Surface Expression Of P-selectin Research Articles

Platelet activation in patients with peripheral vascular disease: Reproducibility and comparability of platelet markers

Background Many markers of platelet activation have been described but their reproducibility and comparability in patient populations are poorly defined. Objectives We sought to compare markers of platelet and monocyte activation with platelet-monocyte aggregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events. Patients/Methods Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme-linked immunosorbant assays. Results Platelet-monocyte aggregation (36.7 ± 7.86%), and platelet surface expression of P-selectin (5.8 ± 1.65%) and CD40L (3.3 ± 1.45%) demonstrated comparable within-day (mean difference ± co-efficient of reproducibility; 0.9 ± 15.4%, 0.21 ± 1.65% and 0.2 ± 2.8% respectively) and between-day reproducibility (2.0 ± 12.4%, 0.10 ± 2.25% and 0.9 ± 6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r = 0.30-0.50, P < 0.02) and monocyte (r = 0.27-0.47, P < 0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility > 40). Conclusions In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation.

In vitro anti‐platelet effects of simple plant‐derived phenolic compounds are only found at high, non‐physiological concentrations

Bioactive polyphenols from fruits, vegetables, and beverages have anti-platelet effects and may thus affect the development of cardiovascular disease. We screened the effects of 26 low molecular weight phenolic compounds on two in vitro measures of human platelet function. After platelets had been incubated with one of 26 low molecular weight phenolic compounds in vitro, collagen-induced human platelet aggregation and in vitro TRAP-induced P-selectin expression (as marker of platelet activation) were assessed. Incubation of platelet-rich plasma from healthy volunteers with 100 μmol/L hippuric acid, pyrogallol, catechol, or resorcinol significantly inhibited collagen-induced platelet aggregation (all p<0.05; n≥15). Incubation of whole blood with concentrations of 100 μmol/L salicylic acid, p-coumaric acid, caffeic acid, ferulic acid, 4-hydroxyphenylpropionyl glycine, 5-methoxysalicylic acid, and catechol significantly inhibited TRAP-induced surface P-selectin expression (all p<0.05; n=10). Incubation with lower concentrations of phenolics affected neither platelet aggregation nor activation. As concentrations of 100 μmol/L are unlikely to be reached in the circulation, it is doubtful whether consumption of dietary phenolics in nutritionally attainable amounts plays a major role in inhibition of platelet activation and aggregation in humans.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Inhibitory effects of lipoteichoic acid from Staphylococcus aureus on platelet function and platelet–monocyte aggregation

Lipoteichoic acid (LTA) from Staphylococcus aureus has been demonstrated to inhibit agonist-stimulated platelet aggregation. However, its effects on platelet inflammatory mediator release and platelet-monocyte aggregation are still unclear. In the present study, LTA is examined for its anti-inflammatory properties and effects on platelet-monocyte aggregation. Blood samples were obtained from 5 healthy volunteers who had taken no medicine in the previous 2weeks. Washed platelets were prepared and incubated with LTA (0.5-2.0μg/mL), then platelet aggregation, P-selectin expression, and soluble CD40L (sCD40L) release were measured by light transmission aggregometry, flow cytometry and enzyme-linked immunoassays, respectively. Platelet-monocyte aggregate formation in whole blood was measured by flow cytometry. Thrombin was used as a stimulant. LTA dose-dependently decreased platelet aggregation from 89.32±10.24% to 36.28±9.01% (P<0.05), sCD40L release from 3.28±0.76 to 1.13±0.45ng/mL (P<0.05) and surface P-selectin expression from 82.01±11.20 to 22.78±6.42% (P<0.05). In human whole blood, 1.0μg/mL LTA inhibited platelet-monocyte aggregation from 78.19±10.94 to 38.24+8.74% (P<0.05). These results indicate that LTA from S. aureus can inhibit platelet-dependent inflammatory mediator release and platelet-monocyte aggregation. These findings suggest that LTA-mediated functional alteration of platelets may contribute to immune evasion of S. aureus.

The Feverfew plant-derived compound, parthenolide enhances platelet production and attenuates platelet activation through NF-κB inhibition

Introduction Few treatments are available that can safely and effectively stimulate new platelet production for thrombocytopenic patients. Additionally, recipients of transfused platelets may experience an inflammatory response due to stored platelets becoming unnecessarily activated, thus creating the need for suitable agents that will dampen undesirable platelet activation. We investigated the effect of the feverfew plant-derived compound, parthenolide on platelet production and platelet activation because of its well-studied ability to induce apoptosis or differentiation in some types of cancer. Methods Parthenolide was used to treat human megakaryoblastic cell lines, primary human and mouse megakaryocytes. Resulting platelet production and function was measured via flow cytometry. The two most common parthenolide signaling mechanisms, oxidative stress and nuclear factor-κB inhibition, were assessed within the megakaryocytes using reactive oxygen species, glutathione and luciferase reporter assays. The influence of parthenolide on ex vivo platelet activation was tested with parthenolide pretreatment followed by collagen or thrombin activation. The resulting P-selectin surface expression and released soluble CD40 ligand was measured. Results Parthenolide stimulates functional platelet production from human megakaryocyte cell lines, and from primary mouse and human megakaryocytes in vitro. Parthenolide enhances platelet production via inhibition of nuclear factor-κB signaling in megakaryocytes and is independent of the parthenolide-induced oxidative stress response. Additionally, parthenolide treatment of human peripheral blood platelets attenuated activation of stimulated platelets. Conclusion Overall, these data reveal that parthenolide has strong potential as a candidate to enhance platelet production and to dampen undesirable platelet activation.

βγ-CAT, a non-lens betagamma-crystallin and trefoil factor complex, induces calcium-dependent platelet apoptosis

In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets. βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, caused several in vivo toxic effects on mammals. Through determined haematological parameters of rabbits, it has been found that βγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect of βγ-CAT on platelets, washed platelets were incubated with various concentrations of βγ-CAT for 30 minutes. We found that βγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarisation of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However, βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation. In addition, we observed that βγ-CAT-induced PS exposure and ΔΨm depolarisation in platelets are Ca2+-dependent. Taken together, βγ-CAT can induce Ca2+-dependent platelet apoptosis but does not cause platelet activation.

Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets

Introduction Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in periodontitis and the effects of periodontopathogens on P-selectin release are unknown. Material and Methods Soluble P-selectin was determined in 26 patients with periodontitis and 19 controls. Furthermore, human endothelial cells and platelets were investigated for their ability to elicit soluble and surface P-selectin in response to periodontopathogens A. actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression as well as NFκB translocation in response to these bacteria were determined on endothelial cells as well as the formation of platelet-leukocyte complexes. Results Plasma levels of soluble P-selectin are significantly elevated in periodontitis and correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets, bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and endothelial cells and results in increased levels of soluble P-selectin. Conclusions Periodontopathogens are able to directly cause activation of endothelial cells and platelets within minutes. Given that transient periodontitis-associated bacteremia commonly occurs after tooth brushing or chewing, our data suggest that reduction of periodontopathogens might result in potential cardiovascular benefits.

Platelet Function and Response to Thrombopoietin Mimetics In Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia.

AbstractAbstract 1429 Introduction:Wiskott-Aldrich syndrome (WAS) is a complex disease in which patients suffer from both bleeding and immunodeficiency. Thrombocytopenia is severe, platelets are small and may be dysfunctional; intracranial hemorrhage occurs in 20% of patients. In patients with the full WAS phenotype, early use of human stem cell transplantation (HSCT) corrects the immune deficiency and thrombocytopenia although platelet transfusions are often required. Bleeding is the main complication of the X-linked thrombocytopenia (XLT) form, whose management may include HSCT, splenectomy, or supportive care; IVIG has limited benefit. Platelet function in WAS/XLT patients with low platelet counts has not been reported because of the inability to accurately perform standard assays in severely thrombocytopenic patients. The present study evaluated platelet function and thrombopoietin (TPO) mimetic (romiplostim or eltrombopag) treatment of thrombocytopenia in patients with WAS/XLT. Methods:Flow cytometry, which enables evaluation of platelet function despite thrombocytopenia, was used to study platelets in 8 WAS/XLT patients and age-matched normal controls. Platelet function was measured by: surface expression of P-selectin and activated GPIIb-IIIa (reported by PAC1) in whole blood following stimulation with low and high dose ADP and thrombin receptor activating peptide (TRAP); annexin V binding, a marker of platelet surface expression of the procoagulant phospholipid phosphatidylserine, in platelet-rich plasma (normalized to 30,000 platelets/μL) following stimulation with convulxin (a specific agonist of the platelet collagen receptor GPVI). The effects of romiplostim (10 μg/kg/wk SQ) or eltrombopag (50-75 mg/day PO) on platelet counts and bleeding were evaluated in 4 patients (3 WAS, 1 XLT). Results:Platelets from WAS/XLT patients showed reduced TRAP-induced platelet surface P-selectin and activated GPIIb-IIIa (p <0.05) compared to age-matched control children (Figure). In contrast, convulxin-induced annexin V binding to platelets was greater than normal controls (p <0.05). These findings were observed in both WAS and XLT platelets and in non-splenectomized (6) and splenectomized (2) patients. As expected, platelet size of WAS/XLT platelets, as judged by forward light scatter, was smaller than that of normal controls. Two infants were treated with romiplostim and 2 older patients were treated with eltrombopag. One infant had 2 intervals of approximately 1 month each in which his platelets were supported entirely by romiplostim and maintained >20-30,000/μL. However, at times of infection with prolonged antibiotics, romiplostim was insufficient although it enabled platelet transfusions to be given weekly. In the other infant, who had both WAS-associated and autoimmune thrombocytopenia, romiplostim had no apparent effect; his platelets were only responsive to the combination of IVIG/methylprednisolone plus platelet transfusion given 2–3 times weekly prior to HSCT. A 25 year old XLT patient received eltrombopag for 4 weeks with a platelet increase from 18 to 33,000/μL. A fourth patient, with WAS, who had failed HSCT was treated with eltrombopag without consistent success. In the first infant on romiplostim and the XLT patient on eltrombopag, clinical bleeding was reduced in conjunction with the increased platelet count. Conclusions:Bleeding in WAS/XLT may be the result of both platelet dysfunction and thrombocytopenia. WAS/XLT platelets are smaller and express less surface P-selectin and less activated GPIIb-IIIa in response to TRAP stimulation than age-matched controls. However, WAS/XLT platelets, when stimulated via the collagen receptor GPVI, express more phosphatidylserine, which supports formation of the prothrombinase complex, than control platelets. The reduced platelet function in WAS/XLT patients resulting from reduced platelet number, size, and surface P-selectin and activated GPIIb-IIIa may be counterbalanced in part by increased GPVI-mediated procoagulant activity. However, increased platelet procoagulant activity may shorten platelet lifespan, contributing to the thrombocytopenia in WAS/XLT. Platelet counts were increased and clinical bleeding was decreased in 2 of 4 WAS/XLT patients treated with TPO mimetics. The possible use of TPO mimetics to increase platelet count and/or function in WAS/XLT patients merits further study. [Display omitted] Disclosures:Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Research Funding, Speakers Bureau; Amgen Inc.: Equity Ownership, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Sysmex: Research Funding. Off Label Use: romiplostim and eltrombopag; increase platelet counts in Wiskott-Aldrich syndrome/X-linked thrombocytopenia patients. Michelson:GlaxoSmithKline: Honoraria.

Von willebrand factor increases endothelial cell adhesiveness for human mesenchymal stem cells by activating p38 mitogen-activated protein kinase

IntroductionDelivered systemically or natively circulating mesenchymal stem cells accumulate in injured tissues. During homing mesenchymal stem cells adhere to endothelial cells and infiltrate underlying tissue. Previously we have shown that adhesiveness of endothelial cells for mesenchymal stem cells correlates with the inhibition of mitochondrial function of endothelial cells and secretion of von Willebrand factor. We hypothesized that von Willebrand factor is an auto/paracrine regulator of endothelial cell adhesiveness and studied the effect of von Willebrand factor on adhesion of mesenchymal stem cells to endothelial cells.MethodsWe used Affymetrix DNA microarrays, human protein phospho-MAPK array, Western blot, cell-based ELISA and flow cytometry analysis to study the activation of endothelial cells by von Willebrand factor. Cell adhesion assay and protein kinase inhibitors were used to evaluate the role of mitogen-activated protein kinases in the regulation of endothelial cell adhesiveness for mesenchymal stem cell.ResultsTreatment of endothelial cells with von Willebrand factor stimulated the mesenchymal stem cell adhesion in a time- and concentration-dependent manner. Mesenchymal stem cells did not adhere to immobilized von Willebrand factor and did not express receptors for von Willebrand factor suggesting that the stimulation of the mesenchymal stem cell adhesion is a result of endothelial cell activation with von Willebrand factor. Treatment of endothelial cells with von Willebrand factor activated ERK-1,2 and p38 MAPK without an effect on gene or cell surface expression of E-selectin, P-selectin, VCAM1 and ICAM1. Inhibition of p38 MAPK, but not ERK-1,2, in endothelial cells completely abrogated the stimulation of the mesenchymal stem cell adhesion by von Willebrand factor.ConclusionsVon Willebrand factor is an auto/paracrine regulator of endothelial cells. Activation of p38 MAPK in endothelial cells by von Willebrand factor is responsible for the regulation of endothelial cell adhesiveness for mesenchymal stem cells.

Effects of various doses of aspirin on platelet activity and endothelial function

Although aspirin has become an established medicine for cardiac and cerebrovascular diseases, the optimal dose remains unknown. We evaluated the optimal dose of aspirin on platelet activity and endothelial function by administering 11 healthy male volunteers (32 ± 6 years of age) doses of aspirin that were increased in a stepwise manner (0, 81, 162, 330 and 660 mg/day) every 3 days. Platelet activity was assessed as surface P-selectin expression (%) measured by flow cytometry and the platelet aggregation ratio. Endothelial function in the brachial artery was assessed by measuring flow-mediated dilation (FMD) before and after reactive hyperemia. Platelet aggregation and P-selectin expression were significantly and dose-dependently suppressed (81-660 mg), and the FMD ratio tended to increase from 0 to 162 mg, but decreased significantly at 660 mg. In conclusion, although aspirin suppressed platelet activity and even surface P-selectin expression, higher doses worsened endothelial-mediated arterial dilation.

Decreased VASP phosphorylation in platelets of male and female smokers of young age

Cigarette smoking represents a major risk factor for atherosclerosis as smoking delivers huge amounts of oxidants and toxins to the human body and elicits an inflammatory response. Notably, oxidative stress and inflammation-derived oxidants are able to modulate platelet function. This is of particular interest as platelets and their activation state are implicated in the very early phases of the development of cardiovascular disease. We therefore investigated the impact of smoking on platelet reactivity and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in a group of 20 young smokers (age 25.7 ± 4.36 years; 10 male/10 female) and an age- and gender-matched control group. After overnight smoking cessation, basal and prostaglandin E1-induced phosphorylation state of intraplatelet VASP was compared between smokers and non-smokers. Furthermore, the ability of several concentrations of ADP to induce surface expression of P-selectin was assessed. Our results show that both the basal as well as prostaglandin E1-induced phosphorylation states of VASP are significantly decreased in the smokers group. These effects can be observed in both male and female smokers to a similar extent. Furthermore, we found significantly enhanced surface expression of P-selectin in response to different concentrations of ADP in smokers; these differences are even more pronounced in the presence of prostaglandin E1. As phosphorylated VASP represents a negative regulator of platelet activation, decreased VASP phosphorylation would be supposed to result in platelet hyperreactivity and pro-coagulant and pro-inflammatory consequences. Therefore, our findings add another piece of evidence to the harmful effects of smoking in both male and female smokers.

Protein phosphatase inhibitor calyculin – A modulates activation markers in TRAP-stimulated human platelets

Platelet activation is accompanied with the phosphorylation of a number of proteins on serine (Ser) and threonine (Thr) residues. The phosphorylation level of these proteins is dependent upon the protein kinase/phosphatase activity ratio. The aim of this study was to investigate the consequences of inhibiting protein phosphatase 1 (PP1) and 2A (PP2A) on platelet functions. Protein phosphatases were inhibited by preincubation of platelet rich plasma (PRP) samples with calyculin-A (CLA). Subsequently, platelets were activated by thrombin-receptor activating peptide (TRAP) and platelet aggregation, platelet-derived microparticle (PMP) formation, surface expressions of P-selectin (CD62), lysosome-associated membrane protein (CD63), glycoprotein Ib and IIb were examined. Phosphatase activity was determined by using phosphorylated 20 kDa myosin light chain (P-MLC20) as substrate. In CLA-treated platelets substantial decrease of P-MLC20 phosphatase activity was observed. CLA significantly suppressed TRAP-induced surface expression of P-selectin and CD63 in a concentration-dependent manner as compared to non-treated samples and moderately decreased platelet aggregation. In TRAP-activated samples, 50 nM of CLA pretreatment completely abolished the level of PMPs and the prevention of GPIb downregulation was also observed; however, no difference was found in GPIIb expression. In conclusion, PP1 and PP2A-catalyzed dephosphorylation processes have crucial roles in PMP formation and in the regulation of alpha-granule and lysosome secretion in human platelets.

Function and activation state of platelets in vitro depend on apheresis modality

In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators. Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry. During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs. In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

α1GT-type calcium channel selectively regulates P-selectin surface expression in pulmonary capillary endothelium

Regulated P-selectin surface expression provides a rapid measure for endothelial transition to a proinflammatory phenotype. In general, P-selectin surface expression results from Weibel-Palade body (WPb) exocytosis. Yet, it is unclear whether pulmonary capillary endothelium possesses WPbs or regulated P-selectin surface expression and, if so, how inflammatory stimuli initiate exocytosis. We used immunohistochemistry, immunofluorescence labeling, ultrastructural assessment, and an isolated perfused lung model to demonstrate that capillary endothelium lacks WPbs but possesses P-selectin. Thrombin stimulated P-selectin surface expression in both extra-alveolar vessel and alveolar capillary endothelium. Only in capillaries was the thrombin-stimulated P-selectin surface expression considerably mitigated by pharmacologic blockade of the T-type channel or genetic knockout of the T-type channel alpha(1G)-subunit. Depolarization of endothelial plasma membrane via high K(+) perfusion capable of eliciting cytosolic Ca(2+) transients also provoked P-selectin surface expression in alveolar capillaries that was abolished by T-type channel blockade or alpha(1G) knockout. Our findings reveal an intracellular WPb-independent P-selectin pool in pulmonary capillary endothelium, where the regulated P-selectin surface expression is triggered by Ca(2+) transients evoked through activation of the alpha(1G) T-type channel.

Influence of glycaemic control on platelet bound CD40–CD40L system, P‐selectin and soluble CD40 ligand in Type 2 diabetes

Type 2 diabetes is a potent cardiovascular risk factor, associated with proinflammatory and prothrombotic processes. The purpose of this study was to investigate whether platelet-bound CD40-CD40L signalling, P-selectin expression and soluble CD40L were increased in patients with diabetes mellitus and can be normalized by improving glycaemic control. Soluble (s) CD40L, platelet surface expression of CD40L, CD40 and P-selectin (CD62P) on platelets were measured by flow cytometry in 71 patients with Type 2 diabetes mellitus and 37 healthy volunteers. In addition, the relationship of HbA1c to CD40-CD40L and P-selectin expression was determined in a longitudinal follow-up. In patients with Type 2 diabetes, platelet membrane CD40 expression (Type 2 diabetes 3.1+/-0.61 vs. controls 2.5+/-0.85 mean fluorescence intensity; P=0.001), platelet-bound CD40L (1.2+/-0.32 vs. 1.1+/-0.14; P=0.034) as well as surface expression of CD62P (0.66+/-0.19 vs. 0.57+/-0.12; P=0.007) were higher than in control subjects. Plasma sCD40L values (3.2+/-1.70 vs. 1.8+/-0.50 ng/ml; P<0.001) were also significantly increased in Type 2 diabetes. After improving glycaemic control in patients with initial HbA1c>8.5% (n=15), platelet P-selectin and CD40-CD40L expression decreased significantly by 54.0%, 36.22% and 16.26%, respectively 1 year later. Type 2 diabetes is associated with up-regulation of the platelet-bound CD40-CD40L system, platelet hyperactivity (enhanced P-selectin expression) and increased sCD40L levels. Improved glycaemic control, however, does help to correct abnormal platelet activation via down-regulation of CD40-CD40L system and P-selectin expression.

New Developments in Lung Endothelial Heterogeneity: von Willebrand Factor, P-Selectin, and the Weibel-Palade Body

Quiescent pulmonary endothelium establishes an antithrombotic, anti-inflammatory surface that promotes blood flow. However, the endothelium rapidly responds to injury and inflammation by promoting thrombosis and enabling the directed transmigration of inflammatory cells, such as neutrophils, into the alveolar airspace. Although the endothelial cell signals responsible for establishing a prothrombotic surface are distinct from those responsible for recognizing circulating neutrophils, these processes are highly interrelated. Von Willebrand factor (VWF)-stimulated secretion plays an important role in thrombus formation, and P-selectin surface expression plays a key role in neutrophil binding necessary for transmigration. Both VWF and P-selectin are located within Weibel-Palade bodies in pulmonary arteries and arterioles, yet Weibel-Palade bodies are absent in capillaries. Despite the absence of the Weibel-Palade bodies, pulmonary capillaries express both VWF and P-selectin. The physiological and pathophysiological significance of these observations is unclear. In this review, we address some anatomical and physiological features that distinguish pulmonary artery, capillary, and vein endothelium. In addition, we review our current understanding regarding the stimulated secretion of VWF and P-selectin in pulmonary artery and capillary endothelium. This information is considered in the context of vasculitis and pneumonia, two pathophysiological processes to which the stimulated secretion of VWF and P-selectin contribute.

Calmodulin antagonists induce platelet apoptosis

Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and α-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca 2+ levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca 2+ chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca 2+ levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis.

C1q induces a rapid up-regulation of P-selectin and modulates collagen- and collagen-related peptide-triggered activation in human platelets

Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin αIIβI was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet–neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5 s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the αIIβI-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90 s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet–neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulation of platelets, and inhibits the formation of platelet–neutrophil aggregates. These immuno-regulatory effects of C1q may have a crucial role in innate immunity and inflammation.

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Delivery of Glycoprotein IIb/IIIa Inhibitor Therapy for Percutaneous Coronary Intervention

A major goal of any antithrombotic regimen administered during percutaneous coronary intervention (PCI) is the preservation of coronary microvascular perfusion, which is critical for myocardial survival. In addition to macrovascular thrombosis, microembolization into the downstream coronary artery bed that occurs during spontaneous plaque rupture and PCI plays a prominent role in the development of microvascular dysfunction that leads to myocardial infarction. In addition to physical obstruction of the lumen by the embolus, vasoconstriction and edema due to inflammation also impair microvascular flow.1 These events likely occur more frequently during PCI performed in the setting of unstable coronary syndromes.2 With Doppler guidewire technology, it was estimated that an average of 25 embolic events occurred during primary PCI for ST-segment elevation myocardial infarction.3 Article see p 784 Platelet aggregates are important components of coronary microemboli, along with leukocytes, erythrocytes, platelet-leukocyte aggregates, cholesterol crystals, and hyalin. Platelets and leukocytes within microemboli play a particularly important role in the pathophysiological changes of blood flow by promoting in situ microvascular thrombosis, vasoconstriction, and inflammation. Interestingly, the composition of the embolized material is similar in the settings of spontaneous and catheter-induced plaque rupture.1 In animal models, distal microembolization of inert microspheres is associated with an immediate reduction in flow due to vessel occlusion, followed by enhanced blood flow due to the effects of adenosine released from the embolized myocardium into the adjacent noninvolved myocardium.4 Microembolization results in overall reduced flow reserve and the progressive loss of contractile function, which often reverses within weeks. The degree of myocardial contractile dysfunction is disproportionate to the degree of infarction and is the result of inflammation.5 However, in the clinical condition, a more complex pathophysiology exists due to the influence of multiple components of the emboli. Platelets aggregate when fibrinogen binds to the active …