Platelet Surface P-selectin Research Articles

The aspirin resistance and platelets` receptors expression in chronic heart failure patients with type 2 diabetes mellitus

Introduction. Chronic heart failure (CHF) is a complex clinical syndrome associated with high mortality rates. The presence of type 2 diabetes mellitus (DM) is an independent unfavourable prognostic factor in heart failure patients. Chronic heart failure and type 2 diabetes mellitus are closely inter-related conditions through different pathological mechanisms, including platelet activation. The presence of DM in CHF patients can dramatically increase platelet dysfunction. The problem is much complicated by high level of aspirin resistance in diabetic patients. The prevalence of aspirin resistance in CHF with DM has never been studied.The aim of our research was to estimate the prevalence of aspirin resistance in heart failure patients with type 2 diabetes mellitus and to clarify platelets` abnormalities according to glycemic control status.Materials and methods. The criteria for study inclusion were: a more than 1-year documented history of CHF due to coronary artery disease, sinus rhythm, type 2 diabetes mellitus, no change in medication for > 3 months, daily aspirin use 100 mg > 6 months. All patients had previous history of myocardial infarction. Exclusion criteria were: type 1 DM, patients with type 2 DM on insulin therapy and/or thiazolidinediones, major bleedings in anamnesis, chronic renal failure, chronic obstructive pulmonary disease, autoimmune diseases, significant liver failure, cancer. We studied 65 patients with heart failure and type 2 diabetes mellitus (mean age 62±8 years). The control population comprised 35 heart failure patients without diabetes mellitus (mean age 60±9 years). Platelet function was evaluated using standard light transmission aggregometry. Aspirin resistance was defined by known criteria: ≥ 70% ADP- and ≥ 40% epinephrine-induced aggregation. Measurements of CD41, CD41a, CD61 and CD62P were carried out by fluorescent microscopy study. The plasma levels of soluble P-selectin and soluble CD40L were measured by ELISA assays.Results. Age, sex, severity of CHF, left ventricular ejection fraction (LVEF), estimated glomerular filtration rate and cardiovascular treatment were comparable between three groups. Hypertension rate was slightly frequent in patients with CHF and decompensated DM. The patients with CHF and decompensated DM had higher fasting plasma glucose, HbA1cand HOMA-IR levels. The duration of the diabetes was similar. Glucose lowering treatment on admission was comparable between groups. The rate of aspirin resistance was tend to be higher in patients with CHF and DM (by ADP-induced aggregation: 23.1% in diabetic patients vs. 14.3% in non-diabetic patients, P=0.43; by epinephrine-induced aggregation: 36.9% in diabetic patients vs. 31.4% in non-diabetic patients, P=0.66), but the difference was not statistically significant. The compensation of DM didn’t influence on aspirin resistance rate also. The compensation of DM didn’t influence on aspirin resistance rate also. No significant changes in platelets’ parameters were observed between CHF patients without DM and patients with CHF and compensated DM. In contrast, the patients with CHF and decompensated DM presented higher platelet surface CD62P, CD41, CD41a and CD61 expression in comparison to CHF patients without DM. The patients with CHF and HbA1c of more than 7 % showed also higher plasma P-selectin and higher platelet surface CD41 and CD61 expression compared with CHF patients with HbA1c of less than 7 %. Plasma sP-selectin correlated positively with FPG, insulin, C-peptide and HOMA-IR. Platelet surface P-selectin correlated only with C-peptide. Both IIb and IIIa glycoprotein receptors correlated with HbA1c. Conclusions. The rate of aspirin resistance varies from 23 to 37% in patients with chronic heart failure and type 2 diabetes mellitus depending on tests used. Platelet reactivity is associated with glycemic control status in heart failure diabetic patients on long-term aspirin therapy.

The association of the Thr715Pro P‐selectin genotype with levels of P‐selectin in platelet concentrates

P-selectin is stored in the alpha granules of platelets and in the Weibel Palade bodies of endothelial cells; upon activation, it is translocated to the cell surface and released into the plasma in soluble form. One variant of the P-selectin gene, the Thr715Pro polymorphism, is strongly associated with the plasma levels of soluble P-selectin. In platelet concentrates soluble P-selectin can be regarded mainly platelet derived. The relation of the genotype with soluble P-selectin, platelet expressed P-selectin and the sum of all forms of P-selectin - comprising soluble P-selectin, platelet surface P-selectin and P-selectin from the alpha granules - was assessed in fresh whole blood and in apheresis platelets suspended in 35% plasma/65% SSP+ obtained from 89 platelet donors. Levels of total P-selectin were genotype associated (P=0·025); likewise, in fresh whole blood there was an association of soluble P-selectin with genotype (P=0·02). In platelets suspended in additive solution, however, levels of the storage-associated or TRAP-6 agonist induced increase of platelets' P-selectin were not associated with the genotype. A correlation between levels of soluble P-selectin and surface expression of P-selectin was observed on day 3 of storage in Thr715Thr individuals (P<0·0001), but not in heterozygotes (Thr715Pro, P=0·2). The donors' genotype has only little influence on levels of soluble P-selectin in apheresis platelets suspended in 35% plasma/65% SSP+.

Increased responsiveness to thrombin through protease-activated receptors (PAR)-1 and -4 in active Crohn's disease

Background and aimsPlatelets are essential in hemostasis and inflammation, thereby linking coagulation with inflammation. Abundant thrombin generation in association with inflammation is considered a major reason for the increased risk for thromboembolic events. We therefore investigated platelet responsiveness to thrombin. MethodsIn this case–control study 85 patients with Crohn's disease (active CD 42, remission 43) and 30 sex- and age-matched controls were enrolled. Clinical disease activity (Harvey–Bradshaw-Index) was assessed and CD-related data were determined by chart review. Platelets' response to protease activated receptor-1 and -4 (PAR-1, -4) was assessed by whole blood platelet aggregometry (MEA), levels of platelets adhering to monocytes (PMA), and platelet surface P-selectin. ResultsPlatelets' aggregation after activation with the specific PAR-1 agonist (SFLLRN) and PAR-4 agonist (AYPGKF) was higher in patients with active CD compared to patients in remission and controls (p=0.0068 and p=0.0023 for SFLLRN, p=0.0019 and 0.0003 for AYPGKF). Likewise, levels of PMA after activation with PAR-1 and PAR-4 receptor agonists were higher in patients with active CD compared to patients in remission and controls (p=0.0001 and p<0.0001 for SFLLRN, p=0.0329 and p=0.0125 for AYPGKF). However, P-selectin expression on human platelets showed heterogeneous results. Only PAR-1 activation of platelets resulted in significant differences between CD patients and controls (p=0.0001 and p=0.0022 for active and inactive CD versus controls, respectively). ConclusionsOur data suggest a new mechanism of platelet activation which has the potential to increase risk for thromboembolism in patients with active CD which might be due to platelets poised for thrombin-inducible activation.

Increased PAC-1 expression among patients with multiple myeloma on concurrent thalidomide and warfarin

Treatment with thalidomide is associated with vascular thrombosis. The effect of thalidomide on platelet activation is unclear, although the use of aspirin is justified for thromboprophylaxis. A study on platelet activation markers was done among multiple myeloma patients receiving thalidomide therapy with warfarin as thromboprophylaxis. Strict criteria and procedure were set to avoid misinterpretation of platelet activation other than due to the thalidomide's effect. Blood specimen pre and post thalidomide therapy were used for flow cytometric analysis. Platelet surface P-selectin, CD62P expression and PAC-1 (antibody that recognizes conformational change of the GPIIb/IIIa complex) were examined by using three-colour flowcytometer. Increased expression marker for PAC-1 was observed after 4 weeks of thalidomide treatment (P < 0.05) indicating one aspect of platelet activation activity seen in these patients. The mechanism of thrombosis by thalidomide is probably multifactorial and one of them is likely through platelet activation. Further study on the affected pathway/s in the platelet activation process would confirm the exact mechanism of thalidomide-induced thrombosis and potential extended usage of this drug in future.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. AimTo determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. MethodsThe relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. ResultsPlatelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. [Display omitted] ConclusionsIn this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures:Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

Effects Of Eltrombopag On Thrombocytopenia, Platelet Function and Bleeding In Patients With Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia

IntroductionPatients with Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) have microthrombocytopenia, and hemorrhage is a major problem. Current management options in WAS/XLT patients include splenectomy, human stem cell transplant (HSCT) and gene therapy. In this study, we asked whether eltrombopag, a thrombopoietin mimetic, would increase platelet counts, improve platelet function, and/or reduce bleeding in WAS/XLT patients. MethodsIn 9 WAS/XLT patients and 8 age-matched healthy control subjects, flow cytometry was used to assess platelet function by surface expression of activated GPIIb-IIIa (reported by PAC1) and P-selectin in whole blood after stimulation with low and high concentrations of ADP or thrombin receptor activating peptide (TRAP), and by annexin V binding (a measure of surface phosphatidylserine) in platelet-rich plasma after stimulation with convulxin. Eltrombopag was administered to 5 WAS and 3 XLT patients (50 mg in 2 adults, and 1 mg/kg in 6 children up to 75 mg/day) with a goal platelet count ≥50k. ResultsHigh concentration ADP- or TRAP-induced PAC1 mean fluorescence intensity (MFI) was significantly reduced in WAS/XLT patients compared to healthy controls (Figure). Platelet surface P-selectin MFI in response to TRAP was also significantly reduced. In contrast, annexin V binding to platelets was not different between WAS/XLT and controls. As expected, platelet size of WAS/XLT patients was smaller than controls. WAS protein (which is deficient in WAS/XLT), is important for cytoskeletal movement and could therefore be involved in trafficking of surface proteins. However, surface expression of activated GPIIb-IIIa and P-selectin were no longer different in WAS/XLT patients vs. controls when corrected for size by platelet surface CD41 MFI. In 3 WAS/XLT patients whose platelet count improved on eltrombopag, platelet function did not improve. The table summarizes the results of eltrombopag treatment in 5 responders (2 WAS, 3 XLT patients) and 3 non-responders (3 WAS patients). Comparison of baseline, peak and change in immature platelet fraction in 5 WAS/XLT responders to eltrombopag vs. 7 pediatric chronic immune thrombocytopenia (ITP) patients responding to eltrombopag showed a significant decrease in all three measures, suggesting that platelet production in WAS/XLT patients is more difficult to increase than in ITP patients. Long term eltrombopag use in WAS/XLT patients showed no tachyphylaxis, transaminitis or induction of malignancy. [Display omitted] Conclusions1) Baseline platelet function in WAS/XLT is reduced compared to healthy age-matched controls, as measured by agonist-induced platelet surface activated GPIIb-IIIa and P-selectin. 2) This reduction is proportional to the reduced platelet size in WAS/XLT compared to controls. 3) In contrast, annexin V binding (a measure of platelet procoagulant activity) showed no differences between WAS/XLT and controls. 4) Eltrombopag has beneficial effects on the thrombocytopenia and bleeding, but not platelet function, in the majority of WAS/XLT patients. 5) This eltrombopag-induced reduction in bleeding is presumably primarily the result of the increased platelet count, but it was also observed in 2 eltrombopag “non-responders” (i.e. patients whose platelet counts did not increase after eltrombopag). 6) The production of new platelets with eltrombopag is less in WAS/XLT than in ITP. Disclosures:Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Michelson:Sysmex: Honoraria. Bussel:GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity's Board of Directors or advisory committees.

TCT-142 Prasugrel 5 mg inhibits platelet GPIIb-IIIa and P-Selectin expression in the very elderly - Results from the GENERATIONS trial, a pharmacodynamic study in stable CAD patients

Platelet surface P-selectin and activated GPIIb-IIIa are markers of platelet activation, degranulation and aggregation. In the TRITON trial prasugrel (Pras) 10 mg reduced ischemic events vs. clopidogrel (Clop) 75 mg but increased bleeding, notably in very elderly (VE) patients. Pras 5 mg is a

In Vitro Shear Stress‐Induced Platelet Activation: Sensitivity of Human and Bovine Blood

As platelet activation plays a critical role in physiological hemostasis and pathological thrombosis, it is important in the overall hemocompatibility evaluation of new medical devices and biomaterials to assess their effects on platelet function. However, there are currently no widely accepted in vitro test methods to perform this assessment. In an effort to develop effective platelet tests for potential use in medical device evaluation, this study compared the sensitivity of platelet responses to shear stress stimulation of human and bovine blood using multiple platelet activation markers. Fresh whole blood samples anticoagulated with heparin or anticoagulant citrate dextrose, solution A (ACDA) were exposed to shear stresses up to 40 Pa for 2 min using a cone-and-plate rheometer model. Platelet activation was characterized by platelet counts, platelet surface P-selectin expression, and serotonin release into blood plasma. The results indicated that exposure to shear stresses above 20 Pa caused significant changes in all three of the platelet markers for human blood and that the changes were usually greater with ACDA anticoagulation than with heparin. In contrast, for bovine blood, the markers did not change with shear stress stimulation except for plasma serotonin in heparin anticoagulated blood. The differences observed between human and bovine platelet responses suggest that the value of using bovine blood for in vitro platelet testing to evaluate devices may be limited.

Stroke with intracranial stenosis is associated with increased platelet activation in sickle cell anemia

Overt stroke in sickle cell anemia (SCA) is associated with intracranial stenosis and thrombus formation. Platelet activation is critical for thrombus formation. Platelet activation studies were performed in 50 subjects: 18 SCA patients with history of stroke or abnormal transcranial Doppler (TCD) and intracranial stenosis seen by magnetic resonance angiogram (MRA), 7 SCA patients with history of stroke or abnormal TCD but no intracranial stenosis, 13 SCA patients with no history of stroke or abnormal TCD, and 12 healthy African-Americans. Of the 18 patients with intracranial stenosis, 11 (61%) had evidence of the moyo-moya phenomenon on MRA. SCA children with intracranial stenosis had a significantly greater total white cell count compared to both healthy African-American controls and SCA patients in the steady-state (P < 0.001). In addition, SCA patients with history of stroke or abnormal TCD had a significantly higher platelet count compared to healthy African-American controls (P < 0.002). The percentage of platelet surface P-selectin expression was significantly greater in patients with intracranial stenosis compared to the other groups (P < 0.05), particularly in individuals that did not have the moya-moya phenomenon seen on MRA. Stroke with intracranial stenosis is associated with increased platelet activation in sickle cell anemia, and further investigation is needed on the role of anti-platelet agents in this high-risk population.

Biocompatibility Assessment of a Long-Term Wearable Artificial Pump-Lung in Sheep

The purpose of this study was to assess the biocompatibility of a newly developed long-term wearable artificial pump-lung (APL) in a clinically relevant ovine animal model. The wearable APL device was implanted in five sheep through left thoracotomy. The device was connected between the right atrium and pulmonary artery and evaluated for 30 days. Three sheep were used as the sham control. Platelet activation was assessed by measuring platelet surface P-selectin (CD62P) expression with flow cytometry and plasma soluble P-selectin with an enzyme-linked immunosorbent assay. Thrombotic deposition on the device components and hollow fiber membranes were analyzed with digital imaging and scanning electron microscopy. Surface P-selectin of the APL and sham groups changed significantly over the study period, but without significant differences between the two groups. Soluble P-selectin for the two groups peaked in the first 24 h after the surgery. Soluble P-selectin of the APL group remained slightly elevated over the study period compared to the presurgical baseline value and was slightly higher compared to that of the sham group. Plasma free hemoglobin remained in the normal ranges in all the animals. In spite of the surgery-related alteration in laboratory tests and elevation of platelet activation status, the APL devices in all the animals functioned normally (oxygen transfer and blood pumping) during the 30-day study period. The device flow path and membrane surface were free of gross thrombus. Electron microscopy images showed only scattered thrombi on the fibers (membrane surface and weft). In summary, the APL exhibited excellent biocompatibility. Two forms of platelet activation, surgery-related and device-induced, in the animals implanted with the wearable APL were observed. The limited device-induced platelet activation did not cause gross thrombosis and impair the long-term device performance.

A double-blind, randomized, multicenter phase 2 study of prasugrel versus placebo in adult patients with sickle cell disease

BackgroundPlatelet activation has been implicated in the pathogenesis of sickle cell disease (SCD) suggesting antiplatelet agents may be therapeutic. To evaluate the safety of prasugrel, a thienopyridine antiplatelet agent, in adult patients with SCD, we conducted a double-blind, randomized, placebo-controlled study.MethodsThe primary endpoint, safety, was measured by hemorrhagic events requiring medical intervention. Patients were randomized to prasugrel 5 mg daily (n = 41) or placebo (n = 21) for 30 days. Platelet function by VerifyNow® P2Y12 and vasodilator-stimulated phosphoprotein assays at days 10 and 30 were significantly inhibited in prasugrel- compared with placebo-treated SCD patients.ResultsThere were no hemorrhagic events requiring medical intervention in either study arm. Mean pain rate (percentage of days with pain) and intensity in the prasugrel arm were decreased compared with placebo. However, these decreases did not reach statistical significance. Platelet surface P-selectin and plasma soluble P-selectin, biomarkers of in vivo platelet activation, were significantly reduced in SCD patients receiving prasugrel compared with placebo. In sum, prasugrel was well tolerated and not associated with serious hemorrhagic events.ConclusionsDespite the small size and short duration of this study, there was a decrease in platelet activation biomarkers and a trend toward decreased pain.

Inhibition of platelet-mediated arterial thrombosis and platelet granule exocytosis by 3′,4′-dihydroxyflavonol and quercetin

Flavonols are polyphenolic compounds with broad-spectrum kinase inhibitory, as well as potent anti-oxidant and anti-inflammatory properties. Anti-platelet potential of quercetin (Que) and several related flavonoids have been reported; however, few studies have assessed the ability of flavonols to inhibit exocytosis of different platelet granules or to inhibit thrombus formation in vivo. 3′,4′-Dihydroxyflavonol (DiOHF) is a flavonol which is structurally related to Que and has been shown to have greater anti-oxidant capacity and to improve the endothelial function in the context of diabetes and ischaemia/reperfusion injury. While the structural similarity to Que suggests DiOHF may have a potential to inhibit platelet function, no studies have assessed the anti-platelet potential of DiOHF. We therefore investigated platelet granule inhibition and potential to delay arterial thrombosis by Que and DiOHF. Both Que and DiOHF showed inhibition of collagen, adenosine diphosphate and arachidonic acid stimulated platelet aggregation, agonist-induced GPIIb/IIIa activation as demonstrated by PAC-1 and fibrinogen binding. While both flavonols inhibited agonist-induced granule exocytosis, greater inhibition of dense granule exocytosis occurred with DiOHF as measured by both ATP release and flow cytometry. In contrast, while Que inhibited agonist-induced P-selectin expression, as measured by both platelet surface P-selectin expression and upregulation of surface GPIIIa expression, inhibition by DiOHF was not significant for either parameter. C57BL/6 mice treated with 6 mg kg−1 IV Que or DiOHF maintained greater blood flow following FeCl3-induced carotid artery injury when compared to the vehicle control. We provide evidence that Que and DiOHF improve blood flow following arterial injury in part by attenuating platelet granule exocytosis.

A Biocompatible Flow Chamber to Study the Hemodynamic Performance of Prosthetic Heart Valves

The goal of this study was to design, fabricate, and characterize a biocompatible flow chamber to study the performance of artificial heart valves. The system consists of a testing fluid chamber and a water chamber (separated by a latex diaphragm), following the design of a left ventricular assist device. Two St. Jude bileaflet mechanical heart valves were placed oppositely in the testing fluid chamber to control flow direction. The flow rate of the testing fluid chamber was set at 5.6 L/min, with a stroke volume of 80 ml. The performance of the system was examined through three-dimensional numerical simulation and in vitro experiments with whole blood and washed platelets. Hemolysis was measured with whole blood using a spectrophotometer. Platelet activation was measured by platelet surface P-selectin expression using flow cytometry. The three-dimensional computational fluid dynamics model demonstrated that the flow field in the chamber was laminar and physiological. Results from in vitro experiments indicated that the flow conditions in the chamber did not induce hemolysis or platelet activation with the presence of St. Jude heart valves. Overall, the flow chamber can provide a feasible environment to study the hemodynamic performance of artificial heart valves.

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.

In vitro effect of anti-β2 Glycoprotein I antibodies on P-selectin expression, a marker of platelet activation

Antiphospholipid antibodies (aPL) associated with thrombembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (β2GPI) represents the major target antigen for aPL, but the pathogenic role of anti-β2GPI antibodies (aβ2GPI) is still unclear. Some authors assume they play a role in activating platelets. The effects of aβ2GPI antibodies on platelet P-selectin expression were evaluated in this study. Aβ2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography during two different stages (catastrophic and quiescent) of the disease. Gel filtered platelets (100,000/μl) from healthy volunteers were incubated with β2-GPI (20 μg/ml) and with different concentrations (5, 25 e 50 μg/ml) of aβ2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. Aβ2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6), a platelet agonist, at a subthreshold concentration. Aβ2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47% versus 15%). TRAP-6 dependent platelet activation by aβ2GPI antibodies is consistent with the “two hit” pathogenetic hypothesis for thrombosis. Aβ2GPI antibodies induce higher platelet P-selectin expression during the active rather than in the acute phases.

Platelet Activation, P-Selectin, and Eosinophil β1-Integrin Activation in Asthma

Eosinophil β1-integrin activation correlates inversely with FEV1 and directly with eosinophil-bound P-selectin in subjects with nonsevere allergic asthma. Determine the relationships between β1-integrin activation and pulmonary function or eosinophil-bound P-selectin in subjects with asthma of varying severity and discern the source of eosinophil-bound P-selectin. Blood was assayed by flow cytometry for P-selectin and activated β1-integrin on eosinophils and platelets. Plasma was analyzed with ELISA for soluble P-selectin, platelet factor 4, and thrombospondin-1. Activated β1-integrin correlated with eosinophil-bound P-selectin among all subjects with asthma even though activated β1-integrin was higher in subjects with nonsevere asthma than severe asthma. Activated β1-integrin correlated inversely with FEV1 corrected for FVC only in younger subjects with nonsevere asthma. Paradoxically, platelet surface P-selectin, a platelet activation marker, was low in subjects with severe asthma, whereas plasma platelet factor 4, a second platelet activation marker, was high. Correlations indicated that P-selectin-positive platelets complexed to eosinophils are the major source of the eosinophil-bound P-selectin associated with β1-integrin activation. After whole-lung antigen challenge of subjects with nonsevere asthma, a model of asthma exacerbation known to cause platelet activation, circulating eosinophils bearing P-selectin and activated β1-integrin disappeared. The relationship between eosinophil β1-integrin activation and pulmonary function was replicated only for younger subjects with nonsevere asthma. However, we infer that platelet activation and binding of activated platelets to eosinophils followed by P-selectin-mediated eosinophil β1-integrin activation occur in both nonsevere and severe asthma with rapid movement of platelet-eosinophil complexes into the lung in more severe disease.

In Vivo Platelet Activation and Its In Vitro Inhibition by Prasugrel's Active Metabolite In Adolescents with Sickle Cell Disease

Abstract 2141 Introduction:In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods:Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results:In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p<0.0001 for both), and in vitro ADP-stimulated platelet surface activated GPIIb-IIIa and P-selectin levels (mean fluorescence, MFI) were significantly lower (128.7 ± 66.2 and 78.1 ± 11.5 vs. 257.3 ± 50.8 and 91.6 ± 5.8, p<0.05 for both). ADP-stimulated platelet aggregation by LTA, VerifyNow and MEA, did not significantly differ between SCD and normal subjects, although whole blood platelet aggregation by MEA and VerifyNow tended to be greater in blood from SCD patients (92.5 vs. 70.4 AU, p=0.064 and 362.9 vs. 314.8 PRU, p=0.488, respectively).Treatment of whole blood in vitro with R-138727 resulted in a concentration-dependent inhibition of platelet function in both SCD patients and normal subjects. However, compared with normal subjects, the IC50 in SCD subjects was significantly lower for LTA but significantly higher for VerifyNow and VASP (Table). R-138727 inhibition of platelet function in SCD patients was similar to normal subjects as judged by MEA, whole blood flow cytometry for ADP-stimulated platelet surface P-selectin and activated GPIIb-IIIa expression, and % PMAs (Table). Sensitivity to R-138727 inhibition in SCD patient blood was greatest as measured by ADP-stimulated platelet surface P-selectin MFI, LTA, and MEA, less with ADP-stimulated platelet surface activated GPIIb-IIIa, and least with VASP, VerifyNow P2Y12 and % P-selectin-positive platelets (Table). Conclusions:1) The markedly higher circulating PMA and PNA levels in SCD patients relative to normal donors demonstrates increased in vivo platelet activation in SCD patients and suggests that PMA and PNA may be useful markers of the in vivo pharmacodynamic effects of antiplatelet therapy in SCD patients. 2) Blockade of platelet P2Y12 with R-138727 produces dose-dependent inhibition of platelet function in SCD platelets. 3) Assay-dependent differences in IC50 values between SCD patients and normal donors suggest the presence of additional variables that affect these measures of platelet function. Further studies are needed to determine the relationship between platelet inhibition measured by these assays and clinical events in SCD patients.TableComparison of R-138727 inhibition of platelet function in blood from SCD adolescents vs. healthy subjects.AssayNormal Donor, n=5 IC50 (95% CI)SCD Patient, n=10 IC50 (95% CI)PLTA, ADP 20 μM2.36 (1.90–2.93)1.59 (1.23–2.05)0.0267VASP P2Y122.14 (1.52–3.02)4.56 (3.90–5.32)0.0013VerifyNow P2Y12, % Inhibition2.09 (1.70–2.56)3.76 (3.28–4.32)<0.0001MEA, 6.5 μM ADP2.43 (1.69–3.49)1.90 (1.50–2.40)0.2479P-selectin, MFI, ADP 20 μM1.08 (0.95–1.24)1.40 (1.15–1.70)0.0503P-selectin, % positive, ADP 20 μM4.25 (3.42–5.27)3.62 (3.15–4.16)0.1891PAC1 MFI, ADP 20 μM2.50 (2.05–3.06)2.45 (2.19–2.73)0.8146% PMAs, ADP 20 μMa2.48a (2.06–3.00)3.30a (1.01–10.8)0.7389Results shown are μM R-138727.aIC50 calculated after subtracting unstimulated values. Disclosures:Frelinger:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Consultancy, Research Funding; Takeda: Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Lilly: Consultancy. Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Monomeric C-reactive protein is prothrombotic and dissociates from circulating pentameric C-reactive protein on adhered activated platelets under flow

We previously reported that C-reactive protein bioactivity on thrombogenesis was based on loss of its pentameric symmetry, resulting in formation of monomeric C-reactive protein. Our purpose was to provide mechanistic information on the direct effects of C-reactive protein isoforms on platelet activation and provide a C-reactive protein dissociation mechanism in circulating blood. C-reactive protein-induced platelet activation was evaluated by flow cytometry. Platelet aggregation, clot properties, and coagulation were also measured. Washed platelets were incubated with C-reactive protein isoforms and vasodilator-stimulated phosphoprotein (VASP) phosphorylation was analysed by western blot and immunofluorescence. C-reactive protein dissociation under flow was evaluated by confocal microscopy on the surface of adhered platelets after perfusing human blood containing pentameric C-reactive protein at different shear rates. Dissociated monomeric C-reactive protein thrombogenicity was measured in flow experiments. Platelet aggregation and flow cytometry analysis revealed that monomeric C-reactive protein significantly induced platelet aggregation, surface P-selectin and CD63 exposure, and glycoprotein IIb-IIIa activation, whereas pentameric C-reactive protein was unable to produce any effect. p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) inhibitors, as well as CD36 blocking antibody partially inhibited monomeric C-reactive protein-induced platelet activation and aggregation. Additionally, monomeric C-reactive protein significantly induced VASP dephosphorylation at serine 239. We found that pentameric C-reactive protein dissociated into monomeric C-reactive protein on the surface of activated adhered platelets under flow conditions and that this generated monomeric C-reactive protein promoted further platelet recruitment. These data indicate that whereas serum pentameric C-reactive protein may not affect platelet activation, monomeric C-reactive protein, which dissociates from pentameric C-reactive protein on the surface of activated platelets, could contribute to atherothrombotic complications by promoting thrombosis.

Thromboembolism and Antithrombotic Therapy in Patients With Heart Failure in Sinus Rhythm

Heart failure (HF) represents a major and growing public health problem because of its prevalence, incidence, morbidity, mortality, and economic costs. The prevalence of HF is 2% to 3% of the general population.1 Five million Americans are affected, with >550 000 cases diagnosed each year.2 The mortality rate from severe HF remains >60% within 5 years of diagnosis, and 50% of hospitalized patients with HF require readmission within 6 months of discharge. In the US estimated costs amount to > $35 billion per year.3 Although several therapies (eg, β-blockers, angiotensin-converting enzyme [ACE] inhibitors, and cardiac resynchronization therapy) have been proven effective in improving HF outcomes, many unanswered questions about optimal treatment remain. One area of ongoing uncertainty is the appropriate role for antithrombotic therapy in patients with HF. Observational data suggest that patients with HF have an increased venous thromboembolism (VTE) risk (deep venous thromboembolism [DVT], pulmonary embolism [PE], peripheral arterial thromboembolism, and stroke).4 These epidemiological findings are supported by multiple mechanisms that can contribute to a hypercoagulable state in patients with HF. Despite this increased risk of VTE, the role of antithrombotic therapy remains unclear. In this article, we provide an overview of epidemiology, pathophysiology, clinical trial data, and therapeutic recommendations for prevention of thromboembolism in HF. We searched PubMed for articles published between 1958 and 2010 using the following search terms: epidemiology of heart failure , thromboembolism and heart failure , thrombogenesis and heart failure , anticoagulation in heart failure , antiplatelet agent and heart failure , aspirin and heart failure , bleeding risk and anticoagulation , and aspirin and angiotensin-converting enzyme inhibitors . We also studied abstracts from national and international cardiovascular meetings to identify unpublished studies using the key words anticoagulation and dilated cardiomyopathy . Data from published observational studies and secondary …

The Influence of Intermittent Hypoxemia on Platelet Activation in Obese Patients with Obstructive Sleep Apnea

Literature regarding platelet function in obstructive sleep apnea (OSA) has considerable limitations. Given the central role of platelets in atherothrombosis and the known cardiovascular risk of OSA, we hypothesized that OSA severity is predictive of platelet function, independent of known comorbidities. Obese subjects, without comorbidities, underwent overnight, in-lab polysomnography. The following morning, 5 biomarkers of platelet activation were measured by whole-blood flow cytometry at baseline and in response to agonists (no stimulation, stimulation with 5 μM ADP agonist, and stimulation with 20 μM ADP agonist): platelet surface P-selectin, activated glycoprotein (GP) IIb/IIIa, and GPIb receptor expression, platelet-monocyte aggregation (PMA) and platelet-neutrophil aggregation (PNA). Of the 77 subjects, 47 were diagnosed with OSA (median apnea-hypopnea index [AHI] of 24.7 ± 28.1/h in subjects with OSA and 3.0 ± 3.9/h in subjects without OSA, p < 0.001). The groups were matched for body mass index, with a mean body mass index of 40.3 ± 9.6 kg/m(2) in subjects with OSA and 38.9 ± 6.0 kg/m(2) in subjects without OSA (p = 0.48). A comparison of time spent with an oxygen saturation of less than 90% showed that subjects who had 1 minute or more of desaturation time per hour of sleep had lower GPIb fluorescence in circulating platelets, as compared with those subjects who had less than 1 minute of desaturation time per hour of sleep; similar findings were observed following 5 μM and 20 μM of ADP stimulation, as compared with control vehicle, suggesting higher levels of circulating platelet activity. In multivariate analyses, only nocturnal hypoxemia and female sex predicted agonist response. Platelet surface P-selectin, platelet surface-activated GPIIb/IIIa, PMA, and PNA were not significantly correlated with markers of OSA. In obese patients with OSA, platelet activation is associated with greater levels of oxygen desaturation, compared with matched control subjects. Metrics other than AHI (e.g., hypoxemia) may determine OSA-related thrombotic risk.