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Synthesis of heparan sulfate-free chains by human keratinocytes is upregulated during terminal differentiation. The cellular location of this product class and the significance of the differentiation effect are unknown. Differential plasma membrane shearing with cationized colloidal silica was used to evaluate the compartmentalization of the heparan and chondroitin sulfate free chains and their respective proteoglycans in 3T3 fibroblasts and human keratinocytes. The method exploits the topologic segregation of plasma membranes of adherent cells into ventral, dorsal, and intracellular domains and the selective binding of the silica to the dorsal membranes, which by shearing can be separated from ventral membranes adherent to the substratum. Analysis of membrane preparations from sheared cells that had been prelabeled with [35S]-sulfate revealed the proteoglycans to be predominantly ventral, at which location a matrix binding function could be accommodated. Proteoglycans were also recovered from dorsal and intracellular membranes, suggesting active trafficking between intra- and extra-cellular sites. In contrast, the major fraction of heparan and chondroitin sulfate free chains was either cytosolic or associated with intracellular membranes, with the remaining approximately 20% segregated to dorsal and ventral membranes. These results suggest different cellular functions for the proteoglycans and glycosaminoglycan free chains. The partial localization of the free chains to peripheral membranes is compatible with our prior hypothesis that they arise by processing of precursor proteoglycans on cell surfaces. Following this origin, the free glycosaminoglycan polymers could be available to bind ligands such as cytokines prior to transport to intracellular sites of action.

Diabetes is accompanied by impaired platelet function and accelerated vascular disease. To find out whether a correlation exists between these two complications, and if modifications occurring in diabetic platelets influence their relationship with endothelium, we have studied the interaction between platelets isolated from plasma of diabetic patients and bovine valvular endothelial cells (VEC), in culture. For quantitative analysis, normal and diabetic [3H]-adenine-labeled platelets were incubated with confluent VEC grown in Dulbecco's modified Eagle medium, containing 4.5 g/l glucose, for 30 min at 37 degrees C. After extensive washing and solubilization of the monolayer, the calculated adhesion index showed a two-fold increased adherence of diabetic platelets to VEC as compared to normal platelets. Statistical analysis (by Pitman randomization test) indicated that the adhesion was significantly higher (p = 0.0003) than that of normal platelets to VEC. To partially identify the membrane components implicated in the adhesion process, either platelets or VEC were treated with neuraminidase, trypsin or heparinase prior to the adhesion assay. Trypsin or neuraminidase treatment of platelets significantly diminished their adherence to VEC, suggesting a role of platelets sialylated glycoproteins in the adhesion process. Neuraminidase or heparinase treatment of VEC increased the adhesion of both normal and diabetic platelets, indicating that the cell membrane sialyl residues and heparan sulfate participate in the normal thromboresistant properties of VEC. Transmission and scanning electron microscopy revealed a close apposition between platelets and VEC with the formation of an adhesion plaque, characterized by fine fibrillar bridges between the plasma membranes of the two cells.(ABSTRACT TRUNCATED AT 250 WORDS)

FN-C/H II is a heparin binding synthetic peptide from the C-terminal cell and heparin binding domain of fibronectin (FN) that mediates neuronal cell adhesion, spreading, and neurite outgrowth. Cellular interactions with FN-C/H II are inhibited by soluble heparin, suggesting that a cell-surface proteoglycan may mediate interactions with FN-C/H II (Haugen et al., 1990). To test this hypothesis further, heparan sulfate (HS) or chondroitin sulfate (CS) was removed from the cell surface by enzyme treatment. Heparitinase but not chondroitinase treatment of cells inhibited rat B104 neuroblastoma cell adhesion and spreading on FN-C/H II. Additionally, heparitinase treatment decreased the spreading of cells on the 33/66 kDa fragments containing the C-terminal heparin binding domain of FN. Furthermore, antibodies generated against a mouse melanoma HS proteoglycan (HSPG) inhibited B104 cell adhesion to FN-C/H II and the 33/66 kDa FN fragments. 35S-HSPG isolated from B104 cells directly bound to FN-C/H II both in solid phase assays and by affinity chromatography, but failed to bind to a control peptide from this region, CS1. The binding of 35S-HSPG was predominantly mediated by the HS and not the core protein of the HSPG. SDS-PAGE of iodinated HSPG demonstrated a single 78 kDa core protein following heparitinase digestion, which migrated at 51 kDa under nonreducing conditions. Anti-HSPG antibodies recognized the 78 kDa core protein by immunoblotting, and stained the surface of rat B104 neuroblastoma cells and cells of the primary neonatal rat nervous system. These results identify a cell-surface HSPG that likely mediates neuronal cell binding interactions with FN-C/H II.

The amyloid precursor protein (APP) is a glycoprotein consisting of at least four isoforms derived from a single gene by a process of alternative splicing. The membrane-bound forms of APP have been suggested to have adhesive properties and to mediate neural cell adhesion. Previous studies have demonstrated the ability of Fab' fragments of antibodies to extracellular domains of APP to inhibit neural cell binding to a collagen substrate, suggesting a physiological role for the collagen-binding properties of APP. The binding of APP has been demonstrated to be specific for type IV collagen, and no binding to other extracellular matrix components, including fibronectin and laminin, was detected. The APP-collagen binding appeared to be mediated by a heparin-bridge mechanism, since the binding was abolished by the addition of excess heparan or heparinase. These results were observed by both a homogenate-collagen binding assay and a cell-surface adhesion assay, thus providing further evidence for the adhesion role of APP. They also pose the question of the possible role of the heparin-binding properties of APP in the genesis of the neuritic plaques characteristic of Alzheimer's disease.

The binding of heparin-binding EGF-like growth factor (HB-EGF) to the epidermal growth factor (EGF) receptor of human endometrial carcinoma cells was compared to that of EGF using an 125I-EGF radioreceptor assay. The inhibitory effect of HB-EGF on 125I-EGF binding was reversed either in the presence of heparin (but not by chondroitin sulfate) or by pre-treating the cells with heparinase. These treatments did not affect the binding of EGF to its receptor. To map potential regions in the HB-EGF molecule that mediate its heparin-dependent interaction with the EGF receptor, HB-EGF peptides were synthesized that were non-homologous to EGF. Accordingly residues 20-25 and 36-41, but not residues 8-19, of HB-EGF were found to be (i) heparin-binding and (ii) modulators of HB-EGF (but not of EGF) binding to the EGF receptor.

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage-mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships.

The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.

A protein kinase capable of phosphorylating basic fibroblast growth factor (FGF) can be localized on the outer cell surface of human hepatoma cells (SK-Hep cells). The addition of [gamma-32P]ATP, but not H3(32)PO4, results in a rapid (less than 10 min) incorporation of 32P into exogenously added basic FGF. The reaction is time and concentration dependent (apparent Km, 170 nM) and is stimulated by the addition of cAMP (EC50, 0.5 microM), but not the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. There is also no tyrosine protein kinase detected on the cell surface. The inhibition of basic FGF binding to its low and/or high affinity sites decreases the phosphorylation of basic FGF by the ecto-protein kinase. Accordingly, pretreatment of cells with heparinase for 30 min or coincubation with heparin (0.1-10 micrograms/ml) decreases phosphorylation in a dose-dependent manner. Furthermore, the addition of a nonphosphorylatable peptide analog of basic FGF ([Val112] basic FGF-(106-146)NH2) that can compete with basic FGF binding to cells prevents the phosphorylation of basic FGF. Together, these observations suggest that 1) exogenous basic FGF must associate with its low and/or high affinity binding sites to be phosphorylated, and 2) the kinase is cAMP dependent and associated with the outer cell surface, and support the hypothesis that phosphorylation may regulate the activity and/or bioavailability of the growth factor.

Basic fibroblast growth factor (bFGF) binds to heparan sulfate proteoglycans at the cell surface and to receptors with tyrosine kinase activity. Prevention of binding between cell surface heparan sulfate and bFGF (i) substantially reduces binding of fibroblast growth factor to its cell-surface receptors, (ii) blocks the ability of bFGF to support the growth of Swiss 3T3 fibroblasts, and (iii) induces terminal differentiation of MM14 skeletal muscle cells, which is normally repressed by fibroblast growth factor. These results indicate that cell surface heparan sulfate is directly involved in bFGF cell signaling.

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.

The properties of two isoforms, alpha and beta, of rabbit antithrombin III (ATIII) were compared in the presence of undamaged or de-endothelialized rabbit aortic wall. Similar quantities of ATIII-alpha and ATIII-beta bound to and rapidly saturated the endothelium in vitro, but the rate of transendothelial passage of ATIII-beta exceeded that of ATIII-alpha by 22%. Furthermore, ATIII-beta was adsorbed approximately twice as rapidly as ATIII-alpha by the subendothelium of the de-endothelialized aorta. Binding of both isoforms was decreased (ATIII-beta more than ATIII-alpha) by pretreating the subendothelial surface with heparitinase. Also, subendothelium-bound ATIII-beta was desorbed more readily than bound ATIII-alpha by thrombin. In vivo, the rate of uptake of iodine-131-labeled ATIII-beta from the circulation by the aortic wall and the major organs was 30-50% faster than that of iodine-125-labeled ATIII-alpha. In contrast, the uptake of 131I-ATIII-beta by the de-endothelialized aorta in vivo was three times faster than that of 125I-ATIII-alpha. By these criteria, ATIII-beta is the more active of the two isoforms. We surmise that plasma and, consequently, vessel wall levels of ATIII-beta may be vital for controlling thrombogenic events caused by injury to the vascular wall.

The gIII glycoprotein of bovid herpesvirus 1 (BHV-1) has been shown to mediate the adsorption of the virions to cells (K. Okazaki, E. Honda, T. Minetoma, and T. Kumagai, 1987, Arch. Virol. 97, 297-307). In this study, the cellular receptor for BHV-1 was investigated. Addition of heparin to the virus inoculum and treatment of the cells with heparinase prevented the virus from adsorbing to and infecting the cells. Of the major glycoproteins of BHV-1 only gIII was found to bind specifically to heparin. The binding of gIII was inhibited by a monoclonal antibody against antigenic site Ia, which interferes with the adsorption of the virus. These findings indicate that the virus adsorption to cells is mediated by interaction of the gIII antigenic site Ia with a heparinlike moiety on the cell surface, which serves as a receptor for BHV-1.

Recently it was demonstrated that growth factors are bound to the extracellular matrix, and can regulate cell behavior. Using three different types of binding assays, we have examined the interaction of interferon-gamma with a basement membrane produced by the Engelbreth-Holm-Swarm tumor. Basement membrane was found to bind interferon-gamma in both a time- and concentration-dependent manner. Equilibrium binding analysis revealed a high-affinity site with a dissociation constant of 1.5 10(-9) M and a maximum binding capacity of 1.6 10(9) sites/mm2 of basement membrane. Competition studies show that the binding is inhibited by heparan sulfate, suggesting that basement membrane-heparan sulfate proteoglycan could be the binding site. This interaction was clearly confirmed by native polyacrylamide gel electrophoresis and dot-blot analysis with purified basement membrane molecules. Furthermore, the carboxy-terminal part of the interferon-gamma molecule contains an amino acid cluster, very closely related to a consensus sequence, present in more than 20 proteins known to bind sulfated glycosaminoglycans such as heparin. These data demonstrate a possible role of extracellular matrix components in storing cytokines and in modulating the cellular response to such factors.

The study of factors affecting phenotypic change and growth of aortic smooth muscle cells (SMC) typically involves either the isolation of SMC by enzymatic dissociation or observation of outgrowth of cells from primary explants of vascular tissue. Explants provide a system in which the growth of cells can be investigated without dissociating them totally from their normal environment and avoids some of the problems of variability associated with enzymatic digestion. We describe here a standardised method for the preparation of medial explants of arterial tissue using a McIlwain tissue chopper, which is both fast and reproducible. Measurement was made of the percentage of explants showing outgrowth and of the distance migrated by cells at various times after plating explants singly into wells of a 96-well plate. Using this method, by 12 days after explanting, more than 95% of explants from normal rabbit aorta had shown outgrowth, in contrast to only 50% of explants prepared using a scalpel blade. Explants from atherosclerotic rabbit aorta showed a shorter lag phase before outgrowth commenced than explants from normal rabbit aorta of a similar age, but the subsequent rate of growth was the same. In contrast, when explants of normal rabbit aorta were grown in hyperlipidic rabbit serum, the lag phase was the same as for normal serum, but the subsequent rate of growth was greater. Explants from normal rabbit aorta treated with heparin showed an increased lag phase but reduced rate of growth. Treatment with heparinase decreased the lag phase and increased the rate of growth as did elastase.

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

Recombinant hirudin (r-hirudin) is currently under development as an anticoagulant for use in surgery, therapeutic anticoagulation, disseminated intravascular coagulation and other pathologic states involving the generation of thrombin. Circulating levels of r-hirudin as an antithrombotic agent range from 2 to 20 micrograms/ml (0.1-1.0 mg/kg) as determined in an animal model of stasis thrombosis. In order to establish a relationship between the r-hirudin circulating level and bleeding, we utilized a rabbit ear blood loss model. r-Hirudin did not produce any loss of blood at dosages up to 20 micrograms/ml i.v. (1.0 mg/kg). When the circulating levels were maintained at 20 micrograms/ml for periods of up to 3 h, no increase in blood loss was observed. At 50 and 100 micrograms/ml initial circulating levels (2.5 and 5.0 mg/kg) a dose-dependent increase in the blood loss was observed which was equivalent to that observed with 1.25 and 2.5 mg/kg i.v. heparin. Such levels of r-hirudin are not expected in clinical usage. In contrast to heparin, the anticoagulant actions of r-hirudin were not neutralized by protamine sulfate, platelet factor 4, other polycationic agents and heparinase. In our studies, the blood loss induced by greater than 2.0 mg/kg i.v. dosages of r-hirudin in an animal model was neutralized by the administration of an activated prothrombin complex concentrate at 25 U/kg. In a similar experimental setting, r-factor VIIa was also partially effective. These studies suggest that r-hirudin anticoagulation may not require neutralization, since bleeding effects are not observed at effective antithrombotic dosages in individuals with normal hemostatic status.(ABSTRACT TRUNCATED AT 250 WORDS)

The process of fusion from without (FFWO) induced by herpes simplex virus (HSV) was analyzed by using various inhibitors and compared to fusion from within (FFWI). The fate of certain elements of the cytoskeleton after FFWO was also investigated. Our experiments demonstrate FFWO as a very suitable system for study of early virus-cell interactions. Zn++ ions proved inhibitory for penetration whilst pretreatment of cells with Ca++ ions before infection enhanced FFWO activity. Dissociation of penetration from the fusion process itself was possible by use of Zn++ ions, low pH-treatment and antiserum on the one hand and N-ethylmaleimide and cytochalasin D on the other. Penetration itself needs only 6 min or less to proceed. FFWO is independent of inhibitors of glycosylation (tunicamycin) and intracellular vesicular traffic (monensin), protein-synthesis (cycloheximide) and energy-delivery (2.4 dinitrophenol and Na-azide). Analyzed strains of HSV-1 and -2 producing FFWI could be subgrouped into three categories: Strain ANG with high, strain HFEM and Lux with low and strains IES, Len, MP, US with no FFWO activity. The results of these experiments indicate that the property of FFWO is not purely a consequence of the number of PFU but depends on certain inherent properties of the virus particles. Addition of heparin as well as treatment of cells with heparitinase effectively prevented FFWO, indicating identical virus receptors for entrance of virus into cells and FFWO. During our studies several calf sera were found to inhibit FFWO-activity. Inhibition of FFWO by a glycoconjugate (ferritin coupled with oleic acid) indicates specific stereochemical hindrance of FFWO by this compound. Shortly after FFWO the actin filaments rearrange to form long fibres and surface fibronectin is being lost from the cell membrane.

Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI2) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI2 was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI2 productions by ATIII and thrombin. ATIII-dependent PGI2 production was significantly inhibited by pretreatment of HUVEC with beta-D-xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI2 production. After treatment of HUVEC with 1 micrograms/ml cycloheximide. ATIII-dependent PGI2 production was completely abolished. These results indicate that the mechanism of the induction of PGI2 production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI2 production. The ATIII-induced PGI2 production is very different from that induced by thrombin.

Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

The main purpose of this work was to identify the macromolecules carrying the surface charge of endothelial cells. This was done by measuring changes in cell electrophoretic mobility caused by enzymatic removal of glycocalyx components. Endothelial cells were removed from the bovine pulmonary artery using nonenzymatic procedures, plated, and identified by immunocytochemical methods and electron microscopy. Cultured cells were suspended in saline and placed in the lumen of a capillary in a Rank Brothers electrophoresis instrument. Voltage was applied between the ends of the capillary, and the velocity acquired by the cells was measured with a microscope. Preincubating the cells in protein-free saline for 1 h reduced the mobility by 25%. This reflects the loss of proteoheparan sulfate from the cell surface. Cell mobility was totally suppressed by exposing the entire cell surface to chondroitin sulfate lyase, but it was only slightly diminished when the enzyme was applied only to the cell side facing the culture medium. A partial decrease in mobility was obtained after enzymatic removal of either heparin, heparan sulfate, or collagen. The results indicate that sulfated glycosaminoglycans are the main carriers of the surface change in vascular endothelial cells. The asymmetrical effect of chondroitinase on the two sides of the cell indicates a distribution polarization for glycosaminoglycans in endothelial cells.

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.

Physiological stimuli induce rapid and unexplained increases in the number of red blood cells within capillaries of skeletal muscle. We hypothesized that such alterations in intracapillary red cell numbers might be due to an undefined interaction between one or more components of blood and the luminal surface of the capillary. This proposition was tested by in situ microperfusion of capillaries with enzymes directed against macromolecules likely to be expressed on the surface of endothelial cells. The instantaneous fractional volume of red blood cells within a capillary (tube hematocrit) was used as an index of a capillary's response to enzyme microperfusion. Five to 8 min of perfusion with enzyme vehicle (0.25% albumin-Ringer solution) produced no significant alteration in capillary tube hematocrit. Perfusion with solutions containing heparinase raised the tube hematocrit at least twofold (P less than 0.05) without a significant change in red cell velocity. Heat-denatured heparinase and other enzymes such as neuraminidase, hyaluronidase, papain, pronase E, and clostripain had no detectable effect on the tube hematocrit (P greater than 0.05). After enzyme treatment, application of adenosine (10(-4) M) or oxygen caused brisk vasomotor responses in arterioles feeding perfused capillary units, but the usual changes in the tube hematocrit were not observed. Thus heparinase treatment results in a sustained elevation in the capillary tube hematocrit and a dissociation of the typical relationship between vasomotor changes and red cell distribution in capillaries. These findings suggest that physiological stimuli which alter the number of red blood cells within capillaries may operate by modifying interactions between plasma and one or more components on the luminal surface of capillaries.

The ability of heparan sulfate, an endogenous component of the glomerulus, to regulate the growth of cultured rat mesangial cells was investigated. Heparan sulfate caused a dose-dependent inhibition of rat mesangial cell growth, 85% inhibition compared with controls at the highest dose (1,000 micrograms/ml). Chondroitin sulfate produced no inhibition. The low-sulfated fraction of heparan sulfate (9%) produced more inhibition than the high-sulfated fraction (13%), 90 +/- 1 vs. 71 +/- 2% (P = 0.002). The effects of the heparan sulfate were completely reversible. Treatment of heparan sulfate with heparitinase increased the degree of inhibition, 71 +/- 1 vs. 84 +/- 1% (P less than 0.001). Four different oligosaccharides derived from heparan sulfate and heparin were tested for their ability to inhibit growth. One of the oligosaccharides, low-sulfated (10%), caused significant inhibition, 76 +/- 2%. Heparan sulfate was also able to inhibit the growth of Swiss 3T3 fibroblasts (63 +/- 5%). This inhibition was less marked than that seen with mesangial cells. Thus heparan sulfate was able to significantly inhibit rat mesangial cell growth in culture. Alterations in glomerular heparan sulfate may play an important role in alterations in mesangial cell growth.

Ruthenium red and toluidine blue O precipitates were described associated with lathyritic elastic fibers in aortas of chickens treated with beta-aminopropionitrile fumarate (I. Pasquali-Ronchetti, C. Fornieri, I. Castellani, G. M. Bressan, and D. Volpin (1981). Alterations of the connective tissue components induced by beta-aminopropionitrile. Exp. Mol. Pathol. 35, 42-56). In this report evidence is given that these precipitates reveal the presence of proteoglycans, as they are completely removed by 5 M guanidine-HCl incubation and by specific enzymatic digestions. In particular, proteoglycans associated with the poorly cross-linked lathyritic elastin can be removed by testicular hyaluronidase, chondroitinase ABC, heparitinase, and nitrous acid treatments, whereas they are rather resistant to streptococcal hyaluronidase and chondroitinase AC. On the contrary, proteoglycans of the matrix or associated with collagen fibers are particularly sensitive to these latter enzymatic treatments. The conclusion is reached that glycosaminoglycans associated with beta-aminopropionitrile-induced lathyritic elastin (i) are different from those of the matrix or associated with collagen, and (ii) include mainly dermatan and heparan sulfates.

Using low magnification Hoffman Modulation Contrast microscopy to rapidly identify precartilage mesenchymal condensations in chick limb bud cultures, we have determined the effect on condensation number of treatments disruptive of the interaction of cell surface components with endogenously produced fibronectin. A monoclonal antibody directed against the amino-terminal heparin-binding domain of fibronectin reduced the number of condensations by more than 50%, as did the oligopeptide gly-arg-gly, which is a repeated motif in that fibronectin domain. In contrast, monoclonal antibodies directed against the collagen- and integrin-binding domains of fibronectin, or oligopeptides containing the fibronectin integrin-recognition sequence arg-gly-asp-ser, had no significant effect on condensation number. Addition of Flavobacterium heparinase to cultures also reduced condensation number by more than 50%. Alcian blue staining of sulfated proteoglycan was greatly reduced in differentiated cultures that had been exposed to treatments that reduced condensation number. Taken together with the accompanying study, which directly demonstrates an adhesive interaction between the amino-terminal domain of extracellular fibronectin and heparin-like molecules on the surfaces of latex bead probes, the data presented here strongly indicate a major role for the corresponding cell-matrix interaction in mediating precartilage condensation in limb mesenchyme.

Recent work suggests that the normal barrier to penetration of the renal glomerular basement membrane by anionic plasma proteins may depend in part on the existence of negatively charged sites within the membrane. We describe an in vitro cytochemical method for the quantitative demonstration of anionic sites in the normal human glomerular basement membrane. In five normal subjects, ranging in age from 10 days to 57 years, the sites were distributed at regular intervals in the lamina rara externa, with a frequency of 23.8 +/- 6.8 sites per 1000-nm length of membrane. A similar distribution was observed in the basement membranes from three normal human fetuses. Ex vivo perfusion of one cadaver kidney revealed a similar distribution of anionic sites. The number of anionic sites in the glomerular basement membranes of five patients with the congenital nephrotic syndrome was reduced to 8.9 +/- 3.7 (P less than 0.001). Prior incubation of sections of normal kidney in purified heparinase resulted in a marked reduction in the number of anionic sites. We conclude that congenital nephrosis results from failure of heparan sulfate--rich anionic sites to develop in the lamina rara externa of the glomerular basement membrane.

A low molecular weight heparin (LMW-heparin) with a mean molecular weight of 4900 dalton was prepared by controlled enzymatic depolymerization of conventional porcine mucosal heparin. The effects of 2,500, 5,000 and 10,000 U (XaI; 29, 58 and 116 mg) on factor Xa inhibition (XaI), factor IIa inhibition (IIaI), APTT, AT III and platelet count were compared to those of 5,000 U (XaI; 26 mg) of conventional heparin given s.c. to 6 healthy volunteers. 5,000 U (XaI; 58 mg) of LMW-heparin was given i.v. A dose related response with regard to the XaI and the IIa-inhibitory activities with peak values at 4 hours after the s.c. injections was obtained. An increase of the XaI/IIaI ratio over the time after injection was seen only after i.v. administration of the LMW-heparin. The APTT was only slightly prolonged and remained within normal range after s.c. injection. AT III and platelet counts were unaffected. The biological half life of the LMW-heparin was 111 minutes if assayed by Xa inhibition, 76 minutes if assayed by IIa inhibition and 40 minutes if assayed by APTT. A strong correlation between the XaI activities obtained and body weight was seen, indicating that LMW-heparin should be administered individually according to body weight.

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.

A stirred blood filter containing an immobilized enzyme, heparinase, has been used to neutralize heparin's anticoagulant activity at the outflow of an extracorporeal circuit in dogs. The hematocrit and red blood cell count remained unchanged throughout the 90-min perfusion period. Platelet and white blood cell counts decreased early in the procedure to approximately 20% of the initial levels, but then returned to 30 and 70%, respectively, of their initial values by the end of the procedure. After 24 h normal levels were reestablished. In vitro experiments with human blood were conducted to determine the principal cause of the observed decrease of formed blood components. An unstirred heparinase filter preserved platelets and white blood cells better than stirred filters possessing higher, the same, or no heparin-degrading capacity, suggesting that most of the loss of formed blood components is due to stirring and not to the heparinase or the Sepharose support on which the enzyme is immobilized.

Adhesion and spreading of cell lines on dishes coated with serum-derived proteins were studied after removal of cell-surface proteoglycans. A mixture of glycosaminoglycans lyases from heparin-induced Flavobacterium heparinum removed 80% of the [35S]sulphate-labelled glycosaminoglycans from the surface of attached cells within 30 min, but this had little effect on cell morphology. The rate of cell attachment to dishes coated with serum was unaffected by prior treatment of cells with this mixture of glycosaminoglycan lyases. While a heparan sulphate lyase preparation abolished cell spreading in response to fibronectin there was no effect of the enzyme on the spreading mediated by vitronectin. These results suggest that, although heparan sulphate is required for spreading on purified fibronectin, the spreading stimulated by serum under routine culture conditions requires neither cellular heparan sulphate nor serum-derived fibronectin.

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.

Fibroblast-like cells were obtained by in vitro cultivation of needle aspirations of human bone-marrow. These cells show a unique composition of coat-associated glycosaminoglycans: 10% chondroitin 4-sulfate, 30% hyaluronic acid and 60% heparan sulfate which were resolved and characterized by electrophoresis, nitrous acid treatment and enzymatic degradation. Chondroitin 4-sulfate is the only glycosaminoglycan detectable on the surface of mature granulocytes, whereas the immature cells do not seem to possess surface glycosaminoglycans. Immature hemopoietic cells can adhere on to marrow-derived fibroblast cells, whereas mature granulocytes cannot. Treatment with mucopolysaccharidases of both mature leukocytes and marrow stromal cells can interfere in these adhesive relationships, suggesting a role of glycosaminoglycans in regulating short-range interactions during hematopoiesis.

Proteoglycans from three cloned, granulated lymphocyte cell lines with natural killer (NK) function (NKB61A2, HY-3, H-1) and one mast cell line (PT-18) were labeled with [35S]sulfate. [35S]proteoglycans were extracted in 1 M NaCl with protease inhibitors to preserve their native structure and were separated from unincorporated [35S]sulfate by Sephadex G-25 chromatography. [35S]proteoglycans from all four cell lines were chromatographed over Sepharose 4B and were found to have a similar range of m.w. The [35S]glycosaminoglycans from each cell line were then separated from parent proteoglycans by treatment with 0.5 M NaOH. The [35S]glycosaminoglycans from the three lymphocyte cell lines exhibited a similar m.w. as assessed by Sepharose 4B gel filtration, whereas the [35S]glycosaminoglycans from the mast cell line chromatographed as a smaller m.w. molecule. [35S )glycosaminoglycan charge characteristics were evaluated with DEAE C1-6B ion exchange chromatography. The consistency of the elution patterns was determined by using [35S]glycosaminoglycans obtained from radiolabelings of each cell line separated by 6 mo in culture. Each NK lymphocyte cell line reproducibly produced two distinct [35S]glycosaminoglycan chains that eluted in two regions well before the commercial heparin marker. The proportions of each chain were dependent upon the specific cell line. The mast cell line produced a single [35S]glycosaminoglycan chain, which eluted overlapping the internal commercial heparin marker, consistent with its higher charge characteristics. [35S]glycosaminoglycans from all cell lines were identified as chondroitin sulfates with the use of specific polysaccharidases. The NK lymphocyte glycosaminoglycans contained chondroitin 4-sulfate disaccharides. The mast cell glycosaminoglycans contained oversulfated disaccharides and chondroitin 4-sulfate disaccharides. Thus, each granulated NK lymphocyte cell line produced chondroitin sulfate glycosaminoglycans that were characteristic of that cell line and of different composition and less charge than those produced by cultured mast cells. These findings demonstrate that glycosaminoglycan profiles are useful biochemical markers in the characterization of diverse granulated cell lines including NK lymphocytes and mast cells.

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.

An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.

The retina is protected from circulating molecules by a blood-retinal barrier. This is comprised of the impermeable apical-lateral junctions of the retinal pigment epithelium and intraretinal blood vessels lined by endothelia that have impermeable junctions and vesicles that do not transport material from the luminal to abluminal front. This study examined the effect of enzyme digestion upon the restrictive properties of the retinal capillary endothelium. Rats were perfused first with enzymes and then by hemoglobin that was visualized by ultrastructural cytochemical methods. After perfusion of buffer alone or buffers containing neuraminidase or heparinase, the cytochemical reaction product was confined to the capillary lumina and to endothelial cell vesicles facing the luminal front. In contrast, after heparitinase or pronase perfusion, reaction product filled the extravascular spaces. Chains of endothelial cell vesicles and patent transendothelial channels were often encountered. Endothelial cell junctions did not appear to be affected by enzyme treatment. These findings indicate that a cell-surface heparan sulfate proteoglycan (or a nonidentified protein removed by proteolysis) is a key molecule required for the maintenance of the blood-retinal barrier.

It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin-binding sites that are not directly related to heparan sulfate.

We have previously demonstrated that confluent fetal fibroblasts migrate into three-dimensional collagen gels to a significantly greater extent than their normal adult counterparts. Recent studies have revealed that this behavioral difference results from the secretion by fetal fibroblasts of a soluble migration-stimulating factor (MSF) which acts on these cells in an autocrine fashion. Adult fibroblasts do not produce MSF but remain responsive to it. Skin fibroblasts from cancer patients resemble fetal fibroblasts (rather than normal adult cells) with respect to their migratory behavior on collagen gels and continued production of MSF. This communication is concerned with elucidating the biochemical basis of MSF activity. Data are presented indicating that a) hyaluronic acid is required for the elevated migratory activity displayed by confluent fetal and breast cancer patient skin fibroblast; b) adult fibroblasts exhibit a bell-shaped dose-response to MSF, with maximal stimulation of migration observed at a concentration of 10 ng/ml; c) the migratory activity of adult fibroblasts pre-incubated with MSF remains high in the absence of additional factor: and d) MSF affects both the quantity and size class distribution of hyaluronic acid synthesized by adult fibroblasts. We have previously speculated that the persistent fetal-like fibroblasts of breast cancer patients play a direct role in disease pathogenesis by perturbing normal epithelial-mesenchymal interactions. The observations reported here suggest that MSF-induced alterations in hyaluronic acid synthesis may contribute to the molecular basis of such perturbations.

Heparan sulphate has been reported to be present in rat embryos. It is covalently linked to a core protein as heparan sulphate proteoglycan (HSPG). Heparitinase specifically degrades heparan sulphate, thus treatment of rat embryos with this enzyme in vitro should result in the perturbation of any tissue interactions which involve heparan sulphate proteoglycan. In this study heparitinase was either added to the culture medium or microinjected directly into the amniotic cavity. Heparitinase treatment resulted in abnormal development of the whole embryo, but the earliest effects were observed in the cranial region. Forebrain development was grossly abnormal: the neural folds remained widely open, with beak-like outgrowths rostrally. Optic sulci failed to develop. The midbrain and rostral hindbrain neural folds also remained widely open. In the trunk, where the pattern of neurulation is less complex than in the cranial region, rostral neural tube closure did occur although the morphology of the closed region was far from normal. These results suggest that heparan sulphate proteoglycan is essential for normal neurulation. Epithelial somite formation was perturbed, but neural crest cell emigration, otic pit formation and pharyngeal arch formation, all important morphogenetic events which occur during this period of development, were not inhibited by heparitinase treatment. Prolonged (44 h) exposure to the enzyme resulted in the conversion of the embryonic structure to a much simpler form: mesenchymal cells (stellate or spindle-shaped) enclosed within a simple epithelial coating.

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.

Finback-whale (Balaenoptera physalus L.) heparin was partially digested with a purified heparinase and an octasaccharide with high affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. This octasaccharide possessed high inhibitory activity for Factor Xa in the presence of antithrombin III, but was essentially inactive for thrombin-antithrombin III reaction. The anticoagulant activity determined by the activated-partial-thromboplastin-time method was very low (40-70 units/mg), although the initial whale heparin exhibited high activity (252 units/mg). On the basis of the results of chemical analyses, 13C n.m.r. spectrum and enzymic studies with purified heparinase, heparitinases 1 and 2, the predominant structure of the octasaccharide was proposed as follows: delta UA(2S) alpha 1 leads to 4GlcNS alpha 1 leads to 4IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4GlcNS(3S) alpha 1 leads to 4IdUA(2S) alpha 1 leads to 4GlcNS. Comparing this structure with those of the heparin octasaccharides so far reported, the presence of the critical structural elements for binding to antithrombin III was suggested in the pentasaccharide region situated at the reducing end of this octasaccharide. Binding to antithrombin III of the critical structural elements alone would appear to elicit the acceleration of the Factor Xa-antithrombin III reaction. Additional structural elements required for the acceleration of the thrombin-antithrombin III reaction and for the manifestation of high anticoagulant activity are discussed.

The regulation of cell growth in the kidney glomerulus plays a key role in many physiologic and pathologic processes. In this communication the authors have examined the possible role of glomerular endothelial cells as potential regulators of mesangial cell proliferation. Conditioned medium was collected from confluent cultures of glomerular endothelial cells and tested for its effects on glomerular mesangial cell and vascular smooth muscle cell growth. When glomerular endothelial cell-conditioned medium was mixed 1:1 with normal growth medium, the growth of these two closely related cell types was inhibited by 60-70%. If the conditioned medium was diluted to 1:9, a stimulation of mesangial and smooth muscle cells growth was seen. Approximately 70% of the antiproliferative activity was destroyed by a highly purified heparinase; the other 30% was sensitive to trypsin. Approximately 90% of the mitogenic activity was protease-sensitive. These results suggest that glomerular endothelial cells may participate in part in mesangial cell growth regulation via a heparin-mediated mechanism.

Anticoagulation with heparin is required during extracorporeal circulation for hemodialysis and cardiopulmonary bypass as well as during vascular surgery. Reversal of anticoagulation with protamine may be associated with hypotension and rebound anticoagulation and requires stoichiometric doses. Heparinase from Flavobacterium heparinum catalytically degrades heparin and reverses its anticoagulant effect. Heparin was administered to New Zealand White rabbits and plasma levels were assayed with the APTT anticoagulant assay and the azure A chemical assay. Heparinase actively degraded heparin both in vitro in rabbit plasma and in vivo in rabbit blood as determined by both the anticoagulant and chemical assays when compared to control heparin disappearance curves. Antibodies to heparinase were demonstrated by the ELISA technique in rabbits receiving i.v. heparinase. These antibodies, however, did not effect the activity of the enzyme in vitro or in vivo. No toxic effects of heparinase were noted in observations of the animals or in blood and histologic studies. Heparinase, either free or immobilized, may be a useful heparin-reversing agent without the drawbacks of protamine.

Human basophils were obtained from three donors with myelogenous leukemia. Proteoglycans were labeled by using [35S]sulfate as precursor and were extracted in 1 M NaCl with protease inhibitors to preserve their native structure. [35S]proteoglycans filtered on Sepharose 4B with an average m.w. similar to that of a rat heparin proteoglycan that has an estimated m.w. of 750,000. The [35S]glycosaminoglycan side chains filtered with an average m.w. slightly smaller than a 60,000-m.w. glycosaminoglycan marker. The [35S]glycosaminoglycans were resistant to heparinase and susceptible to degradation by chondroitin AC lyase and chondroitin ABC lyase. The intact [35S]glycosaminoglycans chromatographed on DEAE Sepharose as a single peak eluting just before an internal heparin marker. These findings indicate that the [35S]glycosaminoglycans were made up only of chondroitin sulfates. No heparin was identified. The chondroitin sulfate disaccharides that resulted from the action of chondroitin ABC lyase on the basophil glycosaminoglycans consisted of 92% delta Di-4S, 6% delta Di-6S, and 2% disulfated disaccharides. The [35S]chondroitin sulfate proteoglycans were susceptible to cleavage with proteases and could be shown to be released intact from basophils during degranulation initiated by the calcium ionophore A23187. The basophil proteoglycans and glycosaminoglycans were capable of binding histamine in water, but not in phosphate-buffered saline, and had no anticoagulant activity.

Recent evidence suggests that heparan sulfate on endothelial cell surfaces acts as a catalyst for the neutralization of thrombin by antithrombin III (AT III). Fibroblasts also produce heparan sulfate which is present on the cell surface and secreted into the extracellular matrix. We evaluated the ability of cultured human fibroblasts to catalyze the interaction between thrombin and AT III and found that heparan sulfate produced by post-confluent fibroblasts was anticoagulantly active. Furthermore, after initial binding of thrombin to cells, thrombin-heparan sulfate appeared in the fluid phase above the cells; this thrombin could be rapidly neutralized by AT III independent of the further presence of cells. These results indicate that fibroblasts do produce an anticoagulantly active species of heparan sulfate and that the normal interaction between AT III and thrombin may be driven by initial release of heparan sulfate from the cell surface by thrombin followed by AT III interaction with the soluble thrombin-heparan sulfate complex.