagnosed patients emphasize that depression is linked to a prothrombotic and proinflammatory state and this possibly contributes to accelerated atherosclerosis.

The basis examination of leukocyte–platelet aggregates with CD45 gating as a novel platelet activation marker

Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 μm thrombin receptor-activated peptide (TRAP) or 2 μg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.

Renal function and aspirin resistance in patients with coronary artery disease

Aspirin resistance and chronic renal failure are both potentially important clinical issues in coronary artery disease. To test the hypothesis of a relationship between the two, we recruited 169 stable outpatients with proven coronary artery disease (myocardial infarction, coronary artery bypass grafting, intra-coronary stents) taking 75mg aspirin daily. Blood was taken for light transmission aggregometry to agonists arachidonic acid (0.5mg/mL) and adenosine diphosphate (10μmol/L), for platelet marker soluble P selectin (enzyme linked immunosorbent assay), resting and stimulated expression of CD62P (flow cytometry) and for renal function (estimated glomerular filtration rate). The estimated glomerular filtration rate was lower when aspirin resistance was defined by response to arachidonic acid after 3, 5 and 7minutes (approximately 30% of patients) (p<0.021), and when defined by response to adenosine diphosphate after 3minutes (approximately 17% of patients)(p=0.015) compared to those who were sensitive to aspirin. Mean [standard deviation] soluble P selectin levels were 57 [23] ng/mL in 49 patients with aspirin resistance, and 50 [15] ng/mL in the 119 aspirin sensitive patients (p=0.02). Estimated glomerular filtration rate correlated inversely with platelet CD62P expression at rest (r=−0.22, p=0.004), and when stimulated by arachidonic acid (r=−0.21, p=0.007) and by adenosine diphosphate (r=−0.17, p=0.023). Aspirin resistance was more than twice as prevalent in those with the greatest renal disease (50% of patients) compared to those with the best renal function (21.4%). Our data point to a weak relationship between worsening glomerular filtration rate and aspirin resistance. Nevertheless, we suspect that failure of patients to be fully responsive to aspirin may be important in the pathophysiology of thrombosis in renal dysfunction.

Treatment with aspirin or clopidogrel does not affect equine platelet expression of P selectin or platelet–neutrophil aggregates

Inflammation-induced P-selectin (CD62P) expression on platelets and endothelial cells facilitates interactions among platelets and polymorphonuclear leukocytes (PMN), and can also promote coagulation. The effects of clopidogrel and aspirin (ASA) on equine platelet CD62P expression were investigated. Six horses were treated in a cross-over design with clopidogrel (2mg/kg PO q 24) or ASA (5mg/kg PO q 24h) for 5 days. Platelets collected at 24, 72, 96, 120, and 168h after the initiation of therapy were stimulated using 0.1μg/mL thrombin, followed by flow cytometric analysis using anti-CD41/61 and anti-equine CD62P antibodies. Platelet–PMN aggregates were also enumerated. Baseline CD62P positive platelet numbers were not different between groups (mean±SD): 4254±1785 (clopidogrel) and 3600±1780 (ASA, P=0. 435). Although expression tended to decrease, there were no significant changes in CD62P+platelets after treatment with either drug (clopidogrel P=0.139, ASA P=0.161). There was also no difference in platelet–PMN aggregates during or after treatment with ASA (P=0.513) or clopidogrel (P=0.543). Due to small numbers of horses, this study may have been underpowered to detect a true decrease in expression, and differences between therapies may have been more pronounced if this study had evaluated horses with systemic inflammation.

Role of mitochondria in the maintenance of platelet function during in vitro storage

Platelets undergo structural and biochemical alterations during in vitro storage and these are collectively called platelet storage lesions (PSL). The mitochondrion is an important cell organelle involved not only in energy production but also in the regulation of cellular functions and viability. This implies that some platelet functions may be regulated by mitochondria; hence, preservation of mitochondrial functions may be important for the maintenance of platelet quality in stored platelet concentrates (PCs). This work describes the effects of various compounds on mitochondrial functions important for the maintenance of platelet quality in in vitro stored PCs. PCs were stored at 22 °C with gentle agitation in the presence or absence of 2,4-dinitrophenol, antimycin A, acetyl-l-carnitine and ascorbic acid. The effects of these products on platelet quality were assessed by analysing glucose and lactate concentrations, pH of the storage medium, shape of the platelets, mitochondrial membrane potential and depolarisation, surface expression of CD62P and collagen-induced platelet aggregation. 2,4-Dinitrophenol and antimycin A increased PSL levels, whereas acetyl-l-carnitine reduced the level of changes in pH and mitochondrial depolarisation. Ascorbic acid in the storage medium resulted in improved levels of collagen-induced platelet aggregation. However, none of the examined reagents suppressed CD62P expression in platelets. These results suggest that preservation of mitochondrial function is fundamental, but not fully sufficient, for the maintenance of platelet in vitro quality during storage. Further research is necessary to develop methods for preserving both mitochondrial and platelet functions in in vitro stored PCs.

Activation of platelets and cellular coagulation in cerebral small-vessel disease

Platelets and monocytes play a pivotal role in the initiation and progression of large-vessel atherosclerosis. An up-regulation of various platelet and coagulation activation markers has been described in cardiovascular diseases and in patients with acute cerebral ischemia. In the present study the role of platelets and cellular coagulation activation in cerebral small-vessel disease (cSVD) was assessed. In 24 patients with cSVD but without established large-vessel disease, whole blood samples were obtained. Patients were divided into three subgroups (Fazekas 1, 2 and 3) according to extent of cSVD based on morphological magnetic resonance imaging criteria. Surface expression of CD40L and CD62P on platelets, tissue-factor exposition on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Plasma levels of soluble CD40L, interleukin (IL)-6 and IL-7 were assessed by ELISA. Patients with cSVD show a significantly elevated expression of platelet CD40L (P < 0.001) and CD62P (P < 0.023), significantly elevated amounts of platelet-monocyte aggregates (P < 0.004), a significantly enhanced tissue-factor exposition on monocytes (P < 0.019) and significantly lower plasma levels of IL-7 compared to 10 healthy controls. However, this platelet and monocyte activation did not correlate with the severity of cSVD. Patients with cSVD show an up-regulation of the platelet CD40L and CD62P system and an activation of cellular coagulation which might contribute to the initiation and progression of cSVD.

Influence of treatment with ultralow-dose aspirin on platelet aggregation as measured by whole blood impedance aggregometry and platelet P-selectin expression in clinically normal dogs

To evaluate the influence of treatment with ultralow-dose aspirin (ULDAsp) on platelet aggregation, P-selectin (CD62P) expression, and formation of platelet-leukocyte aggregates in clinically normal dogs. 18 clinically normal dogs. Studies were conducted before and 24 hours after ULDAsp administration (0.5 mg/kg, PO, q 24 h, for 2 days). Whole blood impedance aggregometry for the assessment of platelet function was performed with sodium citrate-anticoagulated blood and aggregation agonists (ADP at 20, 10, and 5 μmol/L; collagen at 10, 5, and 2 μg/mL). Onset, maximum response, and rate of platelet aggregation were recorded. Flow cytometric assays were configured to detect thrombin-induced CD62P expression and platelet-leukocyte aggregates in EDTA-anticoagulated whole blood. Externalized platelet CD62P and constitutive CD61 (GPIIIa) were labeled with antibodies conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC), respectively. Red blood cell-lysed paraformaldehyde-fixed EDTA-anticoagulated whole blood was dual labeled with CD61-FITC and a panleukocyte antibody (CD18-PE) to characterize platelet-leukocyte aggregates. ULDAsp significantly delayed platelet aggregation onset with ADP at 20 μmol/L by 54% to 104%, attenuated maximum aggregation with various concentrations of ADP and collagen by ≥ 41%, and slowed aggregation rate with the highest ADP and collagen concentrations by ≥ 39%. Depending on the parameter tested, up to 30% of dogs failed to have an ULDAsp effect. Thrombin stimulation significantly increased CD62P expression in platelets and platelet-leukocyte aggregates, but ULDAsp did not alter basal or thrombin-stimulated CD62P expression. ULDAsp treatment of clinically normal dogs impaired platelet aggregation in most dogs, but did not influence CD62P platelet membrane expression.

Exaggerated platelet reactivity to physiological agonists in war veterans with posttraumatic stress disorder

An association between traumatic stress and cardiovascular disease (CVD) is supported by various epidemiological studies. Platelet activation and binding of activated platelets to leukocytes contributes to the pathophysiology of CVD. Evidence of hyperactive sympathetic nervous system, altered expression of platelet α(2)-adrenoreceptors (α(2)AR), and altered platelet adenylate cyclase activity in patients with posttraumatic stress disorder (PTSD) suggest that platelet reactivity in PTSD may be altered as well. We tested whether platelet reactivity to increasing doses of adenosine-diphosphate (ADP), epinephrine (EPI), or their combination differs between war veterans with PTSD (n=15) and healthy controls (n=12). For this purpose, citrated whole blood was incubated with increasing concentrations of ADP (0.1, 1, 10 μM), EPI alone (10 nM, 100 nM, 1000 nM), or EPI (10 nM, 100 nM, 1000 nM) in combination with 0.1 μM ADP. A subset of samples was also incubated with 10 μM yohimbine (YOH), α(2)AR antagonist, to distinguish receptor-specific effects. Platelet CD62P expression and formation of platelet-leukocyte aggregates (PLA) [platelet-monocyte (P-Mo), -lymphocyte (P-Ly), and -neutrophil (P-Ne) aggregates] were measured using three-color flow cytometry. Platelet reactivity was higher in war veterans with PTSD when compared to controls, as determined by greater CD62P expression and formation of PLA in response to ADP alone or in combination with EPI. Platelet reactivity also correlated with the severity of PTSD symptoms. Preliminary experiments with YOH indicate that stress-associated EPI elevations may contribute to platelet activation through a α(2)AR-dependent mechanism. The enhanced platelet reactivity observed in our study may be the underlying mechanism contributing to the development of CVD in PTSD patients.

Might platelet-leucocyte complexes be playing a role in major vascular involvement of Behçet's disease? A comparative study

We determined platelet-leucocyte complexes, which play roles in the thrombosis-inflammation relationship, in Behçet's disease patients with and without major vascular involvement (MVI) and in healthy controls. We included 36 Behçet's disease patients (22 male, 14 female, mean age: 34.4 +/- 8.3 years) and 20 healthy individuals (14 male, six female, mean age: 31.8 +/- 4.4 years). Whole blood count, CRP and ESR were determined in both groups. Clinical data about the patients were obtained from medical charts. Individuals with hypertension, diabetes, coronary artery disease, and smokers were excluded. Behçet's disease patients with MVI were taken as a separate group (8 male, 5 female, mean age: 37 +/- 8 years). MVI was defined as the presence of pulmonary arterial aneurysm, deep venous thrombosis, vena cava inferior or superior thrombosis, or venous sinus thrombosis. Flow cytometry was used to determine platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), basal and adenosine diphosphate (ADP)-stimulated platelet CD62P expression. Behçet's disease patients with MVI had significantly higher PNC than Behçet's disease patients without MVI and healthy controls (P values = 0.01). PMC levels in Behçet's disease patients with MVI were significantly higher than in healthy controls (P = 0.01). The groups were similar in basal and ADP-stimulated platelet CD62P expression (P values >0.05). Basal and ADP-stimulated CD62P expression, PMC and PNC were not significantly different between active Behçet's disease versus inactive Behçet's disease patients. The evaluated parameters were similar in Behçet's disease patients with and without uveitis, and pathergy-positive and pathergy-negative groups. Our results might suggest that the formation of PMC and PNC might play a role in thrombosis and MVI of Behçet's disease.

Expression of EMMPRIN (CD147) on circulating platelets in vivo.

EMMPRIN (CD147) is a matrix metalloproteinase inducer present on leukocytes and recently identified on platelets in vitro. We examined platelet CD147 expression in vivo and in correlation with markers of platelet activation and coronary artery disease (CAD). This prospective observational study involved 70 subjects (55 patients with CAD and 15 controls). Platelet CD62P expression, PAC-1 expression, platelet-leukocyte aggregates and CD147 (both platelet and leukocyte) expression were assessed by flow cytometry, and soluble CD62P expression was assessed by enzyme-linked immunosorbent assay. A full blood count and high-sensitivity C-reactive protein test were performed. CD147 was expressed on 20.45% +/- 1.63% (mean +/- standard error of the mean) of circulating platelets, whereas CD62P and PAC-1 were expressed on 0.87% +/- 0.12% and 0.90% +/- 0.27% of platelets, respectively. Platelet CD147 expression correlated with CD62P expression (r = 0.359, P = 0.002), PAC-1 expression (r = 0.428, P < 0.001), leukocyte CD147 expression (monocyte, r = 0.416, P = 0.001; granulocyte, r = 0.434, P < 0.001), C-reactive protein level and neutrophil/lymphocyte ratio (NLR). CAD patients had significantly higher CD147 mean fluorescence intensity than controls on circulating platelets (2.41 +/- 0.14 vs. 2.87 +/- 0.09, P = 0.014), monocytes (8.57 +/- 1.20 vs. 12.3 +/- 0.57, P = 0.006) and granulocytes (4.30 +/- 0.65 vs. 6.50 +/- 0.34, P = 0.005). Age adjustment eliminated the association between platelet CD147 expression and CAD, but the association between leukocyte CD147 expression and CAD persisted. According to multivariate analysis, the independent predictors of platelet CD147 expression were monocyte CD147 expression, NLR and age. Platelet CD147 expression is evident in vivo and correlates moderately with traditional platelet activation markers and leukocyte CD147 expression. Platelet CD147 expression shows a stronger association with age, and leukocyte CD147 expression a stronger association with clinical CAD, suggesting differences in the regulation of platelet and leukocyte CD147 expression in vivo.

Changes in HDL-associated apolipoproteins relate to mortality in human sepsis and correlate to monocyte and platelet activation

Lipoproteins modulate vascular cell function in inflammation. In this study, we analyzed whether plasma concentrations of lipoproteins and apolipoproteins in human sepsis are related to patient survival and the activation of blood monocytes and platelets. Observational study. Medical and surgical intensive care units (ICU) of a university hospital. 151 consecutive patients after sepsis criteria had been met for the first time. None. Plasma lipoproteins, apolipoproteins, platelet CD62P-expression, monocyte HLA-DR-expression, SAPS II-scores (Simplified Acute Physiology Score) and 30-day-mortality were recorded. Total cholesterol, high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) cholesterol, apolipoprotein (apo)-AI and apo-B were all found to be significantly lower in non-survivors than in survivors. In contrast to other (apo)lipoproteins, apo-AI and HDL cholesterol further decreased in non-survivors during the ICU stay. Logistic regression analysis revealed apo-AI to be an independent predictor of 30-day-mortality. A significant inverse correlation was found for apo-AI/HDL-cholesterol and platelet activation. Later in the course of the disease, HLA-DR expression on monocytes correlated positively to apo-AI and apo-CI concentrations and inversely to the apo-E concentration. Low apo-AI is independently related to 30-day mortality in human sepsis and the decrease in apo-AI/HDL cholesterol correlates to increased platelet activation. Moreover, changes in apolipoproteins supposed to modulate lipopolysaccharide effects, such as apo-CI and apo-E, correlate to monocyte activation.

Platelet Interaction with Bone Marrow Derived CD34+ Stem Cells: Potential Role of Chemoattractant Chemerin

Chemerin is a 16 kDa chemoattractant circulating in blood in an inactive form (prochemerin). Its chemotactic activity is released following proteolytic cleavage of its labile carboxyl-terminal amino acids by proteases of the coagulation, fibrinolytic and inflammatory cascades. Chemokine-like receptor 1 (CMKLR1) is a chemerin receptor expressed on plasmacytoid dendritic cells, natural killer cells and tissue macrophages. We identified the expression of CMKLR1 on mouse CD34 positive (CD34+) bone marrow-derived stem cells (BMSC) using anti-mouse CMKLR1 antibody by FACS. Fluorescein-labeled CD34+/CMKLR1+ BMSC adhered to immobilized chemerin dose-dependently. The EC50 of BMSC adhesion to human and mouse chemerin is 2.5 and 5 ug/mL respectively. BMSCs also adhered to SDF-1 (stromal cell-derived factor-1) with EC50 of 0.3 ug/mL, but did not bind to immobilized fibronectin. We have previously reported that platelets express chemerin and release it upon activation. CMKLR1 is also expressed on platelet surface as demonstrated by FACS. Immobilized chemerin could induce platelet adhesion and CD62P (p-selectin) expression as detected by fluorescein-labeled anti-CD62P antibody. BMSC formed complexes with both resting and activated platelets as demonstrated by fluorescein-labeled washed platelets incubated with unlabeled mouse CD34+/CMKLR1+ BMSCs using FACS analysis. Both anti-chemerin and anti-CD62P antibodies could partially block BMSCs binding to immobilized platelets at 50% and 35% respectively. In conclusion, chemerin directly mediates platelets interaction with BMSCs. Chemerin also facilitates platelet/BMSC complex formation by inducing platelet p-selectin (CD62P) expression. Platelet-derived chemerin, similar to SDF-1, may be involved in tissue regeneration by inducing progenitor cells mobilization and differentiation.

Increased platelet activation markers in rheumatoid arthritis: Are they related with subclinical atherosclerosis?

Atherosclerotic cardiovascular mortality is increased in rheumatoid arthritis (RA) patients. We evaluated the association of inflammatory response with platelet, endothelial, coagulation activation parameters; and subclinical atherosclerosis in RA patients. We included 27 RA patients (21 female; six male) and 19 healthy subjects (14 female; five male). Disease activity score (DAS28) in RA patients was calculated; and patients were divided into two groups as active and inactive. Flow cytometry was used to determine platelet CD62P expression, platelet microparticles (PMP), platelet-monocyte (PMC) and platelet-neutrophil complexes (PNC). Plasma E-selectin, thrombin-antithrombin (TAT) complex, and serum sCD40L levels were determined by ELISA. The intima-media thickness (IMT) of carotid arteries was determined by B-mode ultrasonography. In RA patients, platelet CD62P expression (p < 0.001), PMC (p = 0.037) and sCD40L (p < 0.001) levels were increased when compared to the control group. PNC (p = 0.07) and TAT levels (p = 0.1) were non-significantly higher, and PMP level (p = 0.075) was nonsignificantly lower in RA patients. Soluble E-selectin level was significantly higher in the active RA group than in the inactive RA group (p = 0.009). There was no correlation between carotid IMT and activity markers, the evaluated parameters (p > 0.05).The increase in markers of active platelets, CD62P and sCD40L, and PMC levels might be associated with the increased cardiovascular mortality in RA. Nevertheless, none of these parameters were associated with carotid IMT: this suggests that one cross-sectional value might not be a good marker for atherosclerosis.

Human platelets can activate peripheral blood B cells and increase production of immunoglobulins

Blood platelets represent a link between hemostasis, inflammation, and tissue repair. Their role in immune responses and inflammation mainly involves many molecules, among which Toll-like receptor, major histocompatibility complex class I, CD40 and CD154/CD40 ligand (CD40L). As platelets are the major purveyor of soluble CD40L (sCD40L), we sought to determine their involvement in CD40/CD40L-dependent immune responses and to understand the interactions between platelets and peripheral B lymphocytes. We examined the capacity of platelets to bind nonstimulated B cells, and phenotypic changes by flow cytometry and confocal scanning laser microscopy. Modulation of cytokines/chemokines and total levels of immunoglobulin (Ig) A, IgG, IgM, and IgG subclasses in supernatants of coculture, platelets, and B lymphocytes was performed by sandwich enzyme-linked immunosorbent assay and differential production of cytokine mRNA as determined by reverse transcriptase polymerase chain reaction. In coculture, platelets and B lymphocytes were mutually activated, as demonstrated by the increased expression of platelet CD62p and B-cell CD86. Platelet/B-cell interactions were accompanied by changes in membrane expression of CD40 and CD40L by both platelets and B lymphocytes. IL12p70 and IL8 gene transcription were significantly reduced, which was attributable to B cells. Conversely, there was a significant, platelet-dependent reduction of sCD40L and RANTES mRNA expression. After a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgM. These data emphasize the potentially important role of platelets in the adaptive immune response. Platelets have an immunoregulatory role that might be applied clinically in multitransfused patients (e.g., hematopoietic stem cell transplantation).

Increased circulating platelet–leucocyte complexes in patients with primary Raynaud's phenomenon and Raynaud's phenomenon secondary to systemic sclerosis: a comparative study

Platelet activation and circulating platelet-leucocyte complexes increase in vascular ischemic events and autoimmune inflammatory diseases. Platelet activation markers and platelet-leucocyte complexes were evaluated in primary Raynaud's phenomenon (RP) and in RP secondary to systemic sclerosis (SSc). Whole-blood flow cytometry was utilized to quantify CD62P, platelet microparticles (PMP), platelet-monocyte complexes (PMC) and platelet-neutrophil complexes (PNC) in primary RP and in SSc patients with secondary RP. SSc patients with secondary RP had significantly higher platelet CD62P expression than primary RP patients and controls (P = 0.017 and 0.004, respectively). Primary and secondary RP patients had higher mean PMC and PNC levels than controls (all P < or = 0.001). PMP level in SSc patients with pulmonary hypertension was significantly higher than in others (P = 0.048). All parameters were similar in SSc patients with and without digital ulcers, aspirin-users and nonusers (P > 0.05). CD62P level decreased significantly after iloprost administration in four patients with digital ulcers (16.1 +/- 17.4 vs 7.4 +/- 3.8%, P = 0.03). Our results suggest there is platelet-leucocyte complex formation in RP, and, despite antithrombotic therapy, platelet activation and platelet-leucocyte interaction are ongoing in SSc. This is important as it might have potential therapeutic implications with respect to using antiplatelet drugs in SSc.

PDMS content affects in vitro hemocompatibility of synthetic vascular grafts

An unsolved problem when employing small-diameter vascular grafts for aorto-coronary by-pass and peripheral reconstruction is the early thrombotic occlusion. The PEtU-PDMS is a new elastomeric material, composed of poly(ether)urethane and polydimethylsiloxane, synthesized to realize grafts with improved hemocompatibility characteristics. In order to investigate the effect of PDMS content on hemocompatibility, three different percentages of PDMS containing grafts (10, 25 and 40) were evaluated. Grafts realized with Estane 5714-F1 and silicone medical grade tubes were used as references. The hemocompatibility was investigated by an in vitro circuit in which human anticoagulated blood was circulated into grafts by a peristaltic pump modified to obtain a passive flow. For each experiment, 40 cm length graft was closed into a circular loop and put in rotation for 2 h at 37 degrees C. At the end of the experiments different parameters regarding platelet adhesion and activation were evaluated: circulating platelets count, beta-thromboglobulin release, platelet CD62P expression and amount of monocyte-platelet conjugates. PEtU-PDMS grafts with 25 and 40% of PDMS induced the lowest platelet adhesion, plasma level of beta-TG and amount of monocyte-platelet conjugates. No significative variations were observed in CD62P expression. In conclusion, PDMS content significatively affects blood-graft surface interaction, in fact higher PDMS percentage containing grafts showed the best in vitro hemocompatibility.

The Effects of Glycoprotein IIb/IIIa Antagonist RGDS on Platelet Aggregation and Release Reaction In Vitro.

Platelet activation, including platelet adhesion, platelet aggregation and platelet release reaction, played an important role in thrombogenesis. We all knew that Platelet glycoprotein IIb/IIIa antagonist was the most effective drug for anti-aggregation, while we don't know clearly its effect on platelet release reaction and the relations between its effects on platelet aggregation and release reaction. Platelet release reactions included α-granules and dense granules releasing. When α-granules were released, its membrane glycoprotein CD62p was expressed in the platelet membrane. We used the CD62p expression as the index of platelet release reaction. In the current study, the 4-peptides RGDS (Arg-Gly-Asp-Ser) was used as glycoprotein IIb/IIIa antagonist. We detected the effects of RGDS on platelet aggregation and CD62p expression induced by adenosine diphosphate (ADP) (finial concentration, 5μmol/L) in vitro. 50, 100, 200, 400 and 800μmol/L RGDS were used separately in the test. RGDS of each concentration could significantly inhibited maximal platelet aggregation (PAG(M)) induced by ADP, the 50% inhibiting concentration was approximately 200μmol/L. 800μmol/L RGDS could inhibited PAG(M) by 80.48±8.18%. Only ≥200μmol/L RGDS could significantly inhibited platelet CD62p expression. 800μmol/L RGDS could inhibit platelet CD62p expression by 27.31±9.74%. The inhibiting effect of RGDS on PAG(M) and platelet CD62p expression had significantly correlation (r =0.976, P<0.05). These results indicated that RGDS in low concentration (<200μmol) had little negative effect on platelet release reaction induced by ATP, while in relatively high concentration (≥200μmol) RGDS could inhibit platelet release reaction. When RGDS concentrations were same its effect on platelet release reaction was much less than that on platelet aggregation, which indicated that platelet glycoprotein IIb/IIIa compound could only partly participated in the platelet release reaction but fully participated in platelet aggregation induced by ADP.