Modulation of P-selectin expression on isolated human platelets y an NO donor assessed by a novel ELISA application (original) (raw)
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Clinical & Laboratory Haematology, 2000
P-selectin is an adhesion molecule found in the alpha granules of platelets. Activation occurs in response to a range of in¯ammatory and thrombotic agents resulting in rapid up-regulation. Flow cytometry methods have recently been described for the analysis of platelet P-selectin expression in whole blood. While introducing these methods into our laboratory it was noted that expression could be stimulated, in vitro, in a number of ways. This study shows that red cell lysis, the anticoagulant K 3 EDTA and the time elapse between blood collection and antibody labelling had statistically signi®cant effects on P-selectin expression. Post-labelling ®xation, with CellFIX, caused no signi-®cant effect. We conclude that blood for P-selectin analysis should be collected in sodium citrate and that red cell lysis and centrifugation should be avoided. When comparing samples, the time between collection and labelling should be standardized. The relatively high CV for the assay indicates that all samples should be labelled and analysed in duplicate with the mean level reported.
Plasma P-selectin is increased in thrombotic platelet disorders
Blood
P-selectin is a 140-kD protein found in the a-granules of platelets and the Weibel-Palade bodies o f endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was "3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody, We have measured plasma P-selectin and j3thromboglobulin (BTG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) pa-From the Centre for Thrombosis and Vascular Research, the
Characterization of platelet and soluble-porcine P-selectin (CD62P)
Veterinary Immunology and Immunopathology, 2003
P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine Pselectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release. #
Comparative biochemistry and physiology. Part A, Physiology, 1994
The lectin-like domain of P-selectin, an adhesive receptor (also known as PADGEM, GMP-140 or CD62) is implicated in platelet or endothelial cell interactions with leukocytes. The aim of this study was to characterize the lectin-like domain of rat P-selectin by the use of synthetic peptides. The lectin and EGF-like domains of rat P-selectin were cloned in our laboratory and shown to present very strong homologies to its human counterpart. Peptides corresponding with the lectin-like domain of P-selectin were tested for their ability to inhibit thrombin-activated platelets rosetting to neutrophils. Peptides 23-30 (A) and 76-90 (C), but not peptide 51-61 (B), inhibited thrombin activated rat platelets interactions with rat neutrophils (A = 33%, C = 46%, P < 0.05). Using a combination of peptides (A+B = 35%, P = 0.008 and A+C = 62%, P < 0.001), we observe different degrees of inhibition of platelets binding to neutrophils. The IC50 of peptides A+C was 0.11 mM. LYP-20, an anti-human...
P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation
Thrombosis Research, 2011
Introduction: Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Methods and Results: Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and Pselectin-deficient (CD62P −/− ) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet α IIb β 3 . Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P −/− mice. In addition, CD62P −/− mice exhibited thrombosis abnormalities without an α IIb β 3 activation defect.
P-Selectin Expression on Platelets Determines Size and Stability of Platelet Aggregates
Circulation, 2000
Background —P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells. After platelet activation, P-selectin is translocated from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein (GP) IIb/IIIa between adjacent platelets. Methods and Results —In this study, we define a novel role for P-selectin in platelet aggregation. Expression of P-selectin on the platelet surface correlated strongly with the mean platelet aggregate size. Inhibition of P-selectin binding to its ligand by either monoclonal anti–P-selectin antibodies directed against the lectin domain or soluble human P-selectin reversed platelet aggregation even when added up to 5 minutes after activation; however, fibrinogen binding to platelets was not affected. This deaggregating effect significantly reduced the maximal size and number of platelet aggregates. When added 1 minute after platelet activation, anti–P-selectin antibody achiev...
Thrombosis Research, 2008
Background: Plasma levels of soluble P-selectin (sP-selectin) are often used to demonstrate platelet activation. Methods: We determined sP-selectin in a variety of disorders characterized by high or low platelet counts and compared their levels with those in healthy subjects. Furthermore, we determined the Thr715Pro polymorphism in all subjects. Results: Total concentrations of sP-selectin were clearly associated with levels of platelet counts. Thus, calculation of sP-selectin per platelet showed that these levels in patients with thrombocytopenia due to marrow failure and in patients with increased platelet counts were similar to those in controls. Only patients with an increased platelet turnover had elevated sP-selectin per platelet. While carriers of the Pro715 polymorphism had lower sP-selectin levels than non-carriers, this genetic disposition was overruled in patients with increased platelet turnover. Conclusion: For the demonstration of platelet activation it is preferable to define sPselectin based on platelet counts under the consideration of the Pro715Thr polymorphism.
Comparative biochemistry and physiology. Comparative physiology, 1992
1. Granule membrane protein (GMP-140) is an integral alpha-granule membrane glycoprotein, expressed on the surface of human platelets following degranulation, and is part of a new family of adhesion molecules (selectins) related to the endothelial leukocyte adhesion molecule (ELAM-1) and to the lymphocyte homing receptors in man (Leu-8/TQ1) and in mouse (gp90MEL-14). 2. The cross-reactivity with rat platelets of the monoclonal antibodies (MAb), LYP20 and S12, directed against human GMP-140 was examined, with the purpose of assessing the homology of GMP-140 between human and rat platelets and of using positive MAbs to detect platelet activation in vivo in response to vascular disease in rats. 3. By ELISA technique, LYP20 gave a greater OD reading with thrombin-stimulated rat platelets than with resting platelets. 4. 125I-LYP20 bound significantly more to thrombin-stimulated rat platelets (3875 +/- 750 molecules/platelet) than to resting platelets (645 +/- 240 molecules/platelet, P le...
P-selectin and leukocyte microparticles are associated with venous thrombogenesis
Journal of Vascular Surgery, 2003
Objectives: P-selectin inhibition has been found to limit venous thrombosis. We hypothesize that elevated levels of P-selectin will amplify thrombosis, mediated by procoagulant microparticles (MPs). Methods: Male mice (Mus musculus, n659), 20 to 25 grams, underwent IVC ligation to induce thrombosis. Groups consisted of wild type (WT) C57BL/6 controls, mice with high circulating levels of soluble P-selectin (^CT), P-selectin gene-interrupted knockout mice (PKO), and E-and P-selectin gene-interrupted mice (EPKO). Additional groups were used to evaluate the ability of a P-sel antagonist (rPSGL-Ig) and an antibody directed against PSGL-1 to downregulate the effects of P-sel in^CT mice and WT mice administered soluble P-sel at time of thrombosis. Animals were sacrificed on days 2 and 6 after IVC ligation. Thrombus mass (TM), vein wall morphometrics, and serum leukocyte/platelet microparticles (MPs) were evaluated by means of double-stained fluorescence-activated cell scanning analysis, and soluble P-and E-sel protein determination by ELISA. Results: At days 2 and 6 in phase I of the experiment, significant differences (P < .01) in TM were noted between groups, with^CT animals having the largest thrombi (50% and 57% increase in TM compared to WT at days 2 and 6) while EPKO mice had the smallest thrombi. Statistically, greater levels of neutrophils and total inflammatory cells were noted in the vein walls of^CT animals at day 2 compared with WT and PKO animals. A significant difference was noted between^CT and EPKO for neutrophils, monocytes, and total inflammatory cells, also at day 2. At day 6, the only statistically significant difference was found for monocytes, with a higher number in the^CT animals than in WT animals. The evaluation of MPs revealed that the^CT mice had a mixed leukocyte (MAC-1) and platelet (CD41) MP population that was also present in WT and PKO mice on day 2 and day 6. EPKO mice revealed a primarily platelet-derived MP population. Of interest, the^CT mice with the highest TM showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs.^CT mice revealed statistically higher levels of soluble P-selectin at days 2 and 6. In phase 2, an antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. Conclusions: This study demonstrates that high circulating levels of P-selectin are associated with increased thrombosis, whereas a lack of P-selectin and E-selectin is associated with a lessening of thrombosis. Additionally, leukocyte MPs are associated with venous thrombus formation. These data suggest the importance of selectins to venous thrombogenesis and show that P-selectin and leukocyte-derived MPs should be good targets to limit venous thrombus formation. (J Vasc Surg 2003;38:1075-89.)