Endothelial Cell Glycoprotein Research Articles

Protective effect of Shengmai injection on myocardial endothelial cell glycoprotein detachment after myocardial ischemia-reperfusion injury in isolated rat hearts.

To investigate effects of Shengmai injection (SMI) postconditioning on myocardial ischemia-reperfusion injury (MIRI) in isolated rat hearts. A total of thirty isolated hearts were randomly divided into three groups: Sham group, I/R group and SMI group. Sham group was continuously perfused with K-H solution for 120 minutes. I/R group and SMI group were given balanced perfusion for 30 min followed by reperfusion for 60 min, with an interval of 30 min, and those in the SMI group were given postconditioning with 1% SMI during the first 10 min of reperfusion. The left ventricular function, markers of myocardial injury, endothelial cell injury and oxidative stress injury were measured at 30 minutes after equilibration (t0), 30 minutes after ischemia (t2) and 60 minutes after reperfusion (t3). The results showed that there was no significant difference for all observation indexes at t0. Compared with the Sham group, real portfolio project and coronary arterial flow rate and the activity of superoxide dismutase were significantly decreased in the I/R group, whereas those in the SMI group were significantly higher. Left ventricular end-diastolic pressure, the concentrate of malondialdehyde, lactate dehydrogenase, cTn-I, hyaluronic acid, heparin sulphate, syndecan-1 in the I/R group were markedly higher than those in the Sham group, whereas those in the SMI group were significantly lower. In summary, the present study indicated that 1% SMI postconditioning can alleviate the detachment of endothelial cell glycoprotein envelope induced by myocardial ischemia-reperfusion injury, and its mechanism is probably related to the inhibition of the oxidative stress injury.

Escherichia coli K1 Modulates Peroxisome Proliferator-Activated Receptor γ and Glucose Transporter 1 at the Blood-Brain Barrier in Neonatal Meningitis.

Escherichia coli K1 meningitis continues to be a major threat to neonatal health. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with endothelial cell glycoprotein 96 (Ecgp96) in the blood-brain barrier to enter the central nervous system. Here we show that the interaction between OmpA and Ecgp96 downregulates peroxisome proliferator-activated receptor γ (PPAR-γ) and glucose transporter 1 (GLUT-1) levels in human brain microvascular endothelial cells, causing disruption of barrier integrity and inhibition of glucose uptake. The suppression of PPAR-γ and GLUT-1 by the bacteria in the brain microvessels of newborn mice causes extensive pathophysiology owing to interleukin 6 production. Pretreatment with partial or selective PPAR-γ agonists ameliorate the pathological outcomes of infection by suppressing interleukin 6 production in the brain. Thus, inhibition of PPAR-γ and GLUT-1 by E. coli K1 is a novel pathogenic mechanism in meningitis, and pharmacological upregulation of PPAR-γ and GLUT-1 levels may provide novel therapeutic avenues.

Soluble P-selectin promotes acute myocardial infarction onset but not severity.

P‑selectin, an integral membrane glycoprotein of platelets and endothelial cells, and the soluble form of P‑selectin are hypothesized to play a role in the initiation of atherosclerosis and acute myocardial infarction (AMI). However, limited data are available with which to evaluate the main role of soluble P‑selectin (sP‑selectin) in the onset or the severity of AMI. In the present study, we investigated 15 patients who suffered from angina, 10 patients who underwent percutaneous coronary intervention (PCI) therapy and 10 patients who underwent thrombolysis therapy, compared with 15 volunteers with no cardiovascular disease. We confirmed that the plasma sP‑selectin levels were increased in patients with obesity (particularly pericardial obesity) and hyperlipidemia, positively correlated with plasma tumor necrosis factor (TNF)‑α and strongly negatively correlated with adiponectin in all patients regardless of AMI status. Furthermore, sP‑selectin levels were significantly higher in PCI and thrombolysis patients compared with angina patients and the control cohort. However, we observed that sP‑selectin levels did not change following PCI and thrombolysis therapy. In addition, there was no correlation between sP‑selectin levels and the severity of AMI in the cohort which received PCI or thrombolysis therapy. Therefore, we deduced that sP‑selectin only induced the onset of AMI but did not promote its severity. To confirm this hypothesis, a P‑selectin inhibitor was administered to an atherosclerosis formation model, plaque rapture model and neointimal hyperplasia model. We revealed that atherosclerotic plaque formation and rupture, neointimal formation and neointimal bleeding were suppressed by the sP‑selectin inhibitor. We concluded that sP‑selectin, induced by systemic inflammation in conditions including obesity and hyperlipidemia, promoted atherosclerotic plaque and neointimal formation, plaque rapture and neointimal bleeding, further leading to AMI. We also demonstrated that sP‑selectin had no effect on the severity of AMI.

Multimerization of Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1 Ly6 Domain

GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia.

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Bacterial meningitis is a serious central nervous system infection and Escherichia coli K1 (E. coli K1) is one of the leading etiological agents that cause meningitis in neonates. Outer membrane protein A (OmpA) of E. coli K1 is a major virulence factor in the pathogenesis of meningitis, and interacts with human brain microvascular endothelial cells (HBMEC) to cross the blood-brain barrier. Using site-directed mutagenesis, we demonstrate that two N-glycosylation sites (NG1 and NG2) in the extracellular domain of OmpA receptor, Ecgp96 are critical for bacterial binding to HBMEC. E. coli K1 invasion assays using CHO-Lec1 cells that express truncated N-glycans, and sequential digestion of HBMEC surface N-glycans using specific glycosidases showed that GlcNAc1-4GlcNAc epitopes are sufficient for OmpA interaction with HBMEC. Lack of NG1 and NG2 sites in Ecgp96 inhibits E. coli K1 OmpA induced F-actin polymerization, phosphorylation of protein kinase C-α, and disruption of transendothelial electrical resistance required for efficient invasion of E. coli K1 in HBMEC. Furthermore, the microvessels of cortex and hippocampus of the brain sections of E. coli K1 infected mice showed increased expression of glycosylated Ecgp96. Therefore, the interface of OmpA and GlcNAc1-4GlcNAc epitope interaction would be a target for preventative strategies against E. coli K1 meningitis.

Loss of CD34 Expression in Aging Human Choriocapillaris Endothelial Cells

Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD). In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40), age-matched controls (> age 60 without AMD), or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization). Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I) and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10−6). Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse) or a trend toward decreased CD34 (human) in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

Clinical efficacy of recombinant human soluble thrombomodulin in patients with septic disseminated intravascular coagulation

Thrombomodulin is an endothelial cell cofactor and glycoprotein for thrombin-catalyzed activation of protein C. A recombinant human soluble thrombomodulin (rhsTM) has been recently developed, and this new agent has a unique amino-terminal structure exhibiting anti-inflammatory activity including sequestraction and cleavage of high-mobility group box 1(HMGB-1).

Serum Thrombomodulin as a Newly Identified Biomarker for Postoperative Lung Injury: A Prospective Observational Study

Postoperative lung injury after lung cancer resection is still a difficult problem to be solved. Thrombomodulin (TM) is a membrane-bound glycoprotein of endothelial cells, and its serum level is elevated in patients with acute lung injury or acute respiratory distress syndrome. In fact, TM is abundant in the pulmonary capillary vessels. Lung resection reduces the volume of pulmonary capillary vessels; however, the change in the serum levels of TM after lung resection remains to be investigated. We therefore analyzed the postoperative changes in the serum TM levels in 60 patients who underwent thoracotomy without lung resection (n = 3), partial resection of the lung (n = 15), or lobectomy (n = 42). Preoperative and postoperative day-1 laboratory data including the serum levels of TM and KL-6, a sialylated carbohydrate antigen (a biomarker for pulmonary fibrosis), and oxygenation index were collected. Unexpectedly, the postoperative serum levels of TM were lower than preoperative values in lobectomy group, whereas they remained unchanged after thoracotomy without lung resection and after partial resection of the lung. In addition, the serum TM level decreased proportional to the resected lung volume. Eight out of 42 patients with lobectomy presented high postoperative serum levels of TM, and 3 out of these 8 patients presented postoperative impaired oxygenation. Postoperative impaired oxygenation occurred only in patients with elevated TM levels; namely, an increase of serum TM is associated with impaired oxygenation after lung resection. In conclusion, serum TM is a possible biomarker for predicting the occurrence of postoperative lung injury.

Rare but Relevant Kidney Disorders

The spectrum of predisposing factors to atypical hemolytic uremic syndrome (aHUS) is widening. This syndrome includes cases of HUS that are not linked to Shiga toxin–producing bacteria. Approximately half of the patients with aHUS have mutations in genes that regulate complement system: Either loss-of-function mutations in genes that encode inhibitors of the alternative pathway of the complement system (e.g., factor H or I, CFH or CFI, membrane co-factor protein, factor H–related proteins, C4b-binding protein) or gain-of-function mutations of genes that encode factor B and C3. Antibodies against CFH have been observed in 2 to 10% of patients. Thrombomodulin is a ubiquitous transmembrane endothelial cell glycoprotein that suppresses clot formation, inhibits fibrinolysis, interferes with inflammation, and inactivates some complement-derived anaphylatoxins. Delvaeye et al. studied 152 patients who had aHUS and were recruited from the International Registry of Recurrent and Familial HUS/Thrombotic Thrombocytopenic Purpura and 380 control subjects. They sequenced the entire coding region of the thrombomodulin gene (THBD), which lacks introns. Six heterozygous mutations of THBD were identified in seven (4.6%) unrelated patients. Two of them belonged to families with aHUS. In one of these families, four heterozygous carriers (including adults) were asymptomatic. As in complement defects, the THBD mutation is not sufficient by itself to cause HUS. Low serum C3 levels were measured in four patients. The disease was evident during childhood, from 6 mo to 15 yr of age. Three of seven patients progressed to ESRD. Four had recurrent aHUS. One patient developed a first episode of aHUS after kidney transplantation at 15 yr of age. Further studies will indicate whether there is a risk for recurrence of aHUS after kidney transplantation. In vitro studies on cultured cells showed that thrombomodulin provides protection against complement activation. In addition, thrombomodulin binds to C3 and CFH and negatively regulates complement by accelerating CFI-mediated inactivation of C3b in the presence of co-factors CFH and C4b. Cultured cells that express THBD variants that are associated with aHUS had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and thus were less protected from activated complement. In patients with aHUS, genetic testing should include several genes that encode proteins that regulate complement system, including thrombomodulin.

INFLUENCE OF VERTEPORFIN PHOTODYNAMIC THERAPY ON INFLAMMATION IN HUMAN CHOROIDAL NEOVASCULAR MEMBRANES SECONDARY TO AGE-RELATED MACULAR DEGENERATION

To examine the short- and long-term consequences of verteporfin photodynamic therapy (PDT) on inflammation with regard to infiltration of macrophages and leukocytes and expression of thy-1 in human choroidal neovascularization membranes (CNV) secondary to age-related macular degeneration (AMD). Retrospective review of an interventional case series of 43 patients who underwent removal of CNV. Twenty patients were treated with PDT 3 to 246 days preoperatively. Twenty-three CNV without previous treatment were used as control. CNV were stained for CD34, CD105, cytokeratin 18, Ki-67, thy-1, an endothelial cell glycoprotein known to be upregulated only by inflammatory cytokines, CD68 (macrophages), and CD45 (common leukocyte antigen). Specimens treated by PDT 3 days previously showed significantly reduced endothelial thy-1 expression (P = 0.008), leukocyte (P=0.04) and macrophage (P=0.0063) infiltration, and proliferative activity (P=0.02) compared to control CNV. Specimens at longer intervals after PDT, in contrast, disclosed a significantly increased expression of thy-1 (P=0.004), infiltration with leukocytes (P=0.044) and macrophages (P=0.01), and proliferative activity (P=0.03) compared to CNV excised 3 days after PDT. The rebound effect after PDT seems to be based on an inflammatory response that contributes to enhanced proliferation. These data support the need for an anti-inflammatory therapy as adjuvant to PDT.

Comparison of three commercially available thrombomodulin ELISA kits

Thrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of soluble serum thrombomodulin are regarded as parameters of disease activity in vasculitides and vasculopathies. However, the mean thrombomodulin values of different studies show marked concentration differences of the control values. The purpose of this study was to further investigate these differences. We examined 60 sera of patients with systemic lupus erythematosus (SLE) and 10 of healthy controls with three commercially available thrombomodulin ELISA kits for determination of their thrombomodulin concentration and correlation to disease activity. The disease activity of the SLE patients was determined with the SLAM-score. Raised thrombomodulin values were found in 58% (test A), 55% (test B) and 61.6% (test C). The thrombomodulin values significantly correlated with the SLE disease activity independently of the ELISA kit used (correlation coefficients: r=0.84 (test A), r=0.80 (test B), and r=0.65 (test C)). In addition, the correlation coefficients between the respective thrombomodulin values of the three tests were r=0.86 (test A to B), r=0.73 (test A to C) and r=0.79 (test B to C). However, significant differences between the results of the three ELISA kits were found between the detected thrombomodulin concentrations. The mean thrombomodulin concentrations of the controls were 25.6 ng/ml (test A), 3.53 ng/ml (test B), and 2.52 ng/ml (test C). Our results reveal that the soluble thrombomodulin values of all three commercially available ELISA kits significantly correlate with the disease activity of SLE patients. However, the results show significant differences in the determined thrombomodulin concentrations. A calibration would be required of the different ELISA kits in order to permit a direct comparison of the results of these thrombomodulin ELISAs. A general reference standard would be desirable for this calibration of all thrombomodulin ELISAs. However, this general reference standard has to be adapted to the distinct test conditions of all test kits as well as including all epitopes of thrombomodulin which are recognised by the different antibodies used in the respective test kits. At present, only ELISA kits from the same manufacturer should be used during a single study including any follow-up investigations.

Cloning and expression of the Escherichia coli K1 outer membrane protein A receptor, a gp96 homologue.

Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5' rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.

Kinetics of thrombomodulin release and endothelial cell injury by neutrophil-derived proteases and oxygen radicals.

Thrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of serum thrombomodulin are regarded as a parameter of activity in vasculitides. The latter are pathophysiologically determined by neutrophil-derived inflammation and endothelial cell injury caused by secretion of proteases and hydrogen peroxide. It was the objective of this study to determine whether thrombomodulin is only a late marker of advanced endothelial cell injury or whether it indicates also earlier stages of cell alterations. Over 24 hr endothelial cell cultures were incubated with hydrogen peroxide or the neutrophil proteases proteinase-3, elastase and cathepsin G. The time-dependent increase of thrombomodulin in the supernatant was determined by enzyme-linked immunosorbent assay and immunoblot. In addition the viability (eosin, tetrazolium dye assay), detachment (crystal-violet assay), and apoptosis (4',6-diamine-2'-phenylindole-dihydrochloride assay) of the respective endothelial cells were determined for adherent and non-adherent cells. A rapid thrombomodulin increase was found under all experimental conditions. The additional immunoblotting analysis showed the pattern of proteolytic cleavage caused by the protease reactivity. In case of hydrogen peroxide the thrombomodulin increase was closely correlated with the loss of cell viability and lysis. The incubation of endothelial cells with the different proteases resulted in a time-dependent detachment of primarily viable cells. In addition to cell necrosis apoptotic cell death was found in the subgroup of detached endothelial cells after prolonged incubation over 24 hr with proteinase-3 (23%), elastase (31%), and cathepsin G (19%). In contrast, still adhering cells did not show any signs of necrosis or apoptosis. In summary these studies confirm in vitro that soluble thrombomodulin is not only a parameter of advanced endothelial cell destruction itself but also in addition an early marker of initial endothelial cell membrane changes induced by neutrophil derived proteases and oxygen radicals.

Complexation of VEGF with bevacizumab decreases VEGF clearance in rats.

Angiogenesis plays a critical role in several physiologic and pathologic processes, particularly in tumor growth, invasion, and metastasis (1). New blood vessels provide tumors with nutrients necessary for growth and also remove metabolic waste from the tumor (2). Several growth factors have been implicated in tumor angiogenesis, and one such factor is vascular endothelial growth factor (VEGF), which is a selective mitogen for endothelial cells. VEGF, a 43to 46-kD glycoprotein, induces proliferation and migration of vascular endothelial cells and functions as a vascular permeability factor through two receptors: flt-1 and KDR (3). There are also several isoforms of VEGF (VEGF206, VEGF189, VEGF165, VEGF121, and VEGF110), and in humans, recombinant humanized VEGF165 (rhVEGF) displays nonlinear pharmacokinetics, which is attributed to binding of the drug to endothelial cells (4). In numerous preclinical animal models, administration of an antibody-targeting VEGF has been found to be a potent suppressor of tumor growth and is being considered as a potential, novel anticancer therapy (5). Bevacizumab (AvastinTM, rhuMAb VEGF) is a recombinant humanized monoclonal antibody that binds all isoforms of VEGF and inhibits binding of VEGF to its receptors. The antibody was engineered by combining VEGF-binding residues from a murine-neutralizing antibody with the framework of a human immunoglobulin G (IgG1) (6). Bevacizumab is believed to be cleared through the FcRn system, a MHC class I-related receptor that has been shown to protect circulating IgG1s from catabolism and thereby contribute to the long terminal half-life of antibodies (7). Bevacizumab binds to primate VEGF and to rabbit VEGF (with lower affinity) but does not bind to rodent VEGF (8). The monoclonal antibody has been shown to inhibit tumor growth in a dosedependent manner in various animal models (9). Several clinical studies have also been conducted to characterize the pharmacokinetics, safety, and efficacy of bevacizumab in cancer subjects. In two phase II studies in cancer subjects, bevacizumab, in combination with 5-fluorouracil/leucovorin or carboplatin/paclitaxel, has been shown to be safe and has inhibited tumor growth (10,11). Results from clinical studies have also shown a rise in serum concentrations of endogenous VEGF over baseline after single and multiple intravenous (IV) administration(s) of bevacizumab at doses >1 mg/kg (12). This rise in concentrations was ∼3to 4-fold above baseline and seemed to return to baseline as the antibody cleared systemically. An increase in VEGF synthesis/distribution and/or decrease in VEGF clearance upon complexation with bevacizumab are possible causes for this phenomenon. The latter hypothesis was explored in a pharmacokinetic study conducted in rats where recombinant humanized VEGF165 (rhVEGF) was administered intravenously in the presence or absence of bevacizumab. Rats were selected because (i) bevacizumab does not bind rat VEGF, therefore diminishing any competition for binding of bevacizumab to the administered rhVEGF and (ii) serial sampling was possible in the same animal for measurement of drug concentrations. Results presented here confirm that complexation of VEGF with bevacizumab decreases the clearance of circulating VEGF.

The major surface-labeled α-galactosylated glycoprotein of pig endothelial cells is the β1 integrin subunit

The major surface-labeled α-galactosylated glycoprotein of pig endothelial cells is the β1 integrin subunit

Thrombomodulin with the Asp468Tyr mutation is expressed on the cell surface with normal cofactor activity for protein C activation.

Thrombomodulin (TM) is an endothelial cell glycoprotein that acts as an anticoagulant. Mutation in the TM gene is a potential risk factor for thrombosis. The first TM mutation identified was a heterozygous substitution of T for G at nucleotide position 1456, which predicted Asp468 with Tyr in a Ser/Thr-rich domain. To evaluate the reported TM gene mutation as a possible cause of thrombosis, we transiently tranfected a vector for TM gene carrying the mutation to mammalian COS7 cells. TM antigen levels in lysates of cells transfected with variant TM were comparable to those in preparations of normal TM. The TM cofactor activity for protein C (PC) activation on the variant TM-expressing cells was similar to that of the control. The Michaelis constant Km and Vmax. of variant TM for PC activation were shown to be similar compared to those of normal TM. The affinity of each TM for thrombin in PC activation was also similar. We obtained several stable cell lines expressing normal and variant TM. Lysate of the cell lines with normal and variant TM genes had a similar expression level of TM antigen. Pulse-chase analysis showed that normal and variant TM were glycosylated and resistant to endoglycosidase H, indicating that the variant TM was expressed on the cell surface in a mature form. Variant TM protein is apparently expressed on the cell surface with normal cofactor activity for PC activation. It is unlikely that the TM variant directly causes thrombosis by mechanism of reduced expression or impaired cofactor activity for PC activation, which comprises a major anticoagulant activity of TM.

Increased Serum Levels and Sinusoidal Expression of Thrombomodulin in Acute Liver Damage

Thrombomodulin (TM) is a surface glycoprotein of endothelial cells involved in both anticoagulation and antifibrinolysis. In this study, we assessed the clinical significance of TM in acute liver damage by using a rat model induced by intraperitoneal injection of D-galactosamine (Gal-N). Serum TM levels were measured with enzyme immunoassay utilizing rabbit anti-rat TM antibody. Simultaneously, immunohistochemical examination was performed using the same antibody. Serum TM levels increased significantly after the injection of Gal-N compared with preinjection levels, peaking from 48 to 72 hours after injection and normalizing by 168 hours. Changes in parenchymal damage were synchronized with changes of TM, and changes of TM levels mirrored changes of liver weight. In immunohistochemical examination, TM immunoreactivity was observed only on the endothelial surfaces of both the artery and portal vein within Glisson’s sheath in controls. After injection of Gal-N, TM immunoreactivity was gradually intensified, especially around the necrotic area and the central veins. These findings disappeared with improvement of parenchymal damage. Both the increase of serum TM levels and intensified TM immunoreactivity in the liver were synchronized with acute liver parenchymal damage induced by Gal-N. These findings on TM are related to endothelial damage with parenchymal necrosis and liver regeneration interacting with both homeostasis of microcirculation and healing of parenchymal damage.

The Shape of Thrombomodulin and Interactions with Thrombin as Determined by Electron Microscopy

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of SolulinTM, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human alpha-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.

Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer.

Cell surface antigens of transformed cells are the traditional targets for antibody-guided detection and therapy of solid neoplasms. Alternative targets may be found in the tumor stroma, which contains newly formed blood vessels, reactive fibroblasts, and extracellular matrix proteins. The F19 cell surface glycoprotein of reactive fibroblasts is a prototypic stromal antigen since it is found in the stroma of > 90% of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors. In the present study, we define an additional tumor stromal antigen, FB5, that is selectively expressed in vascular endothelial cells of malignant tumors. Immunohistochemical analysis of 128 tumors identified FB5 in endothelial cells in 67% of the samples (including 41 of 61 sarcomas, 26 of 37 carcinomas, and 18 of 25 neuroectodermal tumors) whereas normal blood vessels and other adult tissues tested lacked FB5 expression. In vitro studies showed that FB5 is a M(r) 165,000 cell surface glycoprotein, comprised of a M(r) 95,000 core polypeptide and highly sialyated O-linked oligosaccharides but few if any N-linked sugars, and that the FB5 gene is located on chromosome 11q13-qter. The restricted tissue distribution of the FB5 protein, which we refer to as endosialin, suggests strategies for tumor imaging and therapy that are aimed primarily at the tumor vasculature.