Functional interplay between platelet activation and endothelial dysfunction in patients with coronary heart disease

Platelet-monocyte binding and surface P-selectin expression are sensitive markers of platelet activation. Endothelium-derived factors are known to inhibit platelet activation and may confer important anti-atherothrombotic effects. We assessed the relationship between platelet activation and endothelium-dependent vasomotion in patients with coronary heart disease (CHD). Twenty male patients with stable CHD were compared with 20 healthy men. Platelet-monocyte binding and platelet surface expression of P-selectin were assessed using two-colour flow cytometry on whole blood. Forearm blood flow was assessed in patients using venous occlusion plethysmography during intra-arterial infusions of substance P, acetylcholine and sodium nitroprusside. Platelet activation was higher in patients than healthy men (platelet-monocyte binding, 27 +/- 3 vs. 20 +/- 1%; P < 0.05). In patients with CHD, there was an inverse correlation between maximal substance P induced vasodilatation and both platelet-monocyte binding (P = 0.003) and P-selectin expression (P = 0.02). A similar correlation was observed between platelet-monocyte binding and the vasomotor response to acetylcholine (P = 0.08) but not with sodium nitroprusside. In patients with stable coronary heart disease, there is a strong inverse relationship between markers of platelet activation and endothelium-dependent vasomotor function. This may explain the pathophysiological mechanism linking endothelial vasomotor dysfunction and the risk of acute atherothrombotic events.     

Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis

Purpose

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation.

Methods

Platelet–monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n = 19), interstitial lung disease (ILD) other than IPF (n = 9), and control subjects without pulmonary fibrosis (n = 14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA.

Results

Significantly elevated levels of platelet–monocyte binding were found in patients with IPF (35.6 ± 4.34 % [mean ± SEM]) compared with patients with non-IPF ILD (23.5 ± 3.68 %) and non-ILD control subjects (16.5 ± 2.26 %; P < 0.01). There was a trend towards increased divalent cation-independent platelet–monocyte binding in IPF (6.0 ± 0.77 % [mean ± SEM]) compared with non-IPF ILD (4.3 ± 1.38 %) and control subjects without ILD (3.1 ± 1.75 %; P = 0.058). There was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet–monocyte binding had no significant correlation with percent predicted TLco or FVC.

Conclusions

Platelet–monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF.     

Platelet reactivity among Asian Indians and Caucasians

Asian Indians are reported to have higher mortality and morbidity from coronary artery disease (CAD) than other ethnic groups. This variation in events cannot be explained only by differences in conventional risk factors. Platelet activation is an important factor in the pathogenesis of CAD, however, there are limited data concerning platelet reactivity in Asian Indians. Therefore, we aimed to examine platelet reactivity in healthy Asian Indians vs. Caucasians. Thirty-five healthy, nonsmoking Asian Indians (mean age 30.1?±?3.6 years, 31.4% women) were matched for age and sex with 35 healthy, nonsmoking Caucasians (mean age 30.8?±?5 years, 31.4% women). Platelet reactivity was evaluated by measuring platelet aggregation, platelet leukocyte aggregates (PLA) formation in response to a 6-mer thrombin receptor agonist peptide (TRAP) at a final concentration of 40?µM and flow cytometry determined P-selectin expression induced by ADP, TRAP and arachidonic acid (AA). In addition, P-Selectin glycoprotein ligand-1 (PSGL-1) density on leukocytes was measured. There were no differences in platelet aggregation, basal PLA or PSGL-1 density on leukocytes between the two groups. AA-stimulated P-selectin expression was significantly higher in Asian Indians than in Caucasians (6.1?±?0.51 vs. 4.2?±?0.41 MFI, P?<?0.02). After stimulation with TRAP, platelets from Asian Indians had increased PLA formation compared with Caucasians (41.6?±?2.9% vs. 31.4?±?2.7%, P?<?0.02). AA induced P-selectin expression and TRAP stimulated PLA formation is increased in Asian Indians compared with Caucasians. These differences indicate an increase in measures of platelet reactivity among Asian Indians and may help elucidate the reported disparity in cardiovascular disease rates between the two ethnic groups.     

Association of Thrombin Generation Potential with Platelet PAR-1 Regulation and P-Selectin Expression in Patients on Dual Antiplatelet Therapy

We studied the association of thrombin generation potential with platelet protease activated receptor (PAR)-1 regulation and platelet activation in 52 stable coronary artery disease patients on continuous therapy with aspirin and clopidogrel (n?=?42) or prasugrel (n?=?10). Compared to controls, peak thrombin generation potential was elevated in only 11 patients (p?>?0.05), while F1.2 was elevated in 26 patients (p?<?0.0001). PAR-1 and thrombin inducible P-selectin expression were significantly elevated in patients compared to controls (p?<?0.001). There were no significant correlations between levels of thrombin generation potential or F1.2 and PAR-1 regulation. However, there was a significant inverse correlation between levels of peak thrombin generation potential and in vitro thrombin-inducible expression of P-selectin (p?=?0.002), suggesting in vivo depletion of platelet alpha granules due to ongoing platelet activation.     

Microparticle-associated P-selectin reflects platelet activation in preeclampsia

Platelet activation in preeclampsia is reflected by elevated levels of platelets exposing P-selectin. In plasma, a non-cell bound (soluble) form of P-selectin is present. Elevated levels of this soluble form have been reported in preeclampsia. Plasma P-selectin may consist of two fractions: microparticle (MP)--associated P-selectin and non-MP--associated P-selectin. In the present cross-sectional study, we investigated to which extent plasma P-selectin is MP--associated and whether such MP are elevated in preeclamptic patients. Preeclamptic patients (n?=?10) were matched with normotensive pregnant women (n?=?10) and non-pregnant controls (n?=?10). Plasma P-selectin was measured by ELISA. MP were isolated, double labelled with anti-CD61 (GPIIIa) and anti-CD62P (P-selectin) and subsequently analyzed with flowcytometry. Plasma P-selectin concentration was elevated in preeclamptic patients compared to non-pregnant controls (p?=?0.007), but not compared to normotensive pregnant women (p?=?0.210). Plasma P-selectin is partially MP--associated (3–5%). In pregnancy, the fraction of P-selectin exposing platelet-derived MP (PMP) (10.9%) was increased compared to non-pregnant controls (8%). This fraction further increased in preeclamptic patients (15.4%), and significantly differed from normotensive pregnant women (p?=?0.02). A minor fraction of plasma P-selectin is associated with PMP. The fraction of PMP exposing P-selectin is increased in preeclamptic patients and to a lesser extent in normotensive pregnancy. Because MP associated P-selectin exclusively originates from platelets, this fraction indicates platelet activation. Platelet activation is prominent in preeclampsia and this study proves that at least a part of the plasma P-selectin originates from platelets.     

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15?µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.     

Platelet-monocyte cross talk and tissue factor expression in stable angina vs. unstable angina/non ST-elevation myocardial infarction

Tissue factor (TF), the major procoagulant in vivo, is usually absent from blood cells. However, since both monocyte TF (MoTF) expression and platelet activation are present in acute coronary syndrome we hypothesized that MoTF expression may in part depend on monocyte platelet aggregate (MPA) formation in coronary artery disease (CAD). Patients with unstable angina/non-ST-elevation myocardial infarction (UA/NSTEMI, n?=?20) had significantly higher levels of MoTF (17.4?±?3.1MFI) and MPAs (CD42b:273?±?183MFI; CD62P:256.3?±?48.5MFI) than patients with stable angina (SA, n?=?40; MoTF:13.2?±?2.2MFI, p?=?0.001; CD42b:160?±?113MFI, p?=?0.025; CD62P:118.7?±?24.5MFI, p?=?0.018) as measured by whole blood flow cytometry on CD14⁺-cells. TF-activity of isolated mononuclear cells (MNC) was elevated in UA/NSTEMI (75?±?27?pg/mL) in comparison to SA (47?±?17?pg/mL, p?=?0.001) as determined by chromogenic assay, and TF mRNA expression in isolated MNC was more frequent in UA/NSTEMI than in SA (50% vs. 18.2%; p?=?0.017). MoTF expression significantly correlated with the constitutive platelet marker CD42b (r?=?0.69, p?<?0.001) and the platelet activation marker CD62P (r?=?0.47, p?=?0.001) on CD14⁺-cells suggesting its association with MPAs in UA/NSTEMI. In addition, MoTF expression correlated with MoTF activity of isolated MNC (r?=?0.41, p?=?0.01) and plasma levels of the F1.2 prothrombin fragment (r?=?0.35, p?=?0.02). In conclusion, MoTF and MPAs are elevated in UA/NSTEMI compared with SA. MoTF expression correlates with platelet mass and activity attached to monocytes.     

Downregulation of platelet activation markers during long-term immobilization

Immobilization and sedentary lifestyle are risk factors for venous thromboembolism and cardiovascular disease, yet little is known about platelet function during long-term physical inactivity. Our aim was to investigate platelet activation markers and their coupling to standardized immobilization: platelet-derived growth factor (PDGF-BB) and P-selectin. We studied 15 healthy females participating in the Women International Space simulation for Exploration study. Following a 20-day ambulatory control period, the subjects underwent 60 days of bed rest in head-down tilt position (?6°) 24 hours a day, finalized by 20 days of recovery. The subjects were randomized into two groups during bed rest: a control group (n?=?8) that remained physically inactive and an exercise group (n?=?7) that participated in both supine resistance and aerobic exercise training. Blood samples for the analysis of platelet activation markers were collected at baseline (5 days before bed rest), after 44 days of bed rest and 8 days into the recovery period. Compared to baseline, the levels of P-selectin and PDGF-BB decreased after bed rest (by 55%, p?=?0.01 and 73%, p?<?0.03, respectively) and remained decreased in the recovery period (by 76%, p?<?0.001 and 78%, p?<?0.02, respectively, compared to baseline). Platelet count (baseline value for the exercise group 260?000/µl?±?34?000 and baseline value for the control group 210?000/µl?±?30?000) did not change during the bed rest study (two-way repeated measurements ANOVA, p?=?ns). There were no statistical differences between the physically inactive and the exercise group. During long-term immobilization, a known risk factor for thrombosis, the levels of P-selectin and PDGF-BB decreased. Our findings indicate downregulation of platelet activation during immobilization.     

Exercise-induced platelet and leucocyte activation is not enhanced in well-controlled Type 1 diabetes,despite increased activity at rest

Aims/hypothesis Stress can evoke a prothrombotic state with activated platelets and leucocytes, and increased platelet activation at rest has been reported for diabetic subjects. Thus, prothrombotic responses to stress may be enhanced in diabetes mellitus. We therefore evaluated platelet and leucocyte responses to exercise in Type 1 diabetic patients.Methods Type 1 diabetes mellitus patients with good metabolic control and healthy subjects matched for age and body mass index (n=15 for both) performed a maximal exercise test. Platelet and leucocyte activation and their heterotypic aggregation were monitored by whole blood flow cytometry.Results Diabetic platelets did not show higher basal levels of P-selectin expression, but were more reactive to ADP and thrombin stimulation. Diabetic patients had increased lymphocyte and monocyte CD11b expression, and increased circulating platelet–monocyte aggregates. Exercise evoked thrombocytosis, increased circulating platelet P-selectin expression, enhanced platelet sensitivity to ADP and thrombin, and elevated plasma levels of soluble P-selectin to a similar degree in diabetic patients and healthy subjects. Exercise induced marked leucocytosis and elevated plasma elastase, but only slightly increased leucocyte CD11b expression and leucocyte reactivity to stimulation by N-formyl-methionyl-leucyl-phenylalanine. In all of these there was no difference between diabetic patients and healthy subjects. The numbers, but not percentages of circulating platelet–leucocyte aggregates were markedly increased by exercise, but the increase was less prominent among diabetic patients.Conclusions/interpretation Strenuous exercise evokes platelet and leucocyte activation in Type 1 diabetic patients and healthy subjects. Platelet and monocyte hyperactivity were found at rest, but responses to stress were not augmented in metabolically well-controlled Type 1 diabetes mellitus patients.Abbreviations FITC fluorescein isothiocyanate - fMLP N-formyl-methionyl-leucyl-phenylalanine - MAb monoclonal antibody - PLA platelet–leucocyte aggregate - P-Lym platelet–lymphocyte aggregate - P-Mon platelet–monocyte aggregate - P-Neu platelet–neutrophil aggregate - RPE R-phycoerythrin     

Increased platelet,leukocyte, and coagulation activation in primary myelofibrosis

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet–neutrophil and platelet–monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients’ clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet–monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.     

Brachial artery endothelial function predicts platelet function in control subjects and in patients with acute myocardial infarction

Platelet activation occurs in an endothelium-dependent flow-mediated dilation (FMD) impairment environment. The aim of this study was to explore the association between platelet reactivity and brachial artery FMD in individuals without established cardiovascular disease (controls) and acute myocardial infarction (AMI) patients. We prospectively assessed brachial artery FMD in 151 consecutive subjects, 104 (69%) controls, and 47 (31%) AMI patients; 115 (76%) men, mean age 53?±?11 years. Following overnight fasting and discontinuation of all medications for?≥?12?h, percent change in brachial artery FMD (%FMD) and endothelium-independent, nitroglycerin-mediated vasodilation (%NTG) were assessed. Platelet aggregation was assessed by conventional aggregometry, and platelet adhesion and aggregation under flow conditions by cone-and-plate(let) technology (Impact-R). Smoking, diabetes, and hypertension were more common in AMI compared to control subjects (p?<?0.01 for all). Furthermore, aspirin, clopidogrel, beta-blockers, angiotensin-converting enzyme inhibitors, and statin administration were more common in AMI compared to controls (p?<?0.01 for all). %FMD but not %NTG was significantly lower in AMI patients compared to controls (10.2?±?4.2% vs. 15.4?±?4.4%; p?<?0.001 and 17.2?±?3.9% vs. 18.0?±?3.7%, p?=?0.803, respectively). %FMD was significantly and inversely associated with all platelet functions tests (p?<?0.001) in all study participants. In a multivariate logistic regression (unadjusted and adjusted for age, gender, smoking status, diabetes mellitus, hypertension, hypercholesterolemia, overweight, family history, and concomitant medications), %FMD remained the best predictor of platelet function, irrespective of group allocation (AMI patients or controls). In conclusion, FMD is inversely correlated to platelet reactivity in both controls and AMI patients.     

Nephropathy in Type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 µM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.     

Platelet and monocyte activation by hyperglycemia and hyperinsulinemia in healthy subjects

Type 2 diabetes mellitus (T2DM) patients have hyperglycemia and hyperinsulinemia and increased risk of atherosclerosis and acute vascular complications. We have reported elevated circulating tissue factor procoagulant activity (TF-PCA) during hyperglycemia (HG) and hyperinsulinemia (HI) in normal subjects. To evaluate the effect of hyperglycemia and hyperinsulinemia on blood cell activation, we assessed platelet CD40L and P-selectin, monocyte tissue factor (TF), and the formation of monocyte-platelet and neutrophil-platelet aggregates. These were assessed in the resting state and following activation with ADP and thrombin (SFLLRN). Healthy individuals were subjected to 24 h of hyperglycemia and hyperinsulinemia, selective hyperglycemia, selective hyperinsulinemia, or normal glucose and insulin. Platelet CD40L expression increased with high glucose/high insulin, selective hyperglycemia and selective hyperinsulinemia. Monocyte-platelet aggregates increased with high glucose/high insulin. Monocyte TF expression increased with high glucose/high insulin and with selective hyperinsulinemia. Upon stimulation with ADP and SFLLRN, monocyte-platelet and neutrophil-platelet aggregates, platelet CD40L and P-selectin, and monocyte TF increased compared to the resting state but was not different between 0 and 24 h, indicating that the responsiveness to those agonists was not altered.Conclusions: Hyperglycemia-hyperinsulinemia in healthy individuals induced platelet activation and monocyte TF expression promoting a procoagulant and proinflammatory state that may contribute to acute vascular events and atherogenesis. Platelet responsiveness to activation with ADP or SFLLRN appears not to be altered by hyperglycemia-hyperinsulinemia.     

Increased soluble glycoprotein V concentration during the acute onset of unstable angina pectoris in association with chronic cigarette smoking

Platelet hyperactivity is important in the pathobiology of acute coronary syndromes. Glycoprotein V (GPV) is an integral membrane protein of platelets in the function of the GPIb-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries. Soluble GPV is a novel marker of platelet activation. The aim of this study is to assess circulating soluble GPV levels in unstable angina pectoris (UA). Twenty-one patients (15 men, six women, aged 52?±?7 years) with UA pectoris were studied. The inclusion criteria were angina at rest lasting >20?min during the preceding 6?h, with transient ST segment depression and/or T wave inversion and no evidence of myocardial infarction detected with the use of cardiac troponin-T. Coronary artery stenosis was angiographically confirmed in all patients. Twenty age- and sex-matched healthy adults (14 men, six women, aged 48?±?7 years) served as controls. There were no significant differences among the studied groups with respect to age, sex, obesity, smoking, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride and platelet counts. Plasma-soluble GPV concentrations were higher in the UA patient group (126?±?46?ng/ml) than those in the healthy controls (82?±?15?ng/ml) (P?=?0.001). There was a significant correlation only between plasma-soluble GPV levels and smoking (r?=?0.526, P?=?0.0001). Smoker UA patients had higher levels of soluble GPV than the non-smoker patients (139?±?40 vs. 113?±?50?ng/ml, respectively, P?=?0.02). However, soluble GPV levels were similar in smoker and non-smoker healthy controls (P?=?0.2). It is concluded that soluble GPV concentrations are significantly increased during the acute clinical course of unstable angina pectoris, indicating that soluble GPV may be useful marker of platelet activation in those patients. The level of the molecule is significantly affected from smoking in those patients.     

Molecular cloning and sequence analysis of alboaggregin B

Platelet flow cytometry is restricted by spontaneous platelet activation in unfixed samples or by significant alterations of platelet performance caused by the use of fixatives. Aim of this study was to evaluate the new platelet stabilizer ThromboFix? for clinical diagnostics.

Methods: Whole blood samples with or without addition of a weak (ADP) or strong (TRAP-6) agonist were fixed with either ThromboFix or paraformaldehyde (PFA) and stored for different periods of time for up to 7 days. Samples were then incubated with either CD41 and CD62P or CD42b and CD45 monoclonal antibodies and analyzed by flow cytometry.

Results: The numbers of platelets, microparticles and aggregates remained stable for 7 days after treatment with ThromboFix but not with PFA due to an increasing aggregate formation after 3 days. Platelet activation was restricted to less than 1% of CD62P positive events in resting samples without a significant difference compared to an unfixed reference sample. Fixation, however, significantly reduced CD62P expression after stimulation (P?<?0.05). Stabilized by ThromboFix, the level of platelet activation remained unchanged in resting and ADP stimulated samples for 7 days but decreased moderately with time after a strong stimulation with TRAP-6 (P?<?0.01). After PFA fixation, intact CD62P antigen disappeared from the platelet surface within hours (P?<?0.01). ThromboFix reduced the formation of platelet–leukocyte conjugates significantly (P?<?0.05) and, in contrast to PFA, failed to stabilize the already formed conjugates.

Conclusion: In clinical situations without immediate access to a flow cytometer, ThromboFix is helpful in the flow cytometric analysis of the platelet activation marker CD62P. It should not be used for the investigation of platelet–leukocyte conjugate formation.     

Patients with systemic lupus erythematosus show increased platelet activation and endothelial dysfunction induced by acute hyperhomocysteinemia

OBJECTIVE: Hyperhomocysteinemia adversely affects the endothelium, although the exact mechanism is unclear. Systemic lupus erythematosus (SLE) is an inflammatory disease with a high atherothrombotic tendency. We examined whether acute hyperhomocysteinemia exacerbates endothelial and platelet dysfunction in patients with SLE. METHODS: Twelve SLE patients and 15 controls were recruited. Oral methionine was used to achieve acute hyperhomocysteinemia. Endothelial function was assessed by flow-mediated dilatation (FMD) of the brachial artery; also assessed were the levels of von Willebrand factor (vWF) and plasminogen activator inhibitor type 1 (PAI-1). Platelet activation was assessed by the levels of beta-thromboglobulin (beta-TG), fibrinogen binding, and P-selectin expression using flow cytometry. RESULTS: After oral methionine loading, vWF levels increased significantly, whereas FMD remained unchanged in both groups. PAI-1 increased significantly only in controls. Fibrinogen binding to platelets increased significantly only in SLE patients. Beta-TG remained unchanged in SLE patients but increased significantly in controls. Platelet P-selectin expression did not change in either group. CONCLUSION: These results suggest that the prothrombotic tendency after acute hyperhomocysteinemia is mediated by endothelial dysfunction and platelet activation in patients with SLE and healthy controls.     

The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection

Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04?mM, 0.2?mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96–29.36] vs. control 2.48[1.56–6.04]; p?<?0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25?±?7.23 vs. control 2.35?±?1.31, p?=?0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31–21.83] vs. control 6.04[1.34–13.15]; p?=?0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09?±?0.64 vs. control 2.44?±?0.11, p?=?0.4591). The HIV group showed higher levels of % CD36 expression post stimulation with 0.04?mM ADP 43.32?±?27.41 vs. control 27.47?±?12.95; p?<?0.0214) and no significant difference at 0.2?mM ADP (HIV % CD36 39.06?±?17.91 vs. control 44.61?±?18.76; p?=?0.3277). Furthermore, the HIV group showed a single phase response to ADP as compared to the control group, which showed a normal biphasic response. We concluded that PLT flow cytometry is valuable in the assessment of levels of PLT activation, and further, that the addition of an endogenous agonist, such as ADP, enabled the measurement of PLT function in HIV infection. We were able to show that, although PLTs are significantly activated in HIV compared to uninfected controls, they retain their functional capacity.     

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads

The aim of the present study was to set up and evaluate a novel method for studies of platelet adhesion and activation in blood and platelet suspensions such as platelet concentrate (PC) samples using protein-coated polystyrene beads and flow cytometry. To demonstrate its usefulness, we studied PCs during storage. PCs were prepared by aphaeresis technique (n?=?7). Metabolic variables and platelet function was measured on day 1, 5, 7 and 12 of storage. Spontaneous and TRAP-6-induced adhesion to fibrinogen- and collagen-coated beads was analyzed by flow cytometry. P-selectin and phosphatidyl serine (PS) expression was assessed on platelets bound to beads as well as on non-adherent platelets. Platelet adhesion to fibrinogen beads had increased by day 12 and adhesion to collagen beads at day 7 of storage (p?<?0.05). TRAP-6 stimulation significantly increased the platelet adhesion to fibrinogen beads (p?<?0.05) as well as the P-selectin and PS exposure on platelets bound to beads (p?<?0.01) during the first 7 days of storage, but by day 12, significant changes were no longer induced by TRAP-6 stimulation. We demonstrate that our adhesion assay using protein-coated polystyrene beads can be used to assess the adhesion properties of platelets during storage without the addition of red blood cells. Therefore it may offer a useful tool for future studies of platelet adhesive capacity in transfusion medicine and other settings.     

Circulated activated platelets and increased platelet reactivity in patients with Beh?et's disease.

Beh?et's disease (BD) is a multisystem disorder. Venous as well as arterial thrombosis is a common complication of BD but exact pathogenetic mechanism of the thrombotic tendency is not well known. This study aimed to evaluate circulating activated platelets and platelet reactivity in Beh?et's patients. Twenty-two Beh?et's patients (4 female, 18 male; mean age 38.6 +/- 10.9 years) and 20 control subjects (8 female, 12 male; mean age 38.8 +/- 9.4 years) were included. Those patients who had hypertension, hyperlipidemia, peripheral or coronary artery disease, hepatic or renal function abnormality, and who were using aspirin and other platelet-active drugs were excluded. Platelet activity and reactivity to adenosine diphosphate (ADP) were measured by whole blood flow cytometry. We assessed markers of platelet degranulation (P-selectin; CD62P) and the activated glycoprotein IIb/IIIa receptor (PAC1 binding to fibrinogen binding site) before and after stimulation with ADP. Platelet P-selectin expression was not significantly different between patients and control subjects both at baseline (p=0.420) and after stimulation (p=0.56). Baseline (p=0.001) and ADP-stimulated (p=0.003) PAC1 binding was significantly higher in Beh?et's patients than in the control group. Clinical activity has no effect on P-selectin expression and PAC1 binding. There is evidence of platelet activity and hyperreactivity in patients with BD and this may contribute to a prothrombotic state. In addition to aspirin, other antiplatelet drugs may be useful in the prevention and treatment of thrombosis in Beh?et's patients.     

Does Hyperconcentration Result in Platelet Activation?

Background and Objectives: Hyperconcentration of platelets may lead to platelet activation and loss of platelet function. Materials and Methods: Platelet activation following hyperconcentration was assessed using flow-cytometric detection of platelet P-selectin expression and platelet swirling. Results: Platelet hyperconcentration led to a minimal increase in P-selectin expression and no differnce in platelet swirling. Conclusion: Hyperconcentration was not associated with a clinically significant change in platelet activation and had no significant effect on platelet quality as detected by pH and platelet yield.