Platelet CD62P Expression Research Articles

Targeting Angiogenesis in Cancer: Bevacizumab-Induced Platelet Activation as a Possible Cause for Unexpected Arterial Thromboembolic Events in Clinical Trials.

The humanized monoclonal VEGF antibody, bevacizumab (Avastin, Genentech), is approved in combination with standard chemotherapy for first-line treatment of patients with metastatic colorectal cancer (CRC) and also shows promising efficacy as anti-angiogenic immunotherapy in patients with non-small-cell lung cancer (NSCLC). A pooled analysis of five randomized, controlled trials (n=1745) showed that, compared to placebo, bevacizumab was associated with an increased risk of arterial thromboembolic events, especially in patients 65 years of age and older (8.5% vs. 2.9%, P<0.01). Because platelets play a crucial role in arterial thrombosis, we hypothesized that bevacizumab has direct platelet-stimulating activity. In a washed platelet system, bevacizumab alone had no effect on platelet aggregation. However, when combined with heparin (0.3 U/ml) and recombinant human VEGF (rhVEGF165 — a homodimeric protein with heparin binding sites) in a 1:2 molar ratio of antibody to antigen to allow optimal formation of immune complexes (ICs) in solution, bevacizumab potently induced platelet aggregation (up to 80–100%; n=5). Bevacizumab-induced platelet aggregation (BIPA) was functionally dependent on Fc domain binding to the platelet low-affinity IgG receptor, FcγRIIA, as demonstrated by an inhibitory monoclonal CD32 antibody (IV.3). BIPA was potentiated in platelets pre-sensitized with low concentrations of ADP or epinephrine. In contrast, BIPA was virtually absent at excess concentrations of heparin (100 U/ml), suggesting that translocation of ICs to the platelet surface via available heparin binding sites (on platelets) was crucial for this platelet response. Unfractionated heparin and the low-molecular-weight heparin, enoxaparin, were equally effective in promoting BIPA. In a manner similar to heparin-PF4 antibodies from patients with heparin-induced thrombocytopenia (HIT), bevacizumab-rhVEGF165-heparin ICs induced significant FcγRIIA-dependent dense granule release (>80%) in a 14C-serotonin release assay (SRA). While strong platelet responses were observed in both SRA and aggregometry, in which platelets were subjected to constant movement and low shear forces, respectively, bevacizumab-rhVEGF165-heparin ICs had only negligible effects on platelet CD62P expression under static conditions, indicating a critical role for platelet-platelet contacts in bevacizumab-mediated FcγRIIA signaling. In summary, our results suggest bevacizumab can induce strong FcγRIIA-dependent platelet activation in vitro when complexed with rhVEGF165 and heparin in an optimal stoichiometry. Due to their analogy to the pathomechanism of HIT, an acquired IgG-mediated disorder potentially associated with deleterious thrombosis, these findings may have direct clinical implications for older cancer patients with cardiovascular comorbidity, especially considering that many patients receive low doses of heparin for thromboprophylaxis and that elevated serum VEGF levels have been demonstrated in various types of malignancy, including CRC and NSCLC.

“Young” Platelets Regulate Thrombus Formation by Expression of Increased Adhesive Properties and Procoagulant Activity.

Previous studies from our lab and others have demonstrated that thrombin mediated activation of platelets results in the formation of a distinct procoagulant subpopulation capable of assembling the components of both the prothrombinase complex and intrinsic tenase complex at its surface. A similar subpopulation has been identified following activation of platelets with the combination of thrombin and convulxin, a glycoprotein VI agonist, and has been found to be enriched in young platelets. In addition, young zebrafish thrombocytes were shown to have a higher density of adhesive receptors and greater functional activity than their more mature counterparts. Since the membrane properties that distinguish the platelet's ability to assemble these procoagulant complexes are unknown, the purpose of this study was to elucidate these important differences. Following their isolation from whole, anticoagulated blood, human platelets were activated with 50 nM thrombin (2 min, ambient temperature) followed by the addition of hirudin (75 nM) to quench the reaction. Equimolar concentrations of factors Va and Xa were added (5 nM, 10 min) to saturate the functional Prothrombinase binding sites determined previously using kinetics of prothrombin activation, prior to platelet fixation with 2% v/v paraformaldehyde. Those platelets capable of binding factors Va and Xa were identified by flow cytometry using specific, non-inhibitory fluorophore-conjugated MoAbs directed against the required cofactor and serine protease, respectively. Analyses of the membrane density of well-known and characterized platelet receptors including glycoprotein IIb/IIIa and glycoprotein Ib-IX-V, were also assessed using specific MoAbs conjugated with the appropriate fluorophore to determine whether expression of any or all of these adhesive receptors defined the platelet subpopulation capable of assembling a competent Prothrombinase. Thiazole orange (TO), a dye frequently used to stain nucleic acid, and previously shown to identify the youngest platelets in circulation, was used to evaluate the effect of platelet age on these various parameters. Using this approach, we demonstrated a 7–8-fold increase in both CD62P (P-selectin) and CD61 (glycoprotein β3) expression in TO-positive platelets, while expression of the platelet marker CD42b (glycoprotein 1bα) increased as much as 14-fold. The subpopulation capable of assembling Prothrombinase likewise demonstrated a 3–4-fold increase in CD62P, CD61, and CD41 (glycoprotein IIb) expression, and a ~7-fold increase in CD42b expression. Dual-labeling studies demonstrated that ~60% of TO-positive platelets are able to bind factor Xa while ~85% of platelets able to bind Xa are TO-positive. Similar results were seen when platelets were evaluated for their ability to bind high levels of factor Va. These data suggest that it is predominantly the younger platelets with increased density of adhesive receptors that are capable of assembling Prothrombinase on their activated surface. Since platelet adhesion is the initiating step in both normal hemostasis and pathological thrombus formation, the observed increase in adhesive receptor density places this platelet subpopulation in a unique position of significance in the regulation of the hemostatic response, and indicates a potential role for these platelets, in the initiation of thrombus formation.

Concentration-dependent effect of abciximab on platelets and neutrophils in a model of cardiopulmonary bypass

Abciximab is a GPIIb/IIIa antagonist used in percutaneous coronary interventions to avoid platelet activation, thrombosis and inflammation. We investigated whether abciximab influenced platelet activation and platelet interaction with neutrophils and polyvinyl chloride (PVC) in a cardiopulmonary bypass (CPB) model. Isolated platelets, preincubated with and without 0.1-20 microg/mL of abciximab, were resuspended with neutrophils in plasma and recirculated by a roller pump. Platelet, but not neutrophil adhesion to PVC was inhibited by abciximab. Only high doses of abciximab reduced platelet aggregation size, but simultaneously increased platelet-neutrophil aggregation. Abciximab had no effect on platelet CD62P expression or degranulation, but platelet activation on platelet-neutrophil aggregates increased with high doses. Only low doses inhibited neutrophil degranulation. The concentration-dependent effect of abciximab on platelet-neutrophil interaction reduces its usefulness and stresses the dependency on experimental design in the evaluation of abciximab. Our study does not support the use of abciximab alone in CPB. However, incorporation of surface-coating the biomaterial with abciximab may be an interesting option.

Increased circulating platelet–neutrophil, platelet–monocyte complexes, and platelet activation in patients with ulcerative colitis: A comparative study

It is reported that the incidence of thromboembolism is increased in ulcerative colitis (UC), and hypercoagulability persists even when patients are in remission. We evaluated the association of inflammatory response parameters with UC activity, and activation parameters of the platelets, endothelium, and the coagulation system in UC. Eighteen UC patients and 19 healthy subjects were included in the study. The patients' clinical features were recorded down; whole blood counts and acute phase parameters were evaluated. UC patients were divided into two as active (9 patients) and inactive (9 patients) according to combined clinical activity index (CAI) and endoscopic activity index (EAI) scores. In all subjects, platelet CD62P expression, platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), and platelet microparticles (PMP) were determined by flow cytometry. E-selectin, thrombin-antithrombin complex (TAT) levels in plasma, and sCD40L levels in serum were determined by ELISA. In both active and inactive UC patients, platelet CD62P expression, the percentages of PMC, and PNC were significantly higher than those in the control group (P< 0.01). PMP level was higher in the control group than in inactive UC patients (P = 0.001). sCD40L level was significantly higher in active UC group than in the control group (P < 0.01). EAI score correlated significantly with PMP (r = 0.5, P = 0.04) and sCD40L (r = 0.48, P = 0.044); CAI score had a negative correlation (r = -0.68, P = 0.002) with sE-selectin level. In addition to increased CD62P expression and sCD40L, increased formation of PMC and PNC suggests a role for platelet-leukocyte complex formation together with platelet activation in thromboembolic events observed in UC.

Platelet Activation in Helicobacter pylori-Associated Idiopathic Thrombocytopenic Purpura: Eradication Reduces Platelet Activation but Seldom Improves Platelet Counts

Introduction: It has been suggested that Helicobacter pylori eradication often increases platelet counts in patients with chronic idiopathic thrombocytopenic purpura (ITP). In addition, H. pylori has been shown to induce platelet activation (CD62p or P-selectin expression) in previous studies. We assessed the response of platelet count and CD62p expression after eradication therapy in patients with ITP and H. pylori infection. Methods and Results: We prospectively studied 15 ITP patients diagnosed with H. pylori infection by serology and breath test. A follow-up breath test was used to document eradication. Two out of 15 patients showed improvement in platelet counts after 6 months, 1 of which may have had drug-induced thrombocytopenia. Overall, certain platelet response rate in our series was 6.7% (1/15). We found that platelet CD62p expression by flow cytometry was elevated in 10/15 (66.7%) H. pylori-infected patients, which is a statistically significant difference when compared with 3/33 (9.1%) control ITP patients seronegative for H. pylori (p = 0.002). In addition, eradication therapy decreased CD62p expression (p = 0.04). However, reduction in platelet activation was not associated with an increase in platelet counts (mean 72.4 × 10⁹/l before and 68.7 after therapy; p = 0.4). Conclusion: In our series, platelet activation was common in ITP patients with H. pylori, and eradication therapy decreased platelet activation but seldom increased platelet counts. Increased platelet CD62p expression is a putative link between chronic infections and atherosclerosis, but further study is needed to clarify the implications of our observation.

Comparison of two sterile connection devices and the effect of sterile connections on blood component quality

Sterile connection devices (SCDs) are used to connect pieces of polyvinylchloride tubing between blood bag systems. After observing a slight decrease in inner diameter of tubing welded with the CompoDock S2 SCD, the effect of welded tubing on storage characteristics of white blood cell (WBC)-reduced red blood cells (RBCs) and platelet (PLT) concentrates was studied. Welds were made with Terumo SCD (T-SCD) or CompoDock S2, and unwelded tubing served as reference. Three WBC-reduced RBC units or 3 PLT concentrates were pooled and divided to prevent donor-dependent differences. The units were transferred 10 times over (1) tubing with a T-SCD weld, (2) a CompoDock S2 weld, or (3) unwelded tubing. RBCs were stored for 42 days and free hemoglobin (Hb) was measured; PLT concentrates were stored for 8 days and CD62P expression was measured, as markers for blood component quality (n = 10 paired experiments). WBC-reduced RBC units had similar hemolysis at the end of storage: 0.47 +/- 0.28, 0.47 +/- 0.35, and 0.49 +/- 0.38 percent of total Hb, for tubing with a T-SCD weld, a CompoDock S2 weld, or no weld, respectively (not significant). CD62P expression of stored WBC-reduced PLT concentrates was not significantly different between the groups: 20.3 +/- 5.1, 19.8 +/- 5.1, and 22.3 +/- 9.8 percent for tubing with a T-SCD weld, a CompoDock S2 weld, or no weld, respectively. The quality of blood components, measured as RBC hemolysis and platelet CD62P expression, is not adversely affected by the presence of a sterile connection in the tubing, made by either the CompoDock S2 or the T-SCD.

Platelet Activity and Serum Homocysteine Levels in Patients with End-Stage Renal Failure with Regard to Dialysis Modality

Background. Recent evidence suggests that the activation of platelets and their interaction with circulating cells are important independent risk factors for atherosclerosis. In non-uremic patients with symptomatic peripheral vascular disease, a relationship between serum homocysteine (Hcy) levels and platelet activity had been reported. The purposes of this study were to evaluate of effects of dialysis modality on platelet activity in patients with end-stage renal failure and to investigate the relationship between platelet activity, Hcy, and left ventricular hypertrophy (LVH). Material and Methods. In age and sex matched 19 healthy subjects, 20 hemodialysis (HD) patients, and 18 continuous ambulatory peritoneal dialysis (CAPD) patients, the expression of platelet surface receptors CD41, CD61, CD42a, and CD62P were investigated. CD62P expression was statistically significantly increased in HD patients compared with CAPD patients and controls (34.4 ± 22.5%; 17.3 ± 19.6%, 12.0 ± 15.6%, respectively, p < 0.05), but not in CAPD patients compared with controls. There was a positive correlation between CD62 expression and duration of dialysis in HD patients (r = 0.498, p = 0.026). Mean plasma Hcy levels in dialysis patients were higher than reference levels. However, we could not find any relationship between CD62 expression, Hcy, and LVH in both groups (p > 0.05). Conclusions. Hemodialysis and peritoneal dialysis (PD) have a different impact on the expression of CD62: peritoneal dialysis seems to have a more favorable effect. It may be possible that the differences in biocompatibility between PD and HD potentially contribute to differences in CD62 expression.

Leukocyte-platelet interaction in patients with essential thrombocythemia and polycythemia vera

Circulating polymorphonuclear leukocyte (PMN) activation occurs in patients with essential thrombocythemia (ET) and polycythemia vera (PV). We want to define whether this phenomenon plays a role in the formation of circulating PMN-platelet aggregates in these conditions. In 80 patients (46 ET and 34 PV) and 50 control subjects, we conducted a flow cytometric analysis to evaluate the levels of PMN-platelet aggregates (defined as the percentage of CD11b-positive PMN coexpressing a platelet-specific marker, i.e., CD42b or CD62P) and the levels of activated PMN and activated platelets. In addition, the in vitro PMN-platelet aggregate formation in response to N-formyl-methionyl-leucyl-phenylalanine (f-MLP)-induced activation of PMN was studied. Significantly high PMN-platelet aggregates in ET and PV patients were found and were associated with increased PMN surface CD11b and surface platelet CD62P expression. In vitro f-MLP stimulation upregulated PMN-CD11b expression and simultaneously increased CD11b/CD42b and CD11b/CD62P aggregates, without affecting platelet surface antigens. In ET patients receiving aspirin, the increments in f-MLP-induced PMN-CD11b and in PMN-platelet aggregates were significantly lower versus ET subjects not treated with aspirin. Our data show that in ET and PV patients PMN activation plays an important role in increasing circulating PMN-platelet aggregates and suggest that aspirin treatment may decrease their formation.

Digoxin use is associated with increased platelet and endothelial cell activation in patients with nonvalvular atrial fibrillation

The purpose of this study was to determine whether digoxin use is associated with increased flow cytometric markers of endothelial cell and platelet activation in patients with nonvalvular atrial fibrillation (AF). Increased intracellular calcium is a key event in platelet activation, and several studies have demonstrated that digitalis activates platelets in vitro. Intracellular calcium also is a key regulator of endothelial cell function, and endogenous digitalis-like substances have been shown to affect biologic processes in endothelial cells. We studied 30 patients with nonvalvular AF. We measured the levels of (1) platelet expression of P-selectin (CD62P), (2) platelet microparticles (PMP); and (3) endothelial microparticles (EMP) identified by anti-CD31 (EMP31) and by anti-E-selectin antibodies (EMP62E). Patients who were taking digoxin (n = 16; mean digoxin level = 0.93 ng/dL) did not demonstrate any significant differences in clinical or echocardiographic characteristics compared with patients not taking digoxin (n = 14). Patients taking digoxin had significantly increased levels of CD62P expression in platelets and platelet-leukocyte conjugates and markedly increased markers of endothelial activation: EMP62E and EMP31. After adjusting for potential confounders (including age, congestive heart failure, coronary artery disease, ejection fraction, antiplatelet, beta-blocker, and calcium channel blocker use), the differences persisted. Digoxin use in patients with AF is associated with increased levels of endothelial and platelet activation. If digitalis activates endothelial cells and platelets at pharmacologic doses, use of digitalis in conditions such as AF could predispose to thrombosis and vascular events.

Increased levels of platelet-derived microparticles in patients with diabetic retinopathy

Diabetic retinopathy is caused by capillary occlusions. Platelet-derived microparticles (PMPs) stimulate the coagulation cascade and increase leukocyte and endothelial cell adhesions, both of which are key events in the development of diabetic retinopathy. However, the correlation between the levels of PMPs and diabetic retinopathy has not been precisely determined. The PMPs levels and the expression of platelet CD62P and CD63 were measured in 92 diabetic patients. The level of PMPs was significantly correlated with the expression of CD62P ( r = 0.76, P < 0.0001) and CD63 ( r = 0.71, P < 0.0001). The mean level of PMPs in diabetics (507 ± 15/10 4 platelets (plt), mean ± S.E.) was significantly higher than that in normal. The PMPs levels increased with the progression of the diabetic retinopathy; 480 ± 28/10 4 plt in diabetic patients without retinopathy ( n = 25), 504 ± 40/10 4 plt with mild or moderate non-proliferative diabetic retinopathy ( n = 13), 512 ± 29/10 4 plt with severe non-proliferative diabetic retinopathy ( n = 25), and 528 ± 25/10 4 plt with proliferative diabetic retinopathy ( n = 29). The PMPs level in patients with non-perfused retinal areas (582 ± 27/10 4 plt, n = 24) was significantly higher than patients without non-perfused areas (469 ± 23/10 4 plt, n = 30; P = 0.0096) and without diabetic retinopathy ( P = 0.024). These high correlations indicate that increased levels of PMPs may accelerate diabetic retinopathy.

SB 497115-GR, a Low Molecular Weight TPOR Agonist, Does Not Induce Platelet Activation or Enhance Agonist-Induced Platelet Aggregation in Vitro.

SB-497115-GR is a low molecular weight, orally active molecule that requires Tpo receptor (TpoR) expession for activity and activates JAK/STAT signalling in platelets. The ability of recombinant Tpo to enhance agonist-induced platelet aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01–10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Citrated whole blood was used directly in platelet activation and P-selectin assays within 15 minutes. Platelet aggregation was assessed by light transmittance aggregometry. Aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 or 1.5 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01–10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115-GR or rhTpo. In experiments investigating the expression of platelet P-selectin, an early marker of platelet activation, platelets labeled with anti-CD62P antibody were analyzed by flow cytometry. Data was expressed as percent of platelets positive for CD62P within the defined platelet gate. Treatment with SB-497115 (0.1 – 10 uM) demonstrated no induction of platelet CD62P expression above background levels. In contrast, rhTpo produced a modest but significant and reproducible increase in the percent of platelets positive for CD62P. This study demonstrates that, in contrast to rhTpo, the orally active small molecular weight agonist of the Tpo receptor, SB-497115-GR, does not mimic the ability of rhTpo to enhance ADP-induced aggregation of platelets. SB-497115-GR also did not directly induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to platelet activation.

Serial changes in platelet activation in patients after ischemic stroke: role of pharmacodynamic modulation.

Enhanced platelet activity has previously been reported in the acute phase after ischemic stroke. We tested the hypothesis that activated platelets (expressed by CD62p) are substantially increased in the acute stage after a stroke and decrease thereafter, and that antiplatelet therapies can suppress CD62p expression. We serially examined platelet CD62p expression using flow cytometry after acute ischemic stroke in 87 consecutive patients. The CD62p expression was also evaluated in 20 healthy volunteers and 33 at-risk control subjects. CD62p expression was significantly higher in the acute phase after ischemic stroke than in normal and at-risk control subjects (both P<0.0001). CD62p expression decreased to a significantly lower level on day 21, and to a substantially lower level on day 90. CD62p expression was not significantly suppressed by warfarin. However, CD62p expression was significantly suppressed by aspirin treatment (P=0.024) and more substantially suppressed by clopidogrel (P<0.0001) on day 90. Furthermore, only clopidogrel treatment (P=0.0016) was significantly independently associated with decreased CD62p expression on day 90. Platelet activation was significantly increased in acute ischemic stroke and substantially decreased thereafter. The lesser long-term pharmacodynamic potency of aspirin relative to clopidogrel raises the prospect of the need for more effective antiplatelet agents or a synergistic combination therapy for stroke prevention in the future.

Effect of clopidogrel pretreatment on inflammatory marker expression in patients undergoing percutaneous coronary intervention

Platelets play an important role in the inflammatory response. In a nonrandomized comparison, we examined the effect of clopidogrel pretreatment on platelet inflammatory marker expression in patients undergoing percutaneous coronary intervention (PCI). Platelet expression of the inflammatory markers CD40 ligand (L) and CD62 P-selectin (P) and serum levels of interleukin-6 and CD40L were compared in patients pretreated (>24 hours before PCI) or not pretreated with clopidogrel. Blood samples were obtained before and after the procedure, and from 18 to 24 hours later. Marker expression in resting and adenosine diphosphate (ADP) (50 μmol/L) and thrombin receptor activating peptide (TRAP) (10 μmol/L) activated samples was quantified by flow cytometry. Serum CD40L and interleukin (IL)-6 levels were determined by enzyme-linked immunosorbent assay. Seventy-nine patients were recruited into the study. Forty-two percent were pretreated with clopidogrel for a median of 5 days (range 1 to 1,325). Clopidogrel pretreatment was associated with lower ADP-activated platelet CD40L expression in baseline and postprocedural samples. Similarly, platelet CD62P expression at all time points in ADP-activated and in baseline and postprocedural TRAP-activated samples was lower in patients pretreated with clopidogrel. These differences remained after multivariate adjustment between the groups. Serum CD40L levels increased from 2.13 ± 2.37 ng/ml at baseline to 4.77 ± 3.86 ng/ml at 18 to 24 hours after the procedure (p <0.0001). Similarly, serum IL-6 levels increased at 18 to 24 hours after the procedure (14.8 ± 42.0 pg/ml before vs 25.5 ± 36.0 pg/ml at 18 to 24 hours after the procedure, p <0.0001). Clopidogrel pretreatment did not affect serum IL-6 or CD40L levels. Thus, clopidogrel pretreatment reduces platelet inflammatory marker expression in patients undergoing PCI.

The influence of VIP and epoprostenol on platelet CD62P expression and primary haemostasis in vitro

Human vasoactive intestinal peptide (VIP) and epoprostenol (prostacyclin) have vasodilatative effects in the pulmonary circulation. Both VIP and epoprostenol are successfully used to treat pulmonary hypertension in humans and experimental animal models. The positive effects of epoprostenol on the course of this disease are achieved through vasodilatation and inhibitory effects on platelet activity. Since VIP also binds specifically to platelets, we compared the in vitro effects of VIP and epoprostenol on platelet P-Selectin (CD62P) expression and primary haemostasis. Anti-aggregative effects of VIP (10−6 mol and 10−8 mol) and epoprostenol (50, 5 and 0.5 ng/ml) on platelets were determined by agonist-induced CD62P expression and in vitro bleeding time (PFA-100™ system). Blood from healthy individuals was either incubated with epoprostenol, VIP or saline control and was analysed by whole blood flow cytometry and the PFA-100™. Prior to flow cytometric analysis, the platelets were stimulated with either arachidonic acid (AA) or adenosine diphosphate (ADP). Whole blood flow cytometry analysis showed that epoprostenol inhibited dose-dependently agonist-induced CD62P expression, whereas VIP did not inhibit CD62P expression. PFA analysis revealed substantial closure time prolongation by epoprostenol and again no effects of VIP. These results indicate that VIP, in contrast to epoprostenol, has no effect on platelet CD62P expression and primary haemostasis.

The effect of hormone replacement therapy on Ca2+ mobilization and P-selectin (CD62P) expression in platelets examined under flow cytometry.

A series of events, such as increase of cytoplasmic free calcium (Ca2+) and expression of P-selectin (CD62P), an adhesion molecule, on the platelet surface, are significant indicators of platelet activation. We have used flow cytometry to examine Ca2+ mobilization and CD62P expression in platelets in whole blood obtained in women prior to, and after, different forms of hormone replacement therapy. Thirty-two women completed a protocol consisting of two consecutive 1-month periods under oestradiol (E2), administered orally (2 mg/day) or transdermally (50 microg/day) in random order, followed by a 4-week transdermal sequential regime, in which, during the last 14 days, either progesterone (300 mg/day) or medroxyprogesterone acetate (5 mg/day) was added to the 50 microg/day E2, administered orally in random order. None of the hormonal combinations determined significant changes in Ca2+ mobilization or CD62P expression in non-stimulated platelets. However, stimulation of platelets with adenosine diphosphate, but not with thrombin, caused a significant increase in cytoplasmic Ca2+ concentration during treatment with transdermal E2 plus progesterone. Also when stimulating with thrombin, transdermal E2 was more active than oral E2 in increasing CD62P expression, a difference that was not reduced by the addition of progestogens. In conclusion, hormone replacement therapy only increased Ca2+ mobilization or CD62P expression in stimulated platelets, and then followed a varied pattern that was dependent on the stimulant and on the specific hormonal formulation.

Severe meningococcal disease is characterized by early neutrophil but not platelet activation and increased formation and consumption of platelet-neutrophil complexes.

Approximately 25% of polymorphonuclear leukocytes (PMNL) circulate in heterotypic complexes with one or more activated platelets. These platelet-neutrophil complexes (PNC) require platelet CD62P expression for their formation and represent activated subpopulations of both cell types. In this study, we have investigated the presence, time course, and mechanisms of PNC formation in 32 cases of severe pediatric meningococcal disease (MD) requiring intensive care. There were marked early increases in PMNL CD11b/CD18 expression and activation, and reduced CD62L expression compared with intensive care unit control cases. Minimal platelet expression of the active form of alphaIIbbeta3 (GpIIb/IIIa) was seen. PNC were reduced on presentation and fell to very low levels after 24 h. Immunostaining of skin biopsies demonstrated that PNC appear outside the circulation in MD. In vitro studies of anticoagulated whole blood inoculated with Neisseria meningitidis supported these clinical findings with marked increases in PMNL CD11b/CD18 expression and activation but no detectable changes in platelet-activated alphaIIbbeta3 or CD62P expression. In vitro PMNL activation with N. meningitidis (or other agonists) potentiated the formation of PNC in response to platelet activation with adenine diphosphate. Therefore, in severe MD, PMNL activation is likely to promote PNC formation, and we suggest that the reduced levels of PNC seen in established MD reflect rapid loss of PNC from the circulation rather than reduced formation.

CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation.

CD40 is a 48-kDa phosphorylated transmembrane glycoprotein belonging to the TNF receptor superfamily. CD40 has been demonstrated on a range of cell types, and it has an important role in adaptive immunity and inflammation. CD40 has recently been described on platelets but platelet activation by CD40 has not been described. In the present study, we use flow cytometry and immunoblotting to confirm that platelets constitutively express surface CD40. CD40 mRNA was undetectable, suggesting that the protein is synthesized early in platelet differentiation by megakaryocytes. Ligation of platelet CD40 with recombinant soluble CD40L trimer (sCD40LT) caused increased platelet CD62P expression, alpha-granule and dense granule release, and the classical morphological changes associated with platelet activation. CD40 ligation also caused beta3 integrin activation, although this was not accompanied by platelet aggregation. These actions were abrogated by the CD40L blocking antibody TRAP-1 and the CD40 blocking antibodies M2 and M3, showing that activation was mediated by CD40L binding to platelet CD40. beta3 integrin blockade with eptifibatide had no effect, indicating that outside-in signaling via alphaIIbbeta3 was not contributing to these CD40-mediated effects. CD40 ligation led to enhanced platelet-leukocyte adhesion, which is important in the recruitment of leukocytes to sites of thrombosis or inflammation. Our results support a role for CD40-mediated platelet activation in thrombosis, inflammation, and atherosclerosis.

Neutrophil CD11b upregulation during cardiopulmonary bypass is associated with postoperative renal injury

Background Renal injury remains a persistent complication of cardiopulmonary bypass (CPB) that, when sufficient to require dialysis, increases mortality eight-fold. The high prevalence of renal failure in sepsis and adult respiratory distress syndrome has been linked to the systemic inflammatory response associated with those disorders. We hypothesized that components of the inflammatory response to CPB may similarly contribute to post-CPB acute renal injury. Methods Markers of leukocyte and platelet activation peri-CPB were measured in 75 patients undergoing cardiac operation with CPB and were correlated with acute renal injury, defined as an increase (≥ 50%) in peak serum creatinine post-CPB. Results Eleven patients sustained post-CPB acute renal injury. This subset of patients demonstrated significantly greater increases in neutrophil CD11b density ( p = 0.01), as well as higher total neutrophil counts ( p = 0.045), compared with patients with preserved renal function. Hemodynamic instability sufficient to require postoperative hemodynamic support also predicted an increased risk of acute renal injury. However, neutrophil CD11b upregulation did not correlate with this or any other clinical variables associated with renal risk, suggesting that this marker of the neutrophil inflammatory response may independently predict renal injury. By contrast other inflammatory markers, neutrophil myeloperoxidase levels, monocyte CD11b, base line C-reactive protein, and platelet CD62P expression did not differ between the two patient groups. Conclusions Upregulation of the neutrophil adhesion receptor CD11b and high circulating neutrophil numbers are associated with acute renal injury after CPB, suggesting a contribution by activated neutrophils to the pathophysiology of this complication.

Platelet CD62p expression and microparticle in murine acquired immune deficiency syndrome and chronic ethanol consumption.

Abnormal platelet counts have been noticed in acquired immune deficiency syndrome (AIDS) patients. However, the actual state of platelets in AIDS is unclear. We hypothesize that platelets are activated and platelet-derived microparticles increase in murine AIDS. To elucidate the ethanol effects on platelets in murine AIDS, we studied four groups: control, murine AIDS, ethanol, and ethanol plus murine AIDS. Platelet CD62p as a platelet activation marker and CD61(+) microparticles as platelet microparticles (PMPs) were measured by flow cytometry. Platelets were significantly activated in mice with murine AIDS and chronic ethanol consumption. Increased platelet CD62p expression and increased PMPs were most pronounced in advanced stages of murine AIDS. Chronic ethanol consumption persistently enhanced platelet activation and PMP formation. Elevated platelet CD62p and PMPs may represent a pro-thrombotic status that have important pathological consequences.

Equine platelet CD62P (P-selectin) expression: a phenotypic and morphologic study

Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet–leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin–PSGL-1 (platelet–leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to investigate phenotypic and morphological properties of equine platelet activation with a focus on CD62P (P-selectin) expression and CD62P mediated platelet–leukocyte interactions. To study high levels of platelet activation, we used 1 U/ml thrombin to induce secondary, irreversible aggregation in both human and equine platelets. Addition of glycyl- l-prolyl- l-arginyl- l-proline amide (GPRP) prior to thrombin activation blocked fibrin polymerization, allowing the use of flow cytometry to study α-granule expression as a measure of platelet activation. Thrombin activation resulted in high levels of activation, measured as P-selectin expression, in both humans and equines. Interestingly, our research illustrates that in healthy horses, P-selectin is also constitutively expressed on 20–25% of resting platelets. This finding is in direct contrast to humans, in which P-selectin expression is negligible (<5%) in the absence of agonist activation. The high baseline level of P-selectin expression among equine platelets may suggest that they are primed for leukocyte adhesion, possibly resulting in prothrombotic conditions. This phenomenon could be of significant clinical relevance, as it may be related to the rapid clinical decline often seen in equine patients with colic and endotoxemia, where vascular injury and thrombotic complications compromise patient survival. Based on these findings, further investigation into the mechanisms of platelet P-selectin-mediated inflammation and platelet–leukocyte mediated vascular injury in the horse appears warranted.