Induced P-selectin Expression Research Articles

Neutrophil Extracellular Traps Induced by Shiga Toxin and Lipopolysaccharide-Treated Platelets Exacerbate Endothelial Cell Damage

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN–Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN–Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN–Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.

Contribution of septins to human platelet structure and function

SummaryAlthough septins have been well-studied in nucleated cells, their role in anucleate blood platelets remains obscure. Here, we elucidate the contribution of septins to human platelet structure and functionality. We show that Septin-2 and Septin-9 are predominantly distributed at the periphery of resting platelets and co-localize strongly with microtubules. Activation of platelets by thrombin causes clustering of septins and impairs their association with microtubules. Inhibition of septin dynamics with forchlorfenuron (FCF) reduces thrombin-induced densification of septins and lessens their colocalization with microtubules in resting and activated platelets. Exposure to FCF alters platelet shape, suggesting that septins stabilize platelet cytoskeleton. FCF suppresses platelet integrin αIIbβ3 activation, promotes phosphatidylserine exposure on activated platelets, and induces P-selectin expression on resting platelets, suggesting septin involvement in these processes. Inhibition of septin dynamics substantially reduces platelet contractility and abrogates their spreading on fibrinogen-coated surfaces. Overall, septins strongly contribute to platelet structure, activation and biomechanics.

Ginsenoside Rb3 attenuates skin flap ischemia-reperfusion damage by inhibiting STING-IRF3 signaling.

We investigate the protective effect of ginsenoside Rb3 on skin flap microvasculature following ischemia-reperfusion (I/R) injury and its regulatory mechanism. We used a rat model of I/R injury with the right iliolumbar artery and oxidative stress model of human dermal microvascular endothelial cells. The effects of Rb3 on skin flap tissue and endothelial cell survival, STING-IRF3 pathway activation, and endothelial cell adhesion were measured. Following reperfusion, the survival rate of rat perforator flaps in the Rb3-treated group gradually increased with increasing Rb3 concentration. The treatment also reduced the amount of STING protein, phosphorylated IRF3, and P-selectin in skin flap tissue, with this change being most obvious in microvascular endothelial cells. In vitro, activated IRF3 binds to the P-selectin promoter and induces P-selectin expression. Our results suggest that Rb3 plays a role in reducing I/R flap damage through negatively regulating STING-IRF3 activation to limit leukocyte-endothelial cell adhesion.

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

DDEL-15. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

OBJECTIVEThe Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents against the SHH pathway have demonstrated efficacy, on-target bone toxicities suggest new therapeutic approaches are needed.METHODSWe investigated the SHH pathway inhibitor, vismodegib, packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin expressed on endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle shows selectivity toward tumor and not normal brain vasculature in a GEM SHH medulloblastoma model as assessed by ex vivo infrared imaging and molecular studies.RESULTSQuantitative RT-PCR analysis of SHH medulloblastoma following single dose XRT and Fi-Vis treatment (10mg/kg) showed synergistic reduction of Gli1 expression (>90% target inhibition). We demonstrate that low-dose XRT (0.25Gy) can induce P-selectin expression specifically on medulloblastoma tumor endothelium and synergize with low-dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p<0.01) compared to radiation or Fi-Vis alone. Assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free vismodegib. Finally, in vitro studies using bEnd.3 brain endothelial cells and in vivo studies using Cav1 knockout mice suggest a caveolin-1 mediated transcytosis mechanism for nanoparticle entry across the blood-brain barrier.CONCLUSIONSThese data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric brain tumors.

New Monoclonal Antibodies Reveal Distinct Specificities for Platelet Factor 4 Bound to Heparin Vs. Platelets: Implications for HIT Pathogenesis

Heparin induced thrombocytopenia (HIT) is a severe adverse reaction to heparin treatment characterized by antibodies to PF4/polyanion complexes. However, HIT antibodies are heterogeneous. Some bind to heparin/polyanion complexes in solid-phase enzyme-linked immunoassay (EIA) but do not activate platelets; other EIA-positive antibodies also activate platelets in assays such as the serotonin release assay (SRA) and in the recently described PF4-dependent p-selectin expression assay (PEA). It is generally thought that only platelet-activating antibodies are pathogenic. On the basis of studies which showed that platelet-activating antibodies, but not those positive only in EIA recognize PF4 bound to platelets (Blood. 2015;125(1):155-61 and others), we hypothesized that pathogenic HIT antibodies may recognize epitopes exposed on PF4 when it binds to a target, possibly chondroitin sulfate, on the platelet surface that are not created when PF4 forms a complex with heparin or other polyanions. Here, we describe studies utilizing newly developed monoclonal antibodies (mAbs) that demonstrate the feasibility of this concept and suggest that HIT antibodies having this specificity could be important in HIT pathogenesis. mAbs were produced in mice immunized with PF4/heparin complexes. We employed a novel dual screening strategy for candidate clones utilizing the PF4/polyanion EIA as well as the PEA. mAbs selected for study were designated 431.3, 431.4 and 447.1 and were compared with KKO, a widely studied HIT-like mAb (generous gift from G Arepally) and an isotype control (IC; Fig 1A). In the EIA, KKO strongly recognized PF4/polyvinylsulfonate (PF4/PVS) but not PF4 alone and was inhibited by high-dose heparin (100U/mL). mAb 447.1 had an EIA profile similar to KKO but with significantly lower optical densities (0.56 vs. 2.43 OD) and 431.3 gave reactions indicating it recognizes PF4 alone but demonstrated much stronger reactivity to PF4/PVS complexes (1.96 OD). However, mAb 431.4, similar to isotype control, was completely non-binding in the EIA (Fig 1A). In contrast, all 4 mAbs strongly recognized PF4-treated platelets (Fig 1B). In addition, all four mAbs produced strong positive reactions in the PEA in which mAbs interact with PF4-primed platelets to induce p-selectin expression (Fig 1C). The EIA requires multiple washing steps, whereas studies performed to assess IgG binding to PF4-treated platelets utilized only a single wash and the PEA is a "no-wash" assay. Thus, failure of 431.4 to react in EIA could be explained by susceptibility of the mAb to being removed by washing, possibly due to low avidity for its target. We therefore studied mAb binding to PF4/heparin complexes in real time using Bio-layer interferometry (Octet system, Pall ForteBio). In this system, mAbs 431.4 and KKO produced a steadily increasing signal, reaching saturation, whereas 431.4 demonstrated no binding to PF4/heparin consistent with results seen in EIA (Fig 1D). These findings indicate that mAb 431.4 binds to PF4 bound to the platelet surface and induces platelet activation as measured by p-selectin expression but fails to react with PF4 in a complex with heparin or PVS. This behavior contrasts strikingly with that of the "HIT-like" mAb KKO that reacts in both assays. Although antibodies from patients with HIT frequently give positive reactions in both platelet-activating assays and in EIA, there are clear examples of antibodies that are positive in one assay and not the other (e.g. EIA-positive, SRA-negative antibodies are common in clinical practice). Thus in HIT patients, multiple antibodies with different specificities are undoubtedly present. Our data support the growing body of evidence that while strength of the reaction in EIA (optical density) is often correlated with platelet-activating antibodies, there are notable exceptions (such as mAb 447.1). These results suggest that studies to isolate and characterize human antibodies according to specificity and reaction strength are needed to define the extent to which antibodies like mAb 431.4 and 447.1 contribute to the pathogenesis of HIT. Disclosures Padmanabhan: Versiti Wisconsin: Patents &amp; Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen R&amp;D: Consultancy. Jones:Retham Technologies: Equity Ownership; Versiti Wisconsin: Patents &amp; Royalties.

SCIDOT-41. NANOTHERAPEUTIC TARGETING OF TUMOR ENDOTHELIUM FOR ENHANCING DRUG DELIVERY PAST THE BLOOD-BRAIN BARRIER

Abstract OBJECTIVE The Sonic Hedgehog (SHH) medulloblastoma subgroup accounts for ~25% of all cases and has an intermediate prognosis. Current conventional therapies result in devastating morbidities including intellectual disability and secondary malignancies. Although molecularly targeted agents that inhibit the SHH pathway have demonstrated clinical efficacy, recent studies have shown on-target secondary toxicities on bone development suggesting new therapeutic approaches are needed. METHODS We investigated the efficacy of the SHH pathway inhibitor, Vismodegib packaged in a fucoidan-based nanoparticle (Fi-Vis) that targets P-selectin, a protein overexpressed on vascular endothelial cells and induced by low-dose ionizing radiation (XRT) in a time- and dose-dependent manner. This P-selectin targeting nanoparticle drug delivery system shows selectivity toward tumor vasculature and not normal brain vasculature in a genetic SHH medulloblastoma mouse model as assessed by ex vivo infrared imaging and two-photon intravital imaging. RESULTS Quantitative RT-PCR analysis of SHH medulloblastoma tissue following single dose XRT and Fi-Vis treatment (10mg/kg) showed a synergistic inhibition of Gli1 expression (&gt;90% target inhibition). Furthermore, we demonstrate that very low dose XRT (0.25Gy) can induce P-selectin expression specifically within MB tumor vasculature and synergize with low dose Fi-Vis (10mg/kg) to significantly enhance mouse survival (p&lt;0.01) when compared to radiation or Fi-Vis alone. Furthermore, assessment of bone toxicity using micro-CT and histological analysis following Fi-Vis administration in postnatal (P10) mice shows no bone toxicity when compared to free Vismodegib. Finally, in vitro studies using mouse brain endothelial cells suggest at least in part a caveolin-1 mediated transcytosis mechanism of crossing the endothelial blood-brain barrier. CONCLUSIONS These data suggest applicability of combined XRT and tumor vasculature-targeted nanotherapeutic dose de-escalation strategies for SHH medulloblastoma with implications for other pediatric and adult brain tumors.

Pharmacokinetics and pharmacodynamics of single dose of inhaled nebulized sodium nitrite in healthy and hemoglobin E/β-thalassemia subjects

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/β-thalassemia (HbE/β-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/β-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FENO), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/β-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (Vd) of 1.3 l/kg and half-life (t1/2) of 0.6 h. CL and Vd of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/β-thal patients had lower nitrite CL and longer t1/2 in RBC than healthy subjects. FENO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in β-thalassemia.

Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage.

Dengue virus (DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1 (NS1), which can be secreted into patients’ blood, can stimulate immune cells via Toll-like receptor 4 (TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine (PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F(ab’)2, anti-TLR4 antibody, a TLR4 antagonist (Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor (TAK242), but not polymyxin B (an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate (ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.

Severe obesity and bariatric surgery alter the platelet mRNA profile

Mechanisms explaining the relationship between obesity and cardiovascular disease (CVD) are needed. Despite growing recognition of the importance of the anucleate platelet transcriptome, low levels of RNA in platelets make assessment difficult. We sought to perform unbiased platelet RNA profiling in obesity by performing a prospective study of severe obesity and weight loss via bariatric surgery on platelet characteristics and mRNA profile in 26 pre-menopausal, non-diabetic women (31.6 ± 8.4 years; BMI 43.0 ± 6.5 kg/m2) who underwent sleeve gastrectomy. Totally, 10 women of similar age with normal BMI served as controls. Platelet activation via flow cytometry was assessed before and after surgery. RNA-sequencing (RNAseq) was performed on platelet isolates from a subset of 13 subjects (eight obese women and five normal-BMI subjects). Platelet count, size, and age did not differ between control and obese women. However, platelet surface P-selectin and CD40 were higher in obesity. RNAseq demonstrated 629 differentially abundant transcripts in obesity. Notably, S100A9 and AGER, established markers of cardiovascular risk, were two of the most highly upregulated transcripts (each > 2.5 fold). At 6 months post-operatively, subjects lost 26.1 ± 5.8% body weight and inducible platelet P-selectin expression was reduced. Expression of 170 transcripts was affected by surgery, but only a small fraction (46/629) were genes found altered in obesity. We demonstrate that obesity is associated with an altered platelet transcriptome and increased platelet activation, which is partly attenuated by bariatric surgery. These observations suggest that platelets may contribute to increased cardiovascular risk in obesity through a variety of mechanisms.

Dyslipidemia-associated atherogenic oxidized lipids induce platelet hyperactivity through phospholipase Cγ2-dependent reactive oxygen species generation

Oxidized low-density lipoprotein (oxLDL) and associated oxidized phosphocholine-headgroup phospholipids (oxPCs) activate blood platelets through ligation of the scavenger receptor CD36. Previously, we found that oxLDL stimulated phosphorylation of phospholipase Cγ2 (PLCγ2). However, the functional relevance of PLCγ2 phosphorylation in oxLDL-mediated platelet hyperactivity remained elusive. Here, we set out to explore the functional importance of PLCγ2 in oxLDL-mediated platelet activation using human and genetically modified murine platelets. The CD36-specific oxidized phospholipid (oxPCCD36) triggered the generation of reactive oxygen species (ROS) in platelets under static and arterial flow conditions. The ROS generation in response to oxPCCD36 was sustained for up to 3 h but ablated in CD36- and PLCγ2-deficient platelets. The functional importance of ROS generation in response to atherogenic lipid stress was examined through measurement of P-selectin expression. OxPCCD36 induced P-selectin expression, but required up to 60 min incubation, consistent with the timeline for ROS generation. P-selectin expression was not observed in CD36- and PLCγ2-deficient mice. The ability of oxPCCD36 and oxLDL to stimulate P-selectin expression was prevented by incubation of platelets with the ROS scavenger N-acetyl-cysteine (NAC) and the NOX-2 inhibitor gp91ds-tat, but not with the NOX-1 inhibitor ML171. In summary, we provide evidence that prolonged exposure to oxLDL-associated oxidized phospholipids induces platelet activation via NOX-2-mediated ROS production in a CD36- and PLCγ2-dependent manner.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes

Background Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin, a commonly used therapeutic agent for diabetes mellitus. Immunization against PRT/heparin complexes is common in diabetic patients. Objectives To investigate the impact of NPH-insulin on the interaction between anti-PRT/heparin antibodies and platelets. Methods The interaction between NPH-insulin and anti-PRT/heparin antibodies was tested using in-house enzyme immunoassays. The ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH-insulin (and heparin) was investigated using flow cytometry. Results Twenty-one out of 80 sera containing anti-PRT/heparin IgG showed binding to NPH-insulin. Anti-PRT/heparin IgG from immunized patients bound to platelets in the presence of NPH-insulin, but not in the presence of native insulin. Anti-PRT/heparin antibodies induced P-selectin expression in the presence of NPH-insulin in a heparin-dependent way (median mean fluorescence intensity in the presence of NPH-insulin: 55, 95% confidence interval [CI] 18.7-100.5 vs. NPH-insulin and heparin: 204, 95% CI 106.5-372.8). The clinical relevance of platelet-activating anti-PRT/heparin antibodies was assessed by investigating a multicenter study cohort of 332 acutely ill medical patients who received heparin. None of the 21 patients with anti-PRT/heparin IgG developed thrombocytopenia or thromboembolic complications. Conclusions Anti-PRT/heparin antibodies activate platelets in the presence of NPH-insulin in a heparin-dependent way. However, results from our preliminary study indicate no major impact of these antibodies on the clinical outcome in medical patients receiving heparin, particularly on thromboembolic complications.

Platelet function in adult ITP patients can be either increased or decreased, compared to healthy controls, and is associated with bleeding risk

Objectives: To test whether, together with platelet count, platelet activity could be an important predictor of bleeding risk in immune thrombocytopenia (ITP) patients.Methods: Platelet activity was tested by flow cytometric measurement of agonist induced P-selectin expression and compared between 23 adult ITP patients and 22 healthy volunteers.Results: Platelet activity could be either increased or decreased in ITP patients, compared to healthy volunteers. In the lowest platelet count category, normal to low platelet activity was associated with the biggest increase in bleeding risk. Risk difference 80% (95% confidence interval: 45–115%) for <32 × 109 platelets/L. For higher platelet counts, there was no association of platelet activity with bleeding risk.Discussion: Increased platelet activity was associated with decreased bleeding risk, but only in patients with low platelet counts.Conclusion: Platelet activity can be a predictor of bleeding risk in ITP patients with low platelet counts.

Supplementation with a 9c,11t-rich conjugated linoleic acid blend shows no clear inhibitory effects on platelet function in healthy subjects at low and moderate cardiovascular risk: a randomized controlled trial.

The 9cis,11trans-conjugated linoleic acid (9c,11t-CLA) is reported to have anti-atherogenic properties in animal models and to modulate protein expression in unstimulated human platelets in vivo. Platelet function was therefore investigated after dietary supplementation with 9c,11t-CLA enriched oil (CLA80:20) in a randomized, baseline-controlled cross-over trial. Forty-three healthy adults at low to moderate risk of cardiovascular disease received 4 g/day of CLA80:20 or placebo for two weeks each. Platelet function, inflammation, and endothelial activation were assessed before and after each phase. Compared with placebo, supplementation had no significant effects on platelet function measured by Platelet Function Analyzer-100. Inhibitory effects on collagen-induced aggregation were sex-dependent (p=0.005) that reached significance only in women (p=0.045). Thrombin receptor-activating peptide (TRAP)-induced P-selectin expression was higher after supplementation in all subjects (p=0.017). TRAP-induced platelet fibrinogen binding was also dependent on sex (p = 0.015), with fibrinogen binding after CLA80:20 being higher in males (p=0.035). Plasma monocyte chemoattractant protein-1 was higher (p=0.041) after CLA80:20. No clear evidence was found for inhibition or activation of platelet function as well as inflammation by CLA80:20 in a low to moderate cardiovascular risk group.

Effects of Selenium in the MAPK Signaling Cascade.

Introduction: This study aimed to discover by which mechanism selenium (Se) suppresses stimulated platelets stimulation in oxidative stress underlying diseases. Methods: Human platelets pretreated with Se and stimulated by Cu2+-oxidized low density of lipoprotein (OxLDL) or thrombin before assessment of P-selectin and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), phosphorylated Jun N-terminal kinase (p– JNK), and phosphorylated extracellular signal-regulated kinases (p-ERK1/2). All variables were measured by solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Results: Se significantly decreased Cu2+-OxLDL induced P-selectin expression, as well as p38 and JNK phosphorylation in platelets, but could not significantly reduce ERK1/2 phosphorylation. Conclusion: Se suppresses inflamed platelets. This effect maybe partly mediated by the p38 or c-JNK signaling pathways. These results create possibility of new co-anti-inflammatory insight for Se in atherosclerosis.

Synthetic analogues of flavonoids with improved activity against platelet activation and aggregation as novel prototypes of food supplements

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1–4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5–7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5–25–50–100μM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP.Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

Carbon‐based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape

Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg(-1) body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg(-1) body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration.

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.