Plasma P-selectin Research Articles

Plasma levels of soluble P-selectin predict survival in CTEPH

Background: The platelet activation marker P-selectin is involved in the pathogenesis of venous thromboembolism (VTE). Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequelae of VTE with obstruction of pulmonary arteries by organizing thrombus. To investigate the role of soluble P-selectin (sP-selectin) as a risk predictor for CTEPH, we performed a prospective cohort study of patients (pts) suffering from CTEPH. Methods: Enzyme-linked immunoassay was used to determine sP-selectin plasma levels in pts with CTEPH at the time of diagnosis. Patients suffering from pulmonary arterial hypertension (PAH), deep venous thrombosis (DVT) and healthy subjects served as controls. Analysis of overall survival/freedom from double lung transplantation was performed using Kaplan-Meier curves stratified by sP-selectin levels above and below median at baseline. Results: Soluble P-selectin plasma levels were studied in 179 pts of whom 95 (53,1%) were classified as operable and 84 (46,9%) as non-operable mainly due to an imbalance between hemodynamics and extent of pulmonary vascular obstructions, or comorbidities. Soluble P-selectin plasma levels (sP-selectin, median ng/ml (range): CTEPH 98.4 (50.7-215)) were significantly higher in CTEPH patients than in controls (sP-selectin, median ng/ml (range): PAH 34.6 (27.1-46.8), DVT 44.3 (36.7-57.6), healthy subjects 35.9 (28.1-45.1); P<0.001), with no difference between operable and non-operable pts. During an observation time of 4.89 years (median; IQR: 2.45-7.67) 83 deaths of any cause occurred and 5 patients underwent double lung transplantation. Patients with sP-selectin levels ≤ 98.4 ng/ml survived longer than those with levels >98.4 ng/ml (P<0.001). Operated pts with sP-selectin levels ≤ 98.4 ng/ml had a better survival than non-operated pts (P<0.001). Conclusion: Soluble P-Selectin is increased in plasma, and predicts survival/freedom from double lung transplantation in operable and non-operable CTEPH. The data suggest that platelet activation is involved in venous thrombosis.

Anti-thrombosis effect of LRRFIP1 shRNA lentivirus in a mouse model of deep vein thrombosis

IntroductionDeep vein thrombosis (DVT) is one of the common complications of orthopedic surgery. Low molecular weight heparin (LMWH) is a usually used agent for DVT, but it would increase the risk of bleeding. LRRFIP1 has been shown to play an important role in the formation of thrombosis. Therefore, we investigated the effect of LRRFIP1 shRNA lentivirus on DVT in mice. Materials and MethodsLentiviral Vectors carrying LRRFIP1 shRNA were constructed and transfected into cultured mouse bone marrow cells (BMCs). Male ICR mice were irradiated with a single dose of 9.5Gy and then were injected with different agents through the tail vein. Stasis venous thrombosis was induced by inferior vena cava (IVC) ligation. Mice were sacrificed on the 1st, 3rd and 7th day post operation and the thrombi were removed, blotted the excess blood on it with filter paper and immediately weighed. P-selectin and d-Dimer were determined by enzyme-linked immunosorbent assay (ELISA). ResultsLRRFIP1 shRNA significantly suppressed the expression of LRRFIP1 in the thrombi. In contrast, low molecular weight heparin (LMWH) and negative shRNA exhibited little effect on the expression of LRRFIP1. LRRFIP1 shRNA, LMWH and negative shRNA inhibited the thrombus formation in vivo significantly. The plasma P-selectin and d-Dimer levels were significantly increased after IVC ligation. LRRFIP1 shRNA significantly decreased the plasma P-selectin and d-Dimer levels. However, LMWH and negative shRNA showed little effects on the levels of plasma P-selectin and d-Dimer. ConclusionLRRFIP1 shRNA might represent a promising prevention strategy for DVT.

Abstract 487: Aspirin Treatment Hampers the Use of Plasma MicroRNA-126 as Biomarker for the Progression of Vascular Disease

Rationale MicroRNA-126 (miR-126) facilitates angiogenesis and regulates endothelial cell function. Recent data suggest that miR-126 can serve as a biomarker for cardiovascular disease. Although endothelial cells are enriched for miR-126, platelets also comprise a major pool of miR-126. We investigated the contribution of platelets to the level of miR-126 circulating in the plasma from patients with type 2 diabetes and how this level is affected by aspirin. Methods &amp; Results In vitro platelet activation resulted in the transfer of miR-126 from the platelet- to the plasma-compartment, which was prevented by aspirin. In vivo platelet activation was studied in patients with type 2 diabetes, who exhibit disease-mediated platelet activation and are prone to develop cardiovascular disease. Platelet activation was monitored by measuring soluble P-selectin in plasma and correlated significantly with circulating levels of miR-126. Administration of aspirin to the patients in a randomized, placebo-controlled cross-over design, resulted in platelet inhibition and concomitantly reduced circulating levels of platelet-derived microRNAs including miR-126. Conclusion The profile of circulating miRs constitutes a fingerprint of activated platelets rather then activated endothelial cells. In particular miR-126 can be derived from activated platelets and the circulating levels of this miRNA can be markedly reduced when aspirin is used in patho-physiological conditions associated with platelet activation such as diabetes type 2. Therefore, the use of platelet inhibitors should be taken into account when using plasma-levels of miR-126 as a biomarker.

Effect of the hydrogen sulfide donor GYY4137 on platelet activation and microvascular thrombus formation in mice

This study evaluates the effect of the H2S donor GYY4137 (GYY) on adhesion molecule expression, protein S-sulfhydration and morphology of platelets in vitro and on kinetics of microvascular thrombus formation in vivo. Using flowcytometry, untreated resting, TRAP-activated, or TRAP-activated and GYY-exposed human platelets were studied for expression of P-selectin, GPIb and GPIIb/IIIa as well as for fibrinogen binding. By means of electron microscopy, platelet morphology and intracellular granule numbers were assessed. Platelet shape change was studied using immunohistochemistry for P-selectin, NSF and F-actin by SR-SIM. Biotin switch assay served for the analysis of platelet protein S-sulfhydration by GYY. Using the FeCl3 and the light/dye model in dorsal skinfold chamber-equipped mice, the effect of GYY and its vehicle DMSO was studied on venular thrombus formation and tail-vein bleeding time. Soluble (s)P-selectin plasma concentrations were measured in GYY- or DMSO-treated animals. Exposure to GYY increased the S-sulfhydration of platelet proteins. GYY reduced dose-dependently the TRAP-induced adhesion molecule expression and attenuated the morphological signs of TRAP-associated platelet activation. In mice, GYY caused a significant prolongation of venular thrombus formation and tail-vein bleeding time. Application of an anti-P-selectin antibody in DMSO-exposed animals prolonged thrombosis formation comparably as GYY did. GYY reversed the TRAP-induced distribution of P-selectin at the plasma membrane of platelets. This indicates reduced exocytosis and shedding of P-selectin, which is supported by significantly lower sP-selectin concentrations in GYY- vs. DMSO-treated mice. H2S acts anti-thrombotic and seems to regulate thrombogenesis by interference with platelet activation and adhesion molecule-mediated aggregation.

Effects of high-amount–high-intensity exercise on in vivo platelet activation: Modulation by lipid peroxidation and AGE/RAGE axis

Physical activity is associated with cardiovascular risk reduction, but the effects of exercise on platelet activation remain controversial. We investigated the effects of regular high-amount, high intensity aerobic exercise on in vivo thromboxane (TX)-dependent platelet activation and plasma levels of platelet-derived proteins, CD40L and P-selectin, and whether platelet variables changes may be related to changes in high-density lipoprotein (HDL) and in the extent of oxidative stress and oxidative stress-related inflammation, as reflected by urinary isoprostane excretion and endogenous soluble receptor for advanced glycation end-products (esRAGE), respectively. Urinary excretion of 11-dehydro-TXB₂ and 8-iso-prostaglandin (PG)F(2α) and plasma levels of P-selectin, CD40L and esRAGE were measured before and after a eight-week standardised aerobic high-amount-high-intensity training program in 22 sedentary subjects with low-to-intermediate risk. Exercise training had a clear beneficial effect on HDL cholesterol (+10%, p=0.027) and triglyceride (-27%, p=0.008) concentration. In addition, a significant (p<0.0001) decrease in urinary 11-dehydro-TXB₂ (26%), 8-iso-PGF(2α) (21%), plasma P-selectin (27%), CD40L (35%) and a 61% increase in esRAGE were observed. Multiple regression analysis revealed that urinary 8-iso-PGF(2α) [beta=0.33, SEM=0.116, p=0.027] and esRAGE (beta=-0.30, SEM=31.3, p=0.046) were the only significant predictors of urinary 11-dehydro-TXB₂ excretion rate over the training period. In conclusion, regular high-amount-high-intensity exercise training has broad beneficial effects on platelet activation markers, paralleled and possibly associated with changes in the lipoprotein profile and in markers of lipid peroxidation and AGE/RAGE axis. Our findings may help explaining why a similar amount of exercise exerts significant benefits in preventing cardiovascular events.

Plasma P-selectin Predicts Long-term Cardiovascular Events in Hospitalized Patients with Suspected Coronary Artery Disease and Preserved Left Ventricular Function: A 10-Year Follow-Up Study

A variety of biomarkers have been investigated on their values to predict cardiovascular outcomes, such as high-sensitivity C-reactive protein (hs-CRP), fibrinogen, troponin-I (TnI), and soluble P-selectin (sP-sel). By a design of head-to-head comparison, this study sought to figure out the long-term prognostic values of these parameters in patients hospitalized with suspected coronary artery disease. A total of 170 patients hospitalized with suspected coronary artery disease were enrolled and followed up for an average of 10 years. sP-sel, hs-CRP, TnI, and fibrinogen levels were measured. During the follow-up period, cardiac events were recorded including cardiac death, non-fatal myocardial infarction, and acute coronary syndromes with hospitalization. For all 170 patients, with a median follow-up time of 9.86 ± 3.87 years, no parameter was able to significantly predict the occurrence of cardiac events. In subgroup analysis, an sP-sel of ≥ 63.5 ng/ml significantly predicted the development of all composite cardiac events only in patients with a left ventricular ejection fraction > 50% (n = 94, p = 0.04). However, the levels of hs-CRP, TnI, and fibrinogen did not have significant predictive values. Multivariate analysis also demonstrated the independent predictive value of sP-sel on all cardiac events (hazard ratio = 5.82, p = 0.02). All parameters, including sP-sel, could not demonstrate prognostic values in patients with a left ventricular ejection fraction ≤ 50% (n = 76). In this 10-year long-term follow-up study, sP-sel was demonstrated to have prognostic values in predicting the cardiac events in patients with preserved left ventricular systolic function.

The Tie2 Receptor Antagonist Angiopoietin-2 in Systemic Lupus Erythematosus: Its Correlation with Various Disease Activity Parameters

Background: Systemic lupus erythematosus is one of the autoimmune diseases characterized by multisystem involvement associated with autoantibody and immune complex vasculitis along with endothelial cell damage. Objective: to study the possible role of Angiopoietin- 2 (Ang-2) as a recently highlighted inflammatory and angiogenic mediator in the pathogenesis of SLE and its correlation with the state of another inflammatory marker, P-Selectin, as well as with various markers of the disease activity. Patients and methods: The present study included 3 main groups: active SLE patients (group I), inactive SLE patients (group II) and healthy normal control subjects (group III). Groups I and II were subjected to disease activity assessment using the SLEDAI scoring system and measurement of plasma Ang-2 and P-Selectin by ELISA in addition to various laboratory investigations to assess disease activity as: Complete blood count, ESR, serum creatinine, C3, C4 and 24-h urinary proteins. Results: The mean level of Plasma Ang-2 and P-selectin showed a high significant increase in active group compared to inactive SLE patients and control subjects (p < 0.001).There was a significant positive correlation between Ang-2, P-Selectin, and each of SLEDAI score and 24-h urinary proteins in all SLE patients as well as in the active group, and Ang-2 was a significant independent marker for proteinuria. A significant negative correlation was found between Ang-2, P-Selectin and each of C3, C4. Ang-2 and P-Selectin showed a high sensitivity and specificity in the patients with SLE. Conclusion: Our study suggests that Ang-2 may be a more useful marker than P-Selectin, C3 and C4 in the assessment of disease activity.

Abstract 74: Endothelial PPARγ Protects Against Vascular Thrombosis by Downregulating P-Selectin-Mediated Leukocyte-Endothelial Interactions

Background and Hypothesis: Tissue-specific effects of peroxisome proliferator-activated receptor-gamma (PPARγ) on vascular thrombosis are not well understood. Using a novel murine model of selective interference with endothelial PPARγ, we tested the hypothesis that endothelial PPARγ protects against vascular thrombosis. Methods: Transgenic mice expressing a dominant negative PPARγ mutant (E-V290M) selectively in endothelium were compared with non-transgenic (non-Tg) littermates. Susceptibility to occlusive thrombosis of the carotid artery was assessed following chemical (7% FeCl3) or photochemical (rose bengal and green laser) injury. Leukocyte rolling on mesenteric venules was measured by intravital microscopy. P-selectin mRNA levels were assayed by real-time PCR and P-selectin protein levels were measured by ELISA and flow cytometry. To assess the role of P-selectin and leukocytes in thrombosis, P-selectin blocking antibodies or leukocyte-depleting antibodies were infused before carotid artery injury. Results: The time to occlusion of the carotid artery was significantly shortened in E-V290M mice compared with non-Tg mice after either chemical (5.1±0.2 vs. 10.1±3.3 vs. minutes; P=0.01) or photochemical injury (48±9 vs. 74±9 minutes; P=0.04). Both plasma P-selectin levels and P-selection mRNA levels in the carotid artery were significantly higher in E-V290M mice than in non-Tg mice (P&lt;0.05), but no differences in platelet surface P-selectin were observed. Increased functional endothelial P-selectin activity was demonstrated by enhanced leukocyte rolling on mesenteric venules in E-V290M mice compared with non-Tg mice (53±8 vs. 25±7 per minute; P=0.025). The shortened occlusion times in E-V290M mice were reversed by P-selectin blocking antibodies or leukocyte-depleting antibodies (P=0.04, and P=0.02 respectively). Gene set enrichment analysis of a gene expression microarray demonstrated significant upregulation of NF-kB target genes, including P-selectin, in endothelial cells from E-V290M mice (P&lt;0.001). Conclusions: We conclude that endothelial PPARγ protects from thrombosis through a mechanism that involves downregulation of NF-kB-mediated P-selectin expression and leukocyte-endothelial interactions.

Number of Microparticles Generated During Acute Myocardial Infarction and Stable Angina Correlates with Platelet Activation

Elevated levels of circulating microparticles (MPs) have been reported in patients with acute myocardial infarction (AMI) and coronary artery disease. Platelet activation and inflammation have been recognized during AMI and stable angina (SA). We hypothesize that the origin and count of MPs in AMI and SA patients are related to markers of inflammation and platelet activation. Platelet, monocytes and endothelial MPs and surface P-selectin were determined in 12 AMI patients, 10 SA patients and 9 controls by flow cytometry. Plasma P-selectin, CD40 ligand (sCD40L) and interleukin 6 (IL-6) levels were evaluated by ELISA methods. The total MP count was compared in control subjects, AMI, and SA patients: 12,765 (8465) vs. 38,750 (11,931) vs. 29,715 (12,072) counts/μl (p = 0.01), respectively. Patients with AMI displayed higher levels of total and platelet origin- tissue factor-positive (CD42/CD142) MPs than patients with SA: 72.8 (6.2) vs. 56.2 (6.4) %, p = 0.001. Levels of soluble P-selectin were significantly elevated in patients with AMI as compared to SA patients: 146 (6.5) vs. 107 (2.7) ng/mL, p = 0.005; significant correlation between total MP count and relative number of CD34, CD51, CD42-positive MPs, and the P-selectin expression was observed in patients with AMI. Platelet activation in AMI is associated with increased generation of MPs not only from platelets, but also monocytes and endothelial cells. It suggests that interactions between platelets, monocytes and endothelial cells play an important role in the pathogenesis of myocardial ischemia.

Endothelial Cell Markers in Patients with Pseudoexfoliation Syndrome

The aim of the study was the assessment of the von Willebrand antigen (vWF Ag), E-selectin, and P-selectin concentration in blood plasma of patients with pseudoexfoliation syndrome (PEX). The group studied comprised 30 patients with PEX, aged from 50 to 86 years (mean 73, SD ± 8 years). Patients with cardiovascular and cerebrovascular diseases, diabetes mellitus, infectious disease, cancer, renal or liver insufficiency, connective tissue disease, current smoking, and hormone, antiplatelet, hypolipidemic, antioxidant, or antihypertensive drug therapy were excluded from the study. Each subject underwent a complete ophthalmological examination. Venous blood samples from the cubital vein were taken into sodium citrate solution. VWF Ag, sP-selectin, and sE-selectin concentration were determined by a commercially available enzyme-linked immunosorbent assay (MedSystems, Diagnostica Stago/Roche, R&D). Concentrations of vWF Ag, soluble E-selectin, and soluble P-selectin in blood plasma in the study group were compared with the levels in blood plasma in the control group. No significant differences were found between the groups. Our results indicate that there might be no correlation between PEX and such endothelial cell markers as vWF Ag, sP-selectin, and sE-selectin concentrations. Since the study size is limited, further investigations to confirm that there is no association between endothelial dysfunction in PEX and risk of future cardiovascular disease are necessary.

The Thr715Pro variant impairs terminal glycosylation of P-selectin

P-selectin variant 715Pro is associated with lower concentrations of plasma P-selectin and reduced risk for thrombosis. We examined the influence of 715Pro on P-selectin synthesis, post-translational processing, surface expression and function using HEK293 cells, which do not express endogenous P-selectin. Mass spectrometry revealed that HEK293 cells produced recombinant P-selectin which has a glycosylation pattern comparable to platelet P-selectin. Compared to wild-type transfectants, 715Pro transfectants have ~50% less terminally glycosylated P-selectin and accumulate more immature P-selectin in Golgi. Following Brefeldin A treatment, the majority of 715Pro P-selectin is not modified by Golgi enzymes, while wild-type P-selectin undergoes complete modification. Flow cytometry revealed that 715Pro transfectants have ~20% less P-selectin on the cell surface compared to wild-type transfectants. Secretion of P-selectin by 715Pro transfectants was about 38% lower compared to wild-type transfectants. Binding of HL-60 cells to 715Pro transfectants was ~29% lower than to wild-type transfectants. This observation was confirmed by the presence of fewer platelet-monocyte aggregates (PMA) in the blood of healthy individuals and patients with angiographically proven atherosclerosis, carrying 715Pro P-selectin compared to individuals with wild-type P-selectin. We conclude that the 715Pro variant impairs terminal glycosylation of P-selectin in Golgi, leading to reduced amounts of mature P-selectin and subsequently less surface expression and secretion of P-selectin. The reduced surface expression of 715Pro P-selectin contributes to inefficient adhesion to HL-60 cells or monocytes.

Development of an immunopredictor for the evaluation of the risk of cardiovascular diseases based on the level of soluble P-selectin

Due to its physiologic role in modulating adhesive interactions between blood cells and the endothelium during inflammatory processes or at injury sites, the adhesion molecule P-selectin is of great interest. The level of soluble P-selectin in plasma or serum can be detected and used as a clinical predictor for adverse cardiovascular events, leading to the presumption that it is secreted, shed or cleaved from the cell membrane during the process of diseases. Increased levels of soluble P-selectin in the plasma have been shown to be associated with a range of cardiovascular disorders, including coronary artery disease, hypertension and atrial fibrillation. Therefore, it is of huge significance to develop simple, rapid and sensitive methods for the detection of such pathological predictors, not only for facilitating the surveillance of cardiovascular mortality/sudden cardiac death, but also for effectively monitoring the drug potency on platelets based on measurement of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting. We herein developed a simple, yet novel and sensitive electrochemical sandwich immunosensor for the detection of P-selectin; it operates through covalent linkage of anti-P-selectin antibody on CNT@GNB nanocomposites-modified disposable screen-printed electrode as the detection platform, with the potassium ferrocyanide-encapsulated, anti-P-selectin-tagged liposomal biolabels as the electrochemical signal probes. The immunorecognition of the sample P-selectin by the liposomal biolabels occurred on the surface of the electrodes; the release of potassium ferrocyanide from the bound liposomal biolabels extensively contributed to the increase in electrochemical signal, which was acquired in HCl solution at +0.32V in square wave voltammetry mode. The resulting sigmoidally shaped dose–response curves possessed a linear dynamic working range from 1×10−13 to 1×10−5g/mL. This liposome-based electrochemical immunoassay provides an amplification approach for detecting P-selectin at trace levels, leading to a detection limit as low as 4.3fg (equivalent to 5μL of 0.85pg/mL solution). A commercially available ELISA kit was used as a reference method to validate the newly-developed assay through the analysis of mouse serum samples. A strong correlation was observed between the two data sets as the R-squared value of 0.997 from the linear regression line. This electrochemical immunosensor will be useful for the detection of P-selectin in biological fluids and tissue extracts.

High levels of platelet-monocyte aggregates after valve replacement for aortic stenosis: Relation to soluble P-selectin and P-selectin glycoprotein ligand-1 genes

BackgroundCalcific aortic valve stenosis is linked to atherosclerosis. The latter is associated with increased levels of platelets adhering to monocytes (PMA). ObjectiveThe hemodynamic impairment in symptomatic aortic valve stenosis can be abated by valve replacement. We investigated the effect of valve replacement on PMA and receptor-ligand axis P-selectin - P-selectin glycoprotein ligand-1 (PSGL-1) in severe aortic valve stenosis. Patients and MethodsPMA, plasma P-selectin (sP-selectin) and polymorphisms within the coding region for PSGL-1 (SELPLG) were determined in 42 patients with severe aortic valve stenosis before and 4 to 8months after valve replacement. Ten patients suffered from significant coronary artery disease and received also a coronary artery bypass graft. Thirty-four patients received a bioprosthetic valve and 8 patients who were <65years old received a mechanical valve. ResultsBefore the intervention, PMA levels were significantly higher in patients with aortic valve stenosis than in two control cohorts, namely healthy indviduals and 88 age- and sex-matched patients with severe atherosclerosis, but without aortic valve stenosis (p<0.001). PMA decreased after surgery, but normalized in only 3 patients, while further increases were noted in 11 patients. sP-selectin was elevated in 3 and 4 patients before and after valve replacement, respectively. sP-selectin increased significantly after surgery, but remained within the normal range. There was no correlation between changes of PMA and sP-selectin or any of the polymorphisms within SELPLG. ConclusionsExceedingly high PMA in aortic stenosis are independent of SELPLG polymorphisms, and largely of the hemodynamic compromise caused by the stenotic valve.

Expression of P-selectin, von Willebrand and endothelin-1 after carotid artery stenting

This study was designed to investigate the impact of carotid artery stenting (CAS) on plasma levels of P-selectin, von Willebrand (vWF) and endothelin-1. Sixty-seven patients who received CAS were divided into group 1 (one stent for a simple lesion, n = 38) and group 2 (two stents for complex lesions, n = 29). The levels of P-selectin, vWF and endothelin-1 were measured before CAS, 1 h, 6h, 24 h and 2 weeks after the stenting. Sixty-one patients completed one-year follow up. Restenosis was noted in 14 (23 %) patients, among these three (4.8 %) had a restenosis of > 50 % of the vascular lumen. In all patients, the levels of P-selectin, vWF and endothelin-1 increased immediately after CAS (P < 0.05 or < 0.01). The levels of vWF and endothelin-1 in group 2 were higher than in group 1 (P < 0.05 or 0.01). There was no significant difference in P-selectin and endothelin-1 between the restenosis and non-restenosis group (P > 0.05). The 24 h vWF in patients with restenosis were higher than in non-restenosis group (P < 0.05). CAS results in a significant increase in plasma P-selectin, vWF and endothelin-1. The post-CAS levels of P-selectin, vWF and endothelin-1 are related to the extent of endothelial injury. Whether they are associated with restenosis 12 months after the treatment requires further investigation.

Male mice have increased thrombotic potential: Sex differences in a mouse model of venous thrombosis

Introduction Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease. Materials and methods Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1β, and tumor necrosis factor-α; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays. Results Male venous thrombi were significantly larger than females at days 2 (13.1 ± 1.0 vs. 6.8 ± 0.5 × 10 -3 grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10 -3 grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10 -3 grams, p < 0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p < 0.05). Males had increased vein wall interleukin-1β, versus females, at 6 hours (180.926 ± 24.596 vs. 60.417 ± 10.478 pg/mL, p < 0.05) and day 6 (76.966 ± 13.081 vs. 33.834 ± 4.198 pg/mL, p < 0.01). Males showed decreased tumor necrosis factor-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α expression at 6 hours (+ 541%) and day 6 (+ 539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p < 0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p < 0.05). Conclusions Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model.

Platelet activation in the pathogenesis of obesity and vascular disease

The pathogenesis of atherosclerotic disease is complex, and very topical, as clearly highlighted in a recent position paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology in this journal, which deals with atherosclerotic plaques and their ‘stabilisation’ per se (1). A number of associations have been observed between lipid abnormalities, markers of metabolic syndrome and platelet activation (1–5). In recent years, many studies have explored associations for platelet activation markers to gain insights on the pathogenesis of obesity and atherosclerotic disease (6–13). In this issue of Thrombosis and Haemo stasis, Csongradi et al. report on a large cross-sectional cohort study that explored relationships for platelet activation markers in obese subjects, including those with and without common co-morbidities for atherosclerotic disease (14). Key findings of their study were that platelet and plasma P-selectin levels, and the plasma levels of platelet microparticles, were associated with carotid intimal medial thickness and a variety of other vascular risk factors, including fasting plasma glucose, insulin levels and triglycerides (14). These observations imply that the extent of atherosclerotic disease, and the metabolic derangements associated with obesity and atherosclerotic disease, influence the generation of platelet microparticles. This possibility is supported by a recent study that showed weight loss-induced improvements in insulin resistance, among women with obesity, reduced markers of platelet activation (6). While association studies cannot determine causation, they are important to developing new hypotheses on the pathogenesis of atherosclerotic disease. Platelet microparticles have attracted much interest both experimentally and clinically (15–19). Indeed, given their significant procoagulant activity (18, 19), their elevation in pathogenic states could contribute to the progression of vascular disease and acute thrombotic events. Nonetheless, uncertainties remain about whether observed associations between elevated levels of platelet-derived microparticles with prothrombotic, metabolic and inflammatory parameters are correlative or causative, and how much platelet microparticle generation, and other aspects of platelet activation, contribute to the pathogenesis of atherosclerotic disease in obese individuals. The “independent” associations that Csongradi et al. found between platelet-derived microparticle levels with body mass index, and between soluble P-selectin and fibrinogen, and platelet P-selectin and both carotid intimal medial thickness and PAI-1 (14) provide further evidence that platelet activation is influenced by the complex metabolic and inflammatory parameters that participate in the pathogenesis of atherosclerotic disease. Despite these advances, uncertainties still remain about whether measuring platelet activation markers has a role in diagnostic or therapeutic decision making for obese and non-obese individuals who have or do not have evidence of atherosclerotic disease. This still remains an exciting and expanding field of research.

Estudo de fatores pró-trombóticos e pró-inflamatórios na cardiomiopatia chagásica

The relationship between inflammatory and prothrombotic activity in chagas cardiomyopathy and in other etiologies is unclear. To study the profile of pro-thrombotic and pro-inflammatory markers in patients with Chagas' heart failure and compare them with patients of non-chagas etiology. Cross-sectional cohort. left ventricle ejection fraction (LVEF) < 45% and onset time to symptoms > one month. The patients were divided into two groups: group 1 (G1) - seropositive for Chagas - and group 2 (G2) - seronegative for Chagas. Pro-inflammatory factor: Ultra-sensitive CRP. Pro-thrombotic factors: thrombin-antithrombin factor, fibrinogen, von Willebrand factor antigen, plasma P-selectin and thromboelastography. Sample calculated for 80% power, assuming a standard deviation difference of 1/3; significant p if it is < 0.05. Fisher's exact test for categorical variables; unpaired Student's t-test for parametric continuous variables and Mann-Whitney test for nonparametric continuous variables. Between January and June 2008, 150 patients were included, 80 in G1 and 70 in G2. Both groups maintained the averages of high sensitivity CRP above baseline values, however, there was no significant difference (p = 0.328). The fibrinogen levels were higher in G2 than in G1 (p = 0.015). Among the thromboelastography variables, the parameters MA (p=0.0013), G (p=0.0012) and TG (p =0.0005) were greater in G2 than in G1. There is no evidence of greater pro-thrombotic status among patients with Chagas disease. The levels of fibrinogen and the MA, G and TG parameters of the thromboelastography point to pro-thrombotic status among non-chagas patients. Both groups had increased inflammatory activity.

Effects of Shenfu injection on coagulation function, D-dimer and plasma p-selectin in elderly patients during total hip replacement surgery

To investigate the effects of SF injection on coagulation function, p-selectin and D-dimer concentrations in plasma of elderly patients undergoing total hip replacement (THR). Twenty-four ASA II/III patients aged 65 to 85 were divided equally into two groups (n = 12). In Group S, SF injection was administered by peripheral intravenous infusion at initially 0.2 ml/kg and then 0.8 ml (kg/h) until the end of operations. In Group N, normal saline was administered similarly. All the patients were given patient-controlled epidural analgesia (PCEA) after operations. Blood samples were taken before operations (T(0)), immediately post-operation (T(1)) and 24 h post-operation to detect the levels of p-selectin, D-dimer, blood platelet count and clotting time at all time points. The concentrations of p-selectin and D-dimer in Group N increased at T(1) and T(2) while those in Group S decreased at T(1) (P < 0.05). Compared with Group N, they were significantly lower in Group S (P < 0.05). There was no significant difference at T(2) between two groups. The plasma concentrations of p-selectin and D-dimer increase obviously in elderly THR patients. SF injection can decrease the plasma concentrations of p-selectin and D-dimer in patients during surgery. Thus it may protect the vascular endothelial cells and attenuate the activation of platelet and fibrinolysis.

Platelet P‐selectin reflects a state of cutaneous inflammation: possible application to monitor treatment efficacy in psoriasis

Haemostasis-maintaining platelets are also recognized as important modulators in the regulation of immune response. Activated platelets expressing P-selectin (CD62P) are involved in the extravasation of leucocytes. This study evaluated platelet P-selectin expression as a biomarker for cutaneous inflammation. P-selectin expression was assessed by flow cytometry in 147 successive patients suffering from an inflammatory or infectious skin condition at the day of admission for in-patient treatment as well as a day prior to demission. Forty-one patients admitted for allergy testing served as controls. A commercially available ELISA was used in 17 patients to determine soluble P-selectin in the plasma. In patients with psoriasis, the Psoriasis Area and Severity Index (PASI) was documented as a measure for disease severity. We observed a significant increase in platelet P-selectin expression in patients with inflammatory or infectious disorders, when compared to the control group (3,01% vs. 1,46%; P < 0.000001). Successful treatment resulted in a significant decrease in P-selectin expression to the level of the control group. In the case of psoriasis (n = 47), we found highly significant correlation between P-selectin and PASI (r = 0.51; P < 0.000001), as well as between the change in the PASI and the change in P-selectin expression (r = 0.4; P = 0.006). Platelet P-selectin expression as determined by flow cytometry correlated well with the results of soluble P-selectin, determined by ELISA (r = 0.63; P < 0.01). Thus, platelet P-selectin expression may be used as an efficacy biomarker to monitor treatment success in psoriasis. As platelet P-selectin correlates with soluble P-selectin in patient plasma, which can be measured by ELISA, the latter is feasible also for routine use.

103 Testing for clopidogrel and aspirin anti-platelet activity in patients with acute coronary syndromes (ACS): should we test, and if so when?

Introduction Oral anti-platelet agents are a cornerstone of treatment for acute coronary syndromes (ACS), and improve outcome after percutaneous coronary intervention (PCI). Low responses to aspirin (ASA) and Clopidogrel place patients at higher risk of subsequent adverse cardiac and cerebrovascular events (ACCE). Near-patient testing devices and markers of platelet activation may identify this risk, but strategies for their use have yet to be developed. Methods Two hundred and sixty nine consecutive patients (68.4% male, 18.6% diabetic) with ACS were studied prospectively. All patients displayed high risk features (elevated serum troponin (Tn), ECG abnormalities or both), received standard therapy and were due to undergo early angiography with a view to PCI. Those with other factors likely to influence platelet function testing were excluded. At angiography, blood was drawn for platelet count and TnT; response to ASA and to clopidogrel using a VerifyNow (VN) near patient testing device; plasma P-selectin levels; and presence of the CYP2 C19 2* allele, GPIIIa PlA1/2 and ABCB1 polymorphisms. Procedural outcomes were recorded and TnT repeated 16—24 h after PCI. At a 30-day visit clinical data were recorded and blood tests repeated. Patients were followed-up for ACCE (death, MI, repeat revascularisation, stroke and unplanned cardiovascular admission) at 30 days, 6 and 12 months. Results VN results were available for 267 patients. For Clopidogrel, P2Y12 reaction units (PRU)>240 and ASA reaction units (ARU) ≥550 indicate a low response to each agent. PRU of >240 was observed in 46.2% of patients, associated with increasing age (p=0.003) diabetes (p=0.027), platelet count (p=0.001) and CYP2 C19 2* status (p 151 (56.1%) underwent PCI, of whom 73(48.7%) demonstrated low response to Clopidogrel at presentation. This was associated with a greater periprocedural rise in Tn (p=0.046) and higher 30 day P-selectin (p=0.014), but did not predict ACCE at 12 months. CYP2 C19 2* status predicted VN PRU at angiography (p=0.001) and 30 days (p=0.022), but not ACCE. Low response to ASA at angiography was also associated with higher plasma P-selectin levels at 30 days (p=0.001), but did not predict ACCE at 12 months. Clopidogrel response at 30 days did predict 12 month ACCE (p=0.017), although P-selectin levels at 30 days were strongly associated (p=0.005). Conclusions Near-patient testing for clopidogrel response identified patients at risk of subsequent events when performed 30 days following PCI, but not at presentation. P-selectin, a marker of residual platelet activation, was associated with low response to each agent when measured at 30 days, and may alone be a better indicator of increased risk.