The contact of fibrin with the apical surface of human umbilical vein endothelial cells (HUVEC) can induce capillary tube formation via the interaction of fibrin beta15-42 with a putative cell receptor (Chalupowicz, D. G., Chowdhury, Z. A., Bach, T. L., Barsigian, C., and Martinez, J. (1995) J. Cell Biol. 130, 207-215). To characterize this interaction, we studied the binding of the thrombin-cleaved N-terminal disulfide knot of fibrin (NDSK II), a dimeric fragment with exposed beta15-42, to HUVEC in three separate assay systems. Time-course binding of 125I-NDSK II to HUVEC monolayers or suspensions revealed that binding was specific at 50-60%, as determined by the addition of unlabeled NDSK II. Specific binding of 125I-NDSK II to HUVEC was 70% reversible by dilution or by competition, and was found to be divalent cation-independent. Binding plateaued after 10 min at a saturation of 15-20 nM. Scatchard analysis using the LIGAND computer program defined a single population of receptors with a KD of 7.7 +/- 1.6 nM and approximately 21,000 +/- 7000 binding sites/cell. N-terminal disulfide knot derivatives in which beta15-42 was absent (NDSK 325) or unexposed (NDSK, NDSK I) did not show specific binding. Specific binding of 125I-NDSK II could not be inhibited by RGDS or by antibodies to the alphavbeta3 or beta1 integrins, PECAM-1, ICAM-1, or N-cadherin. In contrast, a synthetic beta15-42/ovalbumin conjugate inhibited total 125I-NDSK II binding by 47 +/- 19% (corresponding to 95% of specific 125I-NDSK II bound) and a monoclonal antibody to vascular endothelial cadherin (VE-cadherin) inhibited binding by 35 +/- 8% (corresponding to 70% of specific 125I-NDSK II bound). Another assay was based on the capture of cadherins from HUVEC lysates by a polyclonal pan-cadherin antibody immobilized on plastic dishes. Binding of NDSK II to the captured cadherins was 89 +/- 5% specific, while specific binding of NDSK 325 and NDSK was negligible. An immortalized line of human adipose-derived microvascular endothelial cells, which express N-cadherin but not VE-cadherin, demonstrated no specific binding of NDSK II by the capture assay. These data define a novel interaction of fibrin with VE-cadherin, which is mediated by the fibrin N-terminal beta15-42 sequence, and may contribute to the mechanism through which fibrin induces angiogenesis.

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the αvβ3integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.

The subcellular distribution of tissue transglutaminase in human umbilical vein endothelial cells and human arterial and venous smooth muscle cells was examined. Double-immunofluorescence staining of smooth muscle cells and endothelial cells with anti-transglutaminase antisera and rhodamine-tagged phalloidin revealed codistribution of transglutaminase with the stress fibers, with endothelial cells also containing a cytoplasmic pool. This pattern of distribution was confirmed by confocal microscopy. Immunoprecipitation experiments demonstrated that transglutaminase co-immunoprecipitated with myosin in high-molecular-weight complexes, but not with actin, suggesting that the association of transglutaminase with the stress fibers was due to its cross-linking to myosin. About 97% of endothelial cell transglutaminase activity was present in the cytosolic fraction and 3% in the particulate fraction. The detergent-insoluble fraction was practically devoid of activity as measured by the putrescine assay, but was active as evidenced by the covalent cross-linking of 125I-fibronectin. Western blotting with a polyclonal rabbit antiserum raised against human erythrocyte transglutaminase detected high levels of enzyme in endothelial cell cytosol and both detergent-soluble and detergent-insoluble membrane fractions. In contrast, smooth muscle cells contained much less cytosolic transglutaminase, as determined either functionally or antigenically. Furthermore, within the particulate fraction of the smooth muscle cells, most of the enzyme was located in the detergent-insoluble fraction, as assessed by Western blot analysis. Retinoic acid increased the levels of enzyme in the cytosol of all cell types and the increases were correlated with increases in mRNA. Thus, tissue transglutaminase is present in various particulate fractions of vascular smooth muscle cells and endothelial cells and may be present in this cellular fraction by virtue of autocross-linking of the enzyme itself to stress fiber-associated myosin.

We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2-terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.

We studied the interaction of [125I]fibronectin with human umbilical vein endothelial cells. Endothelial cell monolayers cross-linked [125I]fibronectin which had been preadsorbed to gelatin-coated dishes. The cross-linking of the substrate-immobilized [125I]fibronectin was mediated by cell-associated tissue transglutaminase and occurred more rapidly during the first 30 min after endothelial cell seeding but also continued for several hours after the cells were fully spread. The processing of the [125I]fibronectin was associated with the basolateral surface of the endothelial cell, as demonstrated by the finding that cross-linking did not occur when [125I]fibronectin was presented to the apical surface of confluent monolayers. Transglutaminase activity was not necessary for attachment and spreading of HUVEC on a fibronectin/gelatin matrix. The presence of a nonpeptidyl transglutaminase inactivator rendered the cells more susceptible to detachment by trypsin and destabilized the association of fibronectin with the subendothelial extracellular matrix. Thus, endothelial cells process fibronectin into cross-linked multimers due to the expression of tissue transglutaminase at the basal surface of the cell. This process may serve to stabilize the extracellular matrix and to firmly anchor the cells to the basement membrane.

Rabbit hepatocyte surface-expressed tissue (type II) transglutaminase is shown to act as a binding site for fibrinogen or fibronectin and to covalently incorporate these glycoproteins, in addition to itself, into extracellular high molecular weight complexes. This concept is supported by the observation that a nonpeptidyl, active site-directed transglutaminase inactivator (L683685) elicited concentration-dependent (0.1-10 microM) decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen, 125I-fibronectin, or [14C]putrescine by hepatocyte suspensions. In corroboration with these findings, an antiserum against rabbit liver transglutaminase, which did not cross-react with rabbit factor XIII, elicited concentration-dependent decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen or [14C]putrescine by hepatocyte suspensions. Western blots of sodium dodecyl sulfate/Triton-insoluble hepatocyte fractions conducted with this antiserum, with a polyclonal antiserum against human erythrocyte transglutaminase, or with a monoclonal antibody (CUB-7401) against guinea pig liver transglutaminase detected the 80-kDa tissue transglutaminase, as well as tissue transglutaminase-immunoreactive bands of higher molecular mass (range of 90 to greater than 200 kDa). The higher molecular weight species were preferentially incorporated, in a time- and calcium-dependent manner, into very high molecular weight complexes which did not enter the stacking gel. Incorporation of these tissue transglutaminase-containing bands into the high molecular weight complexes was inhibited by L683685, indicating that cross-linking by the enzyme was responsible for the assembly of the complexes of which tissue transglutaminase was itself a component. Cellular integrins did not mediate ligand binding under the experimental conditions, as evidenced by the failure of the Arg-Gly-Asp-Ser tetrapeptide or anti-integrin antibodies to inhibit binding or cross-linking of 125I-fibrinogen or 125I-fibronectin, in the presence or absence of transglutaminase inactivators.

Extracellular sodium is known to influence secretion by certain secretory cells, possibly by mobilizing calcium from cellular stores or by altering intracellular pH via regulation of a Na(+)-H+ antiport system. Using canine tracheal explants, we determined whether agents which alter sodium fluxes are capable of modulating basal or cholinergically-induced secretion of mucus glycoconjugates. Methacholine, a cholinergic agonist, increased mucus secretion from explants incubated in the presence or absence of calcium, but had no effect on secretion when incubated in sodium-deficient media, indicating (a) that cholinergically-induced secretion can be mediated by mobilization of cellular calcium and (b) that extracellular sodium was required for this stimulatory effect. Several agents which increase intracellular sodium were tested for their effect on mucus secretion. Ouabain, a sodium pump inhibitor, and veratridine, a sodium channel activator, did not significantly affect control or methacholine-induced secretion; gramicidin, a sodium ionophore, also had no effect on basal release. Tetrodotoxin, a sodium channel inhibitor, was also without effect on basal or methacholine-stimulated mucus release. Agents which alter intracellular pH were also examined for their effects on basal or methacholine-induced glycoconjugate secretion. Amiloride, which decreases intracellular pH by inhibiting Na(+)-H+ exchange, produced a 19 per cent increase in basal secretion (not statistically significant), but had no effect on methacholine-induced secretion. An agent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which decreases intracellular pH by inhibiting HCO3(-)-Cl- exchange, elicited decreases in both basal and methacholine-induced secretion, but the inhibition did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

In contrast to most plasma glycoproteins, asialofibrinogen and aglycofibrinogen manifest relatively normal in vivo survivals, suggesting that molecular determinants other than the carbohydrate may be involved in their catabolism. To analyse the metabolic properties of fibrinogen further, we analysed the interaction of normal fibrinogen with suspensions of rabbit hepatocytes or human umbilical vein endothelial cells. In so doing, we identified a novel type of fibrinogen - cell interaction which has the distinguishing characteristic of involving covalent transglutaminase-mediated fibrinogen cross-linking. In this report, we highlight some of the major distinguishing characteristics of this novel type of transglutaminase-mediated fibrinogen - cell interaction.

The interaction of endothelial cells with soluble or substrate-immobilized 125I-labeled fibrinogen (125I-FGN) was analyzed. Binding experiments involved incubation of 125I-FGN with cell suspensions at 4 degrees C. Bound ligand was quantitated by centrifugation of cells through silicone oil followed by scintillation analysis of the cell pellet. Calcium-dependent binding of 125I-FGN reached a maximum after 3 h and represented about 60% of the total. Half-maximal saturation occurred at 60 nM, and about 9 x 10(4) molecules were bound/cell at saturation (approximately 100 nM). Calcium-dependent binding was completely inhibited by unlabeled fibrinogen, partially inhibited by a monoclonal antibody (7E3) against glycoprotein IIb-IIIa, but not inhibited by fibrinogen fragments D or E, an anti-glycoprotein IIIa polyclonal antibody, or the Arg-Gly-Asp-Ser tetrapeptide. In contrast, the Arg-Gly-Asp-Ser tetrapeptide as well as the monoclonal antibody 7E3 markedly inhibited attachment of endothelial cells to substrate-immobilized fibrinogen, whereas fragment D or E did not. Both in suspension and monolayer, the 125I-FGN underwent cross-linking involving principally the A alpha chain. The transglutaminase inhibitors putrescine, histamine, and cystamine interfered with 125I-FGN binding and cross-linking by suspended cells. Since cross-linking in suspension was limited to bound 125I-FGN and since transglutaminase activity was not detectable in the binding buffer, cross-linking may have been mediated by a cell-associated transglutaminase.

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.

Fibronectin purified from rabbit plasma was radioiodinated, and its interaction with rabbit hepatocytes in suspension was studied. Iodinated fibronectin interacted in a time-dependent fashion reaching plateau at 3 h. The interaction was greater in the presence of calcium than in the presence of magnesium or EDTA. Saturation occurred at about 140 nM fibronectin with about 1,400,000 molecules bound per cell. The interaction could be inhibited by unlabeled fibronectin or fibrinogen but not by the tetrapeptide Arg-Gly-Asp-Ser or by albumin, transferrin, or fetuin. About 50% of the bound iodinated fibronectin was incorporated, in a calcium-dependent fashion, into cross-linked high molecular weight complexes at the cell surface through a mechanism consistent with a cellular transglutaminase-mediated reaction. Iodinated fibronectin which could be displaced from the cell was monomeric in nature, while the cell-associated material remained in high molecular weight complexes. The role of the interaction is currently under investigation, but it is possible that the binding may promote cellular adhesion or facilitate intercellular interaction.

ZusammenfassungZytostatika werden sehr viel in der Behandlung von Malignomen verwendet. Blutungskomplikationen und thrombotische Ereignisse treten bei Patienten mit Karzinomen entweder durch die Krebserkrankung selbst oder aufgrund der Behandlung der Krebserkrankung auf. Die Mechanismen für die thrombotischen und hämorrhagischen Syndrome sind bisher noch unzureichend untersucht. Diese Arbeit gibt eine Übersicht der wesentlichen thrombotischen und hämorrhagischen Zustände, die sich aus der Anwendung antineoplastischer Substanzen ergeben, und versucht eine Darstellung des gegenwärtigen Wissensstandes der Pathophysiologie dieser Zustände.

We describe a specific fibrinogen-hepatocyte interaction. Rabbit 125I-labeled fibrinogen (125I-FGN) was incubated at 4 degrees C with suspensions of rabbit hepatocytes (approximately 1 X 10(6) cells/ml). Bound ligand was separated from free by centrifugation of cells through oil and quantitated by gamma-scintillation counting. Specific binding, determined by subtraction of nonspecific binding in the presence of 8 mM EDTA from total binding in the presence of 2 mM CaCl2, required 3 h to plateau and represented approximately 70% of total binding. Specific binding was calcium-dependent and was negligible in buffer containing 2 mM MgCl2. Half-maximal saturation occurred at approximately 30 nM 125I-FGN with approximately 480,000 molecules/cell at saturation. Dilution experiments revealed comparable affinities for labeled and unlabeled fibrinogen. Total binding was irreversible as determined by addition of excess unlabeled fibrinogen or EDTA. Specific binding of 25 nM 125I-FGN was inhibited, in a concentration-dependent fashion, by unlabeled fibrinogen or fibrinogen fragment D95 (Mr = 95,000), but not by fibrinogen fragment E or Arg-Gly-Asp-containing peptides. Unlabeled fibrinogen (3.1 microM) completely abolished specific binding, whereas greater than 80% inhibition was achieved with 10 microM fragment D95. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of 125I-FGN bound in the presence of calcium demonstrated disappearance of A alpha chains with formation of products of Mr greater than 200,000; EDTA or unlabeled fibrinogen prevented fibrinogen processing. These data describe a unique fibrinogen-hepatocyte interaction which differs considerably from the platelet-fibrinogen interaction, especially with regard to the processing of the fibrinogen molecule.

Glycoproteins are proteins with covalently attached carbohydrate which is enzymatically added during the biosynthesis of the polypeptide. Attachment of carbohydrate to the polypeptide may involve either an N-glycosidic or an O-glycosidic bond. Most of the plasma glycoproteins have N-linked oligosaccharides attached to appropriate asparagine moieties of the peptide core. The biosynthesis of the N-linked glycoproteins proceeds through three sequential phases which include (a) dolichol-mediated oligosaccharide assembly, (b) oligosaccharide linkage to the polypeptide, and (c) oligosaccharide processing with the addition of peripheral sugars. Much of the diversity in carbohydrate structure of plasma glycoproteins results from variations in processing and addition of peripheral sugars. The terminal monosaccharide of N-linked plasma glycoproteins is usually sialic acid, a negatively charged sugar which markedly affects the physicochemical and biologic characteristics of glycoproteins. Liver disease induces alterations in both the level and structure of many plasma glycoproteins, particularly those which are synthesized by the hepatocyte. Quantitative changes in the concentration of plasma glycoproteins are relatively easy to assess due to the availability of many types of clinical assays. Furthermore, quantitation of the level of a particular glycoprotein in the plasma often provides clinically useful information pertinent to diagnosis and progression of disease. The determination of functional abnormalities of glycoproteins and the correlation of these functional changes with potential alterations in carbohydrate structure is more difficult, however, due to the need for accurate functional assays and sophisticated methods for analysis of the carbohydrate structure of the abnormal molecules, which are themselves often scarce. Liver disease-induced alterations in the oligosaccharide moieties of plasma glycoproteins involve mainly hyperglycosylation or hypoglycosylation defects. These changes can often be traced to increases or decreases in the amount of sialic acid present within the abnormal structure. Since sialic acid plays such a key role in the biology of glycoproteins, changes in the amount of this monosaccharide frequently result in marked alterations in the physicochemical and biologic behavior of the abnormal molecule. The exact mechanisms responsible for the changes in sialic acid are not entirely known, since the complete oligosaccharide structure of any of the abnormal glycoproteins of liver disease has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)