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PubMed2002

Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6' and GSP-6", with three TyrSO3- residues and either difucosylated polylactosamine (C2-O-Le(x)-sLe(x)) or trifucosylated polylactosamine (C2-O-Le(x)-Le(x)-sLe(x)). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6' and GSP-6" bound to P-selectin with low affinity (K(d) approximately 37 microm for GSP-6' and K(d) approximately 50 microm for GSP-6"). Binding of GSP-6' and GSP-6" to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6' and GSP-6". These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.

Amino Sugars↗
PubMed2003

Single molecule characterization of P-selectin/ligand binding.

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.

Actins↗
PubMed2002

Soluble forms of the selectin family in children with Kawasaki disease: prediction for coronary artery lesions.

AIM: To investigate the relationship between the plasma levels of soluble forms of the selectin family and the incidence of coronary artery lesions (CALs) in patients with Kawasaki disease (KD). METHODS: Thirty-three patients with KD, including group A patients (n = 22) who had no CALs and group B patients (n = 11) who had CALs, as well as age-matched febrile (n = 10) and afebrile controls (n = 11), were studied. RESULTS: Peak plasma E-selectin levels (172.0 +/- 58.6 ng ml(-1)) occurred during the acute phase of KD, while peak plasma P-selectin levels (260.3 +/- 43.2 ng ml(-1)) occurred during the subacute phase of the illness (p<0.05). Plasma L-selectin levels (1757.3 +/- 244.3 ng ml(-1)) during the convalescent phase tended to be higher than in either the acute or the subacute phase (not significant). Before intravenous immunoglobulin treatment, the plasma levels of E- (225.1 +/- 46.8 ng ml(-1)) and P-selectin (259.4 +/- 76.2 ng ml(-1)) of patients with CALs (n = 11) were significantly higher than those of patients (n = 22) with no CALs (E-selectin, 131.6 +/- 36.9 ng ml(-1); P-selectin, 184.9 +/- 84.6 ng ml(-1); p < 0.05). When a plasma E-selectin value before immunoglobulin treatment of >184.7 ng ml(-1) was used as the cut-off point, the sensitivity and specificity for the incidence of CALs were 81.8% and 90.9%, respectively. These findings demonstrate the relationship between plasma levels of selectins and disease severity of Kawasaki vasculitis. CONCLUSION: Higher plasma levels of E-selectin may have potential as a predictor of the incidence of coronary artery lesions in Kawasaki disease patients.

Child↗
PubMed2005

Soluble P-selectin levels in diabetes mellitus patients with coronary artery disease.

UNLABELLED: Type 2 (non-insulin-dependent) diabetes is associated with a marked increase in the risk of coronary heart disease. Platelets play a significant role in coronary artery disease. Soluble P-selectin is an index of platelets activation. In this study, Soluble P-selectin levels were measured by ELISA in the peripheral blood of 55 diabetic patients with coronary artery disease [21 acute myocardial infarction (AMI), 20 with unstable angina (UA), 14 with stable angina (SA)], 20 patients with diabetes mellitus without coronary artery disease (DM without), and 10 healthy controls. Soluble P-selectin level was significantly higher in patients with AMI (M+/-SD; 239.3+/-13.0 ng/ml), than those with UA (141.5+/-15.2 ng/ml), SA (92.1+/-7.7 ng/ml), DM without (89.8+/-7.1 ng/ml), and healthy control (86.1+/-4.5 ng/ml) (P < 0.001). In patients with US, sP-selectin was found to be significantly elevated as compared to the SA, DM without and control group. sP-selectin was not significantly different in DM without as compared to healthy controls. The sP-selectin levels was correlated to the duration of diabetes mellitus(R=0.33, P=0.03 ). Moreover, sP-selectin level was significantly higher in AMI patients with recurrent anginal attack as compared to that in those with single attack Multivariate analysis revealed that sP-selectin level at presentation had high adverse influence on coronary artery insult compared to healthy LDL cholesterol level, and the degree of hypertension. IN CONCLUSION: Plasma levels of soluble P-selectin were increased in patients with AMI, and UA compared to patients with SA and normal controls. Measurement of soluble P-selectin may be helpful marker of impending coronary artery insult in diabetic patients.

Biomarkers↗
PubMed1993

Interferon-alpha induces the expression of the L-selectin homing receptor in human B lymphoid cells.

The L-selectin homing receptor expressed by lymphocytes mediates the initial attachment of these cells to high endothelial venules within peripheral lymph nodes. This adhesive interaction is required for the migration of B and T lymphocytes from the blood into peripheral lymph nodes. There is currently little information regarding the nature of the factors involved in the regulation of the synthesis and expression of L-selectin by lymphocytes. In this report, the immunomodulatory cytokine interferon-alpha (IFN-alpha) was shown to markedly upregulate the surface density of L-selectin in the established human B lymphoid Daudi cell line and in a subpopulation of tissue-derived human B lymphoid cells. Other cytokines such as IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-2, IL-4, IL-6, and low molecular weight B cell growth factor did not affect L-selectin surface expression in the model Daudi B cell line. Upregulation of L-selectin surface density in IFN-alpha-treated Daudi B cells correlated directly with an increase in L-selectin mRNA steady state levels and enhanced L-selectin-dependent binding to a carbohydrate-based ligand, phosphomonoester core polysaccharide. Regulation of L-selectin mRNA by IFN-alpha had characteristics similar to that of classical IFN-stimulated genes including rapid kinetics of induction, protein-synthesis-independent induction, and sensitivity to tyrosine-kinase inhibitors. IFN-alpha did not upregulate L-selectin mRNA levels or surface expression in an IFN-resistant Daudi subclone which exhibits a defect in the signal transduction pathway required for the transcriptional induction of IFN-stimulated genes. These data demonstrate a fundamental role for IFN-alpha in regulating L-selectin synthesis and expression in human B lymphoid cells and suggest a mechanism whereby this cytokine regulates the regional trafficking of B cells to peripheral lymph nodes.

B-Lymphocytes↗
PubMed1996

The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.

Antigens, CD↗
PubMed1996

Impaired primary T cell responses in L-selectin-deficient mice.

L-selectin is a homing receptor that mediates the selective attachment of leukocytes to specialized high endothelial venules. To study the potential role of L-selectin in immune responses in intact mice, we generated L-selectin-deficient mice by gene targeting. L-selectin-deficient mice are defective in cutaneous delayed-type hypersensitivity (DTH) responses when tested after conventional intervals of immunization (4 d). Primary T cell proliferative responses and cytokine production (interleukin [IL] 2, IL-4, and interferon gamma) were also compromised when tested after 5 d of immunization, indicating that L-selectin is important for the immune response to antigens. In contrast, after more prolonged immunization protocols (9 d), normal responses were observed, suggesting that L-selectin-independent compensatory mechanisms exist. Interestingly, humoral responses of L-selectin-deficient mice to keyhole limpet hemocyanin are indistinguishable from wild-type control mice, implying that L-selectin plays no rate-limiting role in T cell help of B cell function. Thus, our results suggest that L-selectin plays an important role in the generation of primary T cell responses but may not be essential for humoral and memory T cell responses. L-selectin does not appear to be rate limiting for the events leading to antigen-driven neutrophil recruitment, since normal DTH responses are obtained at late time points after immunization.

Animals↗
PubMed1996

Interleukin-10 induces E-selectin on small and large blood vessel endothelial cells.

In vitro, expression of E-selectin is largely restricted to endothelial cells activated by inflammatory cytokines. Under activated conditions, cytokines such as interleukin (IL) 10, released by keratinocytes in large quantities, may also increase the expression of E-selectin on the dermal microvasculature. The aim of the present study was to investigate the expression of E-selectin on cultured human dermal microvascular endothelial cells (HDMEC) isolated from neonatal foreskins when exposed to IL-10. Expression of E-selectin was determined by immunofluorescence microscopy, FACS analysis, an HL-60 cell-binding assay, and quantitative polymerase chain reaction (PCR) analysis. For comparison with large blood vessel cells, the expression of E-selectin on human umbilical vein endothelial cells (HUVEC) was also determined in parallel by FACS and reverse transcriptase-PCR analysis under identical conditions. These studies demonstrate that IL-10 induces the expression of E-selectin on both HDMEC and HUVEC and that the level of expression of HDMEC is comparable with that induced by IL-1 beta and tumor necrosis factor-alpha. When HL-60 cells are incubated with HDMEC pretreated with IL-10, a consistent increase in adherence of HL-60 to endothelial cells is observed. This adherence was found to be mediated by L-selectin. PCR analysis and the quantification of E-selectin cDNA by a novel, highly sensitive and specific PCR-immunoassay demonstrate the induction of E-selectin mRNA at the transcriptional level. The induction of the expression of E-selectin by IL-10 on HDMEC may provide additional insights into the pathogenic mechanism of neutrophil accumulation at the site of inflammation in inflammatory skin diseases.

Cell Adhesion↗
PubMed2004

CXCR2- and E-selectin-induced neutrophil arrest during inflammation in vivo.

The signaling events leading to the activation of integrins and firm arrest of rolling neutrophils in inflamed venules have yet to be elucidated. In vitro assays suggest that both E-selectin and chemokines can trigger arrest of rolling neutrophils, but E-selectin(-/-) mice have normal levels of adherent neutrophils in inflamed venules. To test whether chemokine-induced neutrophil arrest in vivo can be unmasked by blocking E-selectin, we investigated neutrophil adhesion in inflamed cremaster muscle venules in tumor necrosis factor (TNF)-alpha-treated CXCR2(-/-) or wild-type (WT) mice injected with E-selectin blocking monoclonal antibody (mAb) 9A9. To block chemokine receptor signaling, we investigated E-selectin(-/-) or WT mice treated with pertussis toxin (PTx) intravenously. Neutrophil adhesion was unchanged in CXCR2(-/-), E-selectin(-/-), PTx-treated WT, or mAb 9A9-treated WT mice. However, TNF-alpha-induced neutrophil adhesion was almost completely abrogated in E-selectin(-/-) mice treated with PTx and significantly reduced in CXCR2(-/-) mice treated with the E-selectin blocking mAb. In thioglycollate-induced peritonitis, PTx treatment blocked neutrophil recruitment into the peritoneum of E-selectin(-/-) mice, but had only a partial effect in WT animals. These data show that E-selectin- and chemokine-mediated arrest mechanisms are overlapping in this model and identify CXCR2 as an important neutrophil arrest chemokine in vivo.

Animals↗
PubMed1998

Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion.

P-selectin, an inducible cell adhesion molecule, mediates rolling of neutrophils on activated vascular endothelium. Because rolling is an early event of the inflammatory response, therapeutic applications of selectin antagonists have been of broad interest. There are, however, no truly satisfactory therapeutic candidates among known inhibitors. Consequently, we have used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, a process based on oligonucleotide combinatorial chemistry and in vitro selection, to develop aptamer antagonists of P-selectin. Equilibrium dissociation constants for aptamer/P-selectin binding range from 16 to 710 pM, a 10(5)-10(6)-fold improvement compared with the minimal carbohydrate ligand, sialyl Lewis X (sLeX). Aptamer binding is divalent cation dependent and, unlike sLeX, is specific for P-selectin. The selectivity for human P-selectin relative to human E-selectin or human L-selectin is 10(4)-10(5). In vitro, aptamers bind with subnanomolar affinities to P-selectin expressed on thrombin-activated platelets, inhibit the binding of P-selectin-IgG chimera to sLeX and to neutrophils, and block the binding activated platelets to neutrophils in flow cytometry and in hydrodynamic assays. Extrapolating from their in vitro characteristics, these novel P-selectin-specific antagonists may be suitable candidates for therapeutic development.

Base Sequence↗
PubMed1997

Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1.

Selectins interact with glycoconjugate ligands in important normal and pathological situations. While high affinity recognition of natural ligands is associated with alpha 1-3(4)fucosylated, alpha 2-3sialylated (and/or sulfated) lactosamine sequences, small oligosaccharides that potently inhibit the selectins have not been found. One possibility suggested by other investigators is that high affinity may require unusual sequences not yet tested, for example, the "major capping group" (6'-sulfo-sialyl Le(x)) of the L-selectin ligand GlyCAM-1. To explore this possibility, we synthesized a spectrum of novel synthetic and semisynthetic oligosaccharides related to those on natural ligands. In studying these molecules, we noted that binding of recombinant soluble selectins to immobilized sialyl Le(a) or 3'-sulfo-Le(x) is markedly inhibited by concentrations of chloride above the physiological range. This indicates the ionic nature of the interactions, and shows that buffers typically used in screening assays for inhibitors are not optimal. Using parameters that more closely approximate physiological conditions, we confirmed that alpha 2-3-linked sialic acids, and alpha 1-3(4)fucosylation are important for recognition. Similar results obtained with both types of immobilized targets for the three selectins indicated that the binding sites for sialic acid and sulfate are very close, or identical. While O-sulfate esters mostly improved L- and P-selectin recognition, effects depended upon their position and number. Furthermore, sulfation can also impart some "negative" specificity: the major capping group does not interact with E-selectin. The branched Core 2 sequence seemed to enhance L- and P-selectin binding, however, the best inhibitors still appeared to be sialyl Le(a) and 3'-sulfo-Le(x), with the aglycone group of the latter affecting binding. Of particular note, the "major capping group" of GlyCAM-1 was not an unusually potent nor highly selective inhibitor of L-selectin, even when studying the interaction of L-selectin with native GlyCAM-1 itself.

Carbohydrate Sequence↗
PubMed2000

Redistribution of selectin counter-ligands induced by cytokines.

Soluble recombinant (r) P-selectin and rP-selectin immobilized on plastic surfaces were tested for their capacity to activate neutrophils to produce superoxide anion. Soluble rP-selectin was incapable of activating leukocytes, whereas immobilized rP-selectin was able to induce leukocyte activation. When neutrophils were pretreated with a low dose of IL-8, granulocyte colony stimulating factor or granulocyte macrophage colony stimulating factor, soluble rP-selectin was no longer inert. These cytokine-primed leukocytes produced superoxide anion in the presence of soluble rP-selectin. During this priming period, sialyl Lewis X (sLe(X)) epitopes redistributed to one end of the leukocytes. Similar polarization of sLe(X) epitopes was observed at the attachment site of cells that adhered to immobilized rP-selectin. Cap formation and superoxide anion production induced by solid-phase P-selectin or by IL-8 and soluble rP-selectin treatment were inhibited by treatment of the leukocytes with cytochalasin B. These observations suggest that the redistribution of the carbohydrate ligands and the polarization of the leukocyte surface through an active process is a prerequisite but not sufficient to leukocyte superoxide production through P-selectin.

Cytokines↗
PubMed2004

Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling.

Lymphocyte homing to lymph nodes is regulated by transient but specific interactions between lymphocytes and high endothelial venules (HEVs), the initial phase of which is mainly governed by the leukocyte adhesion molecule L-selectin, which recognizes sulfated and sialylated O-linked oligosaccharides displayed on sialomucin core proteins. One of the sialomucin proteins, endomucin, is predominantly expressed in vascular endothelial cells of a variety of tissues including the HEVs of lymph nodes; however, whether it functions as a ligand for L-selectin remains to be formally proven. Here we show that the endomucin splice isoform a is predominantly expressed in PNAd+ HEVs and MAdCAM-1+ HEVs, as seen in non-HEV-type vascular endothelial cells. Using affinity purification with soluble L-selectin, we found that HEV endomucin is specifically modified with L-selectin-reactive oligosaccharides and can bind L-selectin as well as an HEV-specific mAb, MECA-79. Our results also indicated that a 90-100 kDa endomucin species is preferentially decorated with L-selectin-reactive sugar chains, whereas an 80 kDa species represents conventional forms expressed in non-HEV-type vascular endothelial cells in lymph nodes. Furthermore, a CHO cell line expressing endomucin together with a specific combination of carbohydrate-modifying enzymes [core-2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), alpha-1,3-fucosyltransferase VII (FucTVII) and L-selectin ligand sulfotransferase (LSST)] showed L-selectin-dependent rolling under flow conditions in vitro. These results suggest that when endomucin is appropriately modified by a specific set of glycosyltransferases and a sulfotransferase, it can function as a ligand for L-selectin, and that the endomucin expressed in HEVs may represent another sialomucin ligand for L-selectin.

Animals↗
PubMed2004

Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process.

Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 h post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge for the first time, the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation.

Amino Acid Sequence↗
PubMed2003

Activation of calcium-dependent calmodulin by calcium(II)3(3,5-diisopropylsalicylate)6(H2O)6 decreases thrombin receptor activating peptide-induced P-selectin expression.

We examined the influence of 3,5-diisopropylsalicylic acid (3,5-DIPS) and calcium(II)3 (3,5-diisopropylsalicylate)6 (H2 O)6 [Ca(II)3 (3,5-DIPS)6 ], a new activator of calcium-dependent calmodulin-triggered nitric oxide synthase, on thrombin-induced platelet P-selectin expression. Citrated whole blood samples were incubated with either ethanol vehicle, 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6. These whole blood samples were also co-incubated with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), to up-regulate P-selectin (CD62P) on platelets. Both TRAP and ADP up-regulated P-selectin on platelets compared with platelets in whole blood samples that were not incubated with either platelet activator. Co-incubation of whole blood samples with TRAP, ADP together with 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6 revealed that Ca(II)3 (3,5-DIPS)6 caused a decrease in platelet P-selectin expression for TRAP, ADP, and no-activator co-incubated samples of whole blood. Incubation of platelets with 3,5-DIPS also caused a decrease in ADP-induced up-regulation of P-selectin but failed to affect TRAP or no-activator-treated platelets. Incubation of whole blood with Ca(II)3 (3,5-DIPS)6 induced some hemolysis. We found that hemolysis increases basal P-selectin expression on platelets. We therefore conclude that Ca(II)3 (3,5-DIPS)6 decreased not only basal, but also hemolysis-induced P-selectin expression on platelets. In contrast, incubation of haemolysed whole blood with SIN-1 (standard nitric oxide-releasing drug) had no effect on P-selectin expression. In summary, Ca(II)3 (3,5-DIPS)6, a new calmodulin-dependent nitric oxide synthase activator, decreases P-selectin expression of human platelets in response to thrombin receptor activation. Improved calcium-dependent calmodulin activators may become useful drugs for the treatment of disorders associated with platelet activation, and P-selectin may decrease expression due to hemolysis.

Adenosine Diphosphate↗
PubMed2003

Plasma P-selectin levels are elevated in patients with chronic liver disease.

P-selectin is a leukocyte receptor and platelet activation marker that has been shown to be involved in thrombogenesis as well as bleeding disorders and may represent a possible link between inflammation and thrombosis. In animal models, high plasma levels correlated with a procoagulant tendency. In acute liver damage models such as hepatic ischaemia-reperfusion-injury, P-selectin was found to be a key mediator of liver injury. In order to investigate the clinical and pathogenetic role of P-selectin in chronic liver diseases, plasma P-selectin levels were measured in 111 patients with chronic liver diseases. P-Selectin was significantly elevated in patients (median 56 ng/ml, range 0-180) compared with controls (n = 38, median 20 ng/ml, range 3.3-42, P < 0.001). Current clinical bleeding symptoms were common, whereas thrombotic events occurred rarely. P-selectin levels were not associated with haemorrhagic or thromboembolic complications. P-selectin correlated with platelet and white-blood-cell counts, but not with endothelial injury markers thrombomodulin and tissue factor or coagulation factors. Interestingly, P-selectin levels were not associated with Child's stage of cirrhosis or disease aetiology, but were generally elevated in chronic liver diseases. Severe hepatic leukocyte infiltration in liver histology was associated with a tendency towards higher P-selectin levels. In line with its role in acute liver damage, P-selectin elevation in chronic liver disease may suggest a possible pathogenetic role in the course of liver cirrhosis.

Adolescent↗
PubMed2000

Role of porcine P-selectin in complement-dependent adhesion of human leukocytes to porcine endothelial cells.

BACKGROUND: Rapid leukocyte adherence to donor organ vasculature is a hallmark of hyperacute xenograft rejection. However, the molecular interactions required for leukocyte binding to vascular endothelium have not been characterized. METHODS AND RESULTS: Binding assays performed between human neutrophils and porcine aortic endothelial cells (PAEC) after exposure to human complement demonstrated that adhesion was mediated by both surface-bound C3b and C5b-9 activity. C5b-9-dependent adhesion was blocked by neuraminidase treatment of the neutrophils, suggesting that this binding was mediated by porcine P-selectin. Porcine P-selectin was isolated from a PAEC cDNA library. The porcine P-selectin primary sequence contained an open reading frame encoding 646 amino acids with 82% identity to human P-selectin. Recombinant soluble porcine P-selectin specifically bound to human neutrophils and HL-60 cells. Transfection of COS cells with the full-length porcine P-selectin cDNA resulted in surface expression of the protein and markedly increased the binding of human neutrophils to these cells. The binding of both soluble and COS-expressed porcine P-selectin to human neutrophils was blocked by pretreatment of the neutrophils with neuraminidase or the addition of EDTA. Finally, treatment of PAEC with human thrombin or normal human serum but not purified human C5a- or C8-deficient human serum resulted in the rapid expression of porcine P-selectin on the cell surface. CONCLUSIONS: This report establishes that porcine P-selectin supports the binding of human neutrophils to PAEC in vitro. Further, these data suggest that sublytic deposition of C5b-9 during hyperacute rejection results in the expression of porcine P-selectin, which may contribute to the rapid adhesion of neutrophils to porcine xenografts.

Amino Acid Sequence↗
PubMed2000

Kinetics of P-selectin expression in regional vascular beds after resuscitation of hemorrhagic shock: a clue to the mechanism of multiple system organ failure.

Leukocyte-endothelial cell interactions play an important role in mediating organ dysfunctions observed after hemorrhagic shock. P-selectin is the first endothelial cell adhesion molecule to be upregulated after an ischemic insult. The objective of this study was to define kinetics of P-selectin expression in different regional vascular beds of mice exposed to hemorrhagic shock. In-vivo P-selectin expressions were determined using dual radiolabeled monoclonal antibody technique in lungs, heart, liver, kidneys, intestinal mesentery, stomach, small bowel, and colon 0.5, 1, 2, 5, 10, and 24 h after resuscitation of 40 mmHg hemorrhagic shock. In another group, P-selectin expression was determined in same organs 5 h after resuscitation of 30 mmHg hemorrhagic shock. Hemorrhagic shock of 40 mmHg caused significant upregulation of P-selectin in lungs and liver at 30 min after resuscitation (P < 0.001). There was a second and more pronounced upregulation of P-selectin in lungs and liver at 5 h after resuscitation (P < 0.001). In heart, intestinal mesentery, stomach, small bowel, and colon, P-selectin was not upregulated until 5 h after resuscitation from 40 mmHg hemorrhagic shock (P < 0.001). While hemorrhagic shock of 40 mmHg did not cause P-selectin upregulation in kidneys, hemorrhage to 30 mmHg did elicit a significant increase at 5 h after resuscitation (P < 0.001). We conclude that P-selectin is upregulated after resuscitation of hemorrhagic shock in lungs, liver, heart, stomach, and intestines. P-selectin upregulation in kidneys only takes place after more severe hemorrhagic shock.

Animals↗