Enhanced expression of a peanut agglutinin reactive O linked oligosaccharide on fibronectins from the synovial fluid of patients with rheumatic disease: quantitation,domain localization,and functional significance

OBJECTIVE: To characterize the domain localization, quantitation, and functional binding consequences of an O linked oligosaccharide expressed on synovial fluid (SF) fibronectin (Fn). METHODS: Identification and localization of the O linked oligosaccharide was performed by limited digestion of isolated SF Fn with a series of proteolytic enzymes followed by Western blotting with peroxidase labeled peanut agglutinin. Binding affinity to denatured collagen was performed utilizing a solid phase gelatin-binding assay. Quantitation was performed by measuring purified Fn in an antibody-lectin sandwich binding assay. RESULTS: A desialyated O linked oligosaccharide was identified on the C-terminal 18 kDa segment of the SF Fn collagen-binding domain. These SF collagen-binding Fn fragments were more basic and had higher gelatin-binding affinities than corresponding plasma fibronectin fragments. Expression of this O linked oligosaccharide was highest on Fn isolated from osteoarthritic SF, followed by Fn isolated from rheumatoid arthritis SF, and finally normal human plasma. CONCLUSION: Fn isolated from SF have glycosylation alterations that may influence their biologic properties in the diseased joint.     

Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).

Carboxypeptidase N (kininase I, arginine carboxypeptidase; EC 3.4.17.3) cleaves COOH-terminal basic amino acids of kinins, anaphylatoxins, and other peptides. The tetrameric enzyme of Mr 280,000 was purified from human plasma by ion-exchange and arginine-Sepharose affinity chromatography. Treatment with 3 M guanidine dissociated the enzyme into subunits of 83,000 and 48,000 molecular weight, which were separated and purified by gel filtration or affinity chromatography. When tested with hippurylarginine, hippurylargininic acid, benzoylalanyllysine, or bradykinin, the Mr 48,000 subunit was as active as the intact enzyme and was easily distinguished from human pancreatic carboxypeptidase B (EC 3.4.17.2). However, the Mr 48,000 subunit was less stable at acid pH or at 37 degrees C than the intact enzyme was. The carbohydrate-containing Mr 83,000 subunit was enzymatically inactive but stabilized the Mr 48,000 subunit at 37 degrees C. Trypsin, plasmin, and plasma or urinary kallikrein cleaved carboxypeptidase N into lower molecular weight active fragments, which were unstable at 37 degrees C. Cleavage of the Mr 48,000 subunit with the same enzymes increased activity and yielded fragments of Mr 29,000 or less. Antibodies to the Mr 83,000 of Mr 48,000 subunits crossreacted with the intact enzyme, and antibody to carboxypeptidase N also recognized both subunits. However, antibody to the Mr 83,000 subunit did not recognize Mr 48,000 subunit and antibody to the Mr 48,000 subunit did not crossreact with the Mr 83,000 subunit. Thus, this study indicates that carboxypeptidase N is composed of two immunologically distinct subunits, a Mr 48,000 subunit that is responsible for the enzymatic activity and a Mr 83,000 subunit that may stabilize the enzyme in blood.     

Evaluation of the developmental and nutritional changes in porcine insulin-like growth factor-binding protein-1 and -2 serum levels by immunoassay.

Porcine serum contains five insulin-like growth factor-binding proteins (IGFBPs), whose regulation has been studied by ligand blotting. To more accurately quantify changes in two specific forms of IGFBP a heterologous RIA for porcine (p) IGFBP-2 was developed, and IGFBP-1 levels were analyzed by immunoblotting. By RIA, postnatal hypophysectomy caused a 7-fold increase in serum pIGFBP-2 levels compared to controls (2,622 +/- 378 vs. 382 +/- 10 ng/ml, respectively). Fetal pIGFBP-2 levels were higher at 110 vs. 45 days gestation (1,074 +/- 214 vs. 418 +/- 30 ng/ml, respectively), rose to 1,905 +/- 167 ng/ml within 12 h after birth, then decreased to 1,010 +/- 10 ng/ml at 48 h. By immunoblot analysis, bovine IGFBP-2 antiserum reacted with a 34,000 mol wt (Mr) IGFBP and did not react with other forms of IGFBP detected by ligand blotting. Serum levels of the 34,000 Mr IGFBP, as detected by ligand blot analysis, are decreased when neonatal pigs are fasted for 48 h. In contrast, by RIA, pIGFBP-2 concentrations increased 4-fold. Immunoblots of these sera showed two lower Mr (22,000 and 14,000 Mr) bands that did not bind either [125I]IGF-I or [125I]IGF-II and were distinct from a smaller (20,000 Mr) IGFBP which bound only [125I]IGF-II. These two bands were increased in serum of 48-h fasted compared to fed piglets, suggesting that they are proteolytic fragments of pIGFBP-2. In vitro incubation of 48-h fasted pig serum with intact IGFBP-2 failed to reveal proteolytic fragments, indicating that the IGFBP-2 fragments were not generated by a protease that was released into the serum. Analysis performed with human IGFBP-1 antiserum revealed a 29,000 Mr immunoreactive band whose abundance was increased by either postnatal hypophysectomy or fasting. No fragments of IGFBP-1 were found in any serum tested. We conclude that heterologous antibodies can be used to identify and quantify IGFBP-1 and IGFBP-2 in porcine serum. Changes in pIGFBP-2 levels measured during fasting are due to the combination of changes in intact 34,000 Mr IGFBP-2 and smaller non-IGF-binding fragments. Changes in levels of specific forms of IGFBP as well as the presence of fragments have the potential to modulate the transport of IGF-I and -II out of the vasculature.     

Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.     

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting.

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synovial fluid fibronectin fragments: No evidence for a mitogenic effect on fibroblasts

Fragments of bovine plasma fibronectin produced by cathepsin D digestion are reportedly mitogenic for hamster fibroblasts. Rheumatoid arthritis synovial fluid contains many fibronectin fragments, which may contribute to the proliferation of synovial cells. We have therefore investigated the potential of fibronectin fragments to stimulate proliferation of synovial fibroblast-like cells using human material. Affinity-purified human plasma and synovial fluid fibronectin was digested with cathepsin D at pH 3.5 for 0-18 h and proteolysis stopped with pepstatin. A variety of fragments were produced ranging from 50 to 200 kDa when analysed by SDS-PAGE. The proliferative activity of various test preparations was studied using quiescent human skin and synovial fibroblasts. Tests were applied for 24 h to 10(4) cells and DNA synthesis measured by tritiated thymidine incorporation. Both undigested and peptides of fibronectin consistently failed to stimulate DNA synthesis in fibroblasts at all concentrations tested, compared with a phosphate-buffered saline control. This was in marked contrast to human synovial fluid from either rheumatoid arthritis or osteoarthritis patients, which stimulated DNA synthesis in the same system (P less than 0.01). Therefore, our data do not confirm the findings of previous studies in which animal materials were used. We can find no evidence that fibronectin fragments play a role in stimulating synovial proliferation in inflammatory arthritis.     

Fibronectin dependent macrophage fibrin binding

Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.     

Characterization of the molecular forms of fibronectin in fulminant hepatic failure

The plasma levels of the opsonic glycoprotein fibronectin are decreased in patients with fulminant hepatic failure, which may be an important factor in their impaired host-defense. Twenty-nine patients in fulminant hepatic failure were studied on admission, and the mean fibronectin level in Grade 0-2 encephalopathy was 82 micrograms per ml (range = 0 to 150) and in Grade 3-4 encephalopathy 61 micrograms per ml (range = 5 to 158) as compared to normal controls (268 micrograms per ml, range = 178 to 380, n = 62). No fibronectin degradation products could be detected in fulminant hepatic failure plasma by sodium dodecyl sulfate-gel electrophoresis on a polyacrylamide gradient (5 to 15%) followed by immunoblotting onto nitrocellulose with detection using a rabbit antihuman fibronectin antiserum visualized with a peroxidase conjugate. The plasma levels of the marker proteolytic enzyme cathepsin D were significantly elevated in fulminant hepatic failure (120 +/- 31 mU per ml per hr) as compared to the normal controls (18 +/- 2.1 mU per ml per hr, n = 10, p less than 0.01). Cross-immunoelectrophoresis of fulminant hepatic failure plasma for fibronectin on agarose plates gave an additional slower migrating peak in 15 of the 29 patients, as well as that of fibronectin, which corresponded to the fibronectin complex reported by other workers in leukemia. An intermediate gel containing antihuman fibrinogen demonstrated fibrinogen to be one component of this complex. Binding of other substances to fibronectin will reduce its apparent biological activity and may be the result of their lack of clearance by the damaged liver.     

Potential proteolytic activity of human plasma fibronectin.

Evidence is presented that fibronectin (FN) polypeptide chain contains a latent proteinase. Human plasma FN was cleaved with cathepsin D into three main fragments: 140-kDa and 70-kDa single-chain and 140-kDa double-chain polypeptides. Their separation was achieved according to their affinity for heparin-Sepharose. A single-chain 140-kDa fragment (H-1) was eluted in the first peak. This peptide corresponds to the already described fragment that originates from the central part of FN; it contains a low-affinity heparin-binding site, one free SH group, and a cell-binding site. After reduction and further purification by preparative polyacrylamide gel electrophoresis, this fragment revealed a spontaneous decomposition, which could be attributed to proteolytic degradation. The subfragments, ranging from 25 to 95 kDa, yielded the same proteolytically active doublet of 28-30 kDa when tested by NaDodSO4/polyacrylamide gel electrophoresis in a gel containing copolymerized gelatin or fibrinogen. The proteolytic activity was inhibited by specific SH proteinase inhibitors. The proteinase forms a labeled complex after its incubation with 125I-labeled cystatin. Neither FN, cathepsin D, nor any products from previous purification steps were proteolytically active under the conditions of the assay. It was suggested that the same fragment may also yield an inhibitor, since structural analogies were found between the cell-binding region of FN and SH proteinase inhibitors.     

The immunoreactivity, ligand, and cell binding characteristics of rheumatoid synovial fluid fibronectin

Fibronectin promotes macrophage adherence and expression of Fc receptors, is chemotactic for fibroblasts, and is an opsonin for fibrin and denatured collagen. These properties suggest a role for fibronectin in the modulation of joint inflammation. Since structural modification of the fibronectin molecule has been shown to result in loss or de novo acquisition of opsonic and chemotactic activity, we determined the functional and immunochemical properties of fibronectin isolated from the inflamed joint. Eighty-six percent of synovial fluids obtained from patients with active rheumatoid arthritis (RA) contained fibronectin fragments, and 39% of the fluids no longer displayed the dimeric form. Compared with native fibronectin, RA peptides were as active in promoting synoviocyte chemotaxis and in glycosaminoglycan binding, but displayed lower affinity for fibrin and gelatin. Although comparable with intact protein in augmenting monocyte attachment to gelatin, the RA synovial fluid peptides did not augment monocyte attachment to fibrin. Analysis of whole synovial fluid and isolated fibronectins by enzyme immunoassay showed that the increased fibronectin immunoreactivity, previously reported in RA synovial fluid, measures intact and nearly intact protein and does not measure extensively degraded fragments.     

Plasma fibronectin in French centenarians

Plasma fibronectin was shown to increase with age, the difference between individuals (the SD of the mean) also increases with age. Fibronectin is highly sensitive to proteolytic degradation and several of the degradation products were shown to have noxious effects as proper proteolytic activity, activation of IL-1 and collagenase expression and also activation of fibronectin biosynthesis. It was therefore interesting to compare the plasma fibronectin values of centenarians in relatively good health with an elderly population in a geriatric hospital, somewhat younger (70-96 years) but with a variety of pathologies. A third population of men and women between 59 and 70 in good health (the EVA-epidemiological study) was also used for comparison. Plasma fibronectin was determined by a specific and highly sensitive Elisa assay. Fibronectin fragments were characterized by immunoblot. It could be shown that plasma fibronectin in centenarians had a lower distribution with lower average values than the geriatric population. Fibronectin fragments could be demonstrated in the plasma of a selection of geriatric patents but not in the plasma of centenarians. These results suggest a more moderate increase of plasma fibronectin in the relatively healthy centenarians as compared to a younger but pathological population. They also show that the plasma fibronectin of the investigated centenarians was better protected from proteolytic degradation than in the geriatric population. The above results also confirm the contention that epigenetic mechanisms such as an age-dependent increase of fibronectin synthesis and degradation and the potential noxious effects of degradation products may well play an important role in the age-dependent decline of physiological functions.     

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Progressive Loss of Fibronectin-Mediated Opsonic Activity in Plasma Cryoprecipitate with Storage

Septic injured patients often manifest a deficiency of plasma fibronectin. Several studies have shown improvements in organ function in such patients following infusion of fibronectin-rich plasma cryoprecipitate, while other studies found no improvement. One explanation for these differences may be the use of plasma cryoprecipitate which has been stored for various time intervals prior to its use as a source of fibronectin. This investigation tested the hypothesis that the opsonic activity of fibronectin in cryoprecipitate may decline with increased storage duration. Using a bioassay of opsonic activity, we evaluated human plasma cryoprecipitate that was stored at either -20 or -80 degrees C for various intervals (2 weeks to 12 months) after its preparation from fresh donor plasma. Our findings demonstrated that the opsonic activity of fibronectin in cryoprecipitate declined with increasing time of storage. Significant loss (p less than 0.05) of opsonic activity was first evident after 2 months of storage. Storage at -80 degrees C did not prevent this decline in opsonic activity as compared to storage at -20 degrees C. Immunoblot analysis revealed extensive fragmentation of the dimeric fibronectin (440 kdaltons) and the presence of lower molecular weight fragments in 4- to 12-month-old plasma cryoprecipitate. Therefore, plasma cryoprecipitate of varying ages (storage time) when used as a source of fibronectin for replacement therapy to support phagocytic function in septic injured patients may result in different fibronectin-mediated responses. The decline in activity may be due, in part, to fragmentation of the fibronectin molecule.     

Subtilisin and cyanogen bromide cleavage products of fibronectin that retain gelatin-binding activity.

The gelatin-binding region of fibronectin has been obtained by subtilisin digestion and cyanogen bromide cleavage of the molecule. Enzymatic digestion yielded two fragments of molecular weights 50,000 (S50K) and 30,000 (S30K) which were isolated by elution from gelatin-Sepharose affinity columns. Because the S50K fragment also mediated the adhesion of fibroblasts to collagen, it contains both the collagen and cell binding sites on the fibronectin molecule. Both fragments had valine as the NH2-terminal residue, were enriched in half-cystine and methionine residues compared to the whole molecule, and were identical by immunodiffusion. The S50K fragment begins with the sequence Val-Tyr-Gln-Pro-Gln-Pro-His-Pro-Gln-Pro-(Pro)-(Gly)-Tyr-Gly-His-( )-Val, a region with an extended conformation which is susceptible to proteolysis and connects this domain to the remainder of the fibronectin molecule. The S50K fragment appears to be located in the COOH-terminal one-third of the fibronectin molecule but does not contain the interchain disulfide bridge(s); the S30K fragment is probably derived from the NH2-terminal region of S50K.     

Decreased gelatin-binding fibronectin in patients with chronic inflammatory bowel diseases

The plasma fibronectin concentration (PF) was measured in 48 patients with chronic inflammatory bowel disease (CIBD) and in 48 age- and sex-matched healthy controls, using electroimmunoassay and a functional (gelatin-binding) assay. Whereas no difference in immunochemically measured PF was found between the two groups, patients with CIBD had significantly lower gelatin-binding PF than healthy controls (p less than 0.001). Immunochemically measured PF increased, whereas functionally measured PF tended to decrease with increasing disease activity.     

Incorporation of cellular and plasma fibronectins into smooth muscle cell extracellular matrix in vitro.

Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.     

Decreased Gelatin-Binding Fibronectin in Patients with Chronic Inflammatory Bowel Diseases

The plasma fibronectin concentration (PF) was measured in 48 patients with chronic inflammatory bowel disease (CIBD) and in 48 age- and sex-matched healthy controls, using electroimmunoassay and a functional (gelatin-binding) assay. Whereas no difference in immunochemically measured PF was found between the two groups, patients with CIBD had significantly lower gelatin-binding PF than healthy controls (p < 0.001). Immunochemically measured PF increased, whereas functionally measured PF tended to decrease with increasing disease activity.     

Blood-borne fragments of fibronectin after thermal injury

Fibronectin is an adhesive protein that can promote phagocytosis and endothelial cell adhesion. Plasma fibronectin declines following burn in animals and patients, potentially due to its complexing with circulating collagenous debris as well as its rapid binding to sites of tissue injury. Such depletion of fibronectin initiates an opsonic deficiency of the plasma. In view of the sensitivity of fibronectin to proteolytic enzymes, an additional factor that could contribute to the decrease of plasma opsonic activity after burn is the proteolytic fragmentation of fibronectin in the blood. In the current study, we determined if fibronectin fragments appear in the blood of anesthetized rats after a sublethal full-thickness skin burn of 15% to 16% of body surface. Plasma fibronectin concentration was quantified by enzyme-linked immunosorbent assay and the presence of fibronectin fragments in plasma was determined by immunoblot analysis. All blood was collected in an antiprotease mixture to yield final plasma concentrations of 0.15% EDTA, 3mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L iodoacetate to prevent degradation of fibronectin after sampling. Plasma fibronectin decreased 60% to 70% within 30 minutes post-burn, and this low level lasted for at least 4 hours. Within 30 minutes post-burn, two prominent fragments of fibronectin with a molecular weight of 110 +/- 2.2 kd and 122 +/- 3.3 Kd, respectively, were also detected in the plasma. Peak concentration of these fragments was detected at 60 minutes post-burn, but their level declined by 4 hours. By 4 hours, both bands appeared to resolve into doublets. To rule out the possibility that the fragments of fibronectin detected in the plasma were actually generated by coagulation enzymes activated at the site of peripheral blood sampling, rapid direct inferior vena cava sampling was performed, which also yield the presence of the fragments. Thus, fibronectin fragments exist in the plasma following thermal injury. Because fragments of fibronectin can compete with the intact fibronectin molecule with respect to its ability to stimulate macrophage phagocytosis, such fragments may contribute to altered systemic phagocytic host defense following thermal injury. Furthermore, because fibronectin peptides can compete with matrix fibronectin and impair adhesion of cultured endothelial cells, such circulating fragments may also influence the integrity of the vascular barrier.     

Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells

The cysteine protease cathepsin L is one of the most potent mammalian elastases and collagenases, widely expressed at basal levels in most tested tissues and cell types, and regulated by pro-inflammatory stimuli. The inflammatory arterial diseases abdominal aortic aneurysm (AAA) and atherosclerosis involve extensive vascular remodeling that requires elastolysis and collagenolysis. This study examined the hypothesis that cathepsin L is over-expressed in human AAA and atherosclerotic lesions and its expression in vascular cell types found in these lesions is regulated by pro-inflammatory cytokines. Immunohistochemical and tissue extract immunoblot analysis demonstrated increased expression of cathepsin L in human AAA and atheromata and localized its expression to lesional smooth muscle cells (SMC), endothelial cells (EC), and macrophages. In primary cultured human SMC, EC, and monocyte-derived macrophages, pro-inflammatory cytokines or growth factors induced the expression of cathepsin L and its activity against extracellular collagen and elastin. Patients with coronary artery stenosis (n=65) had higher serum cathepsin L levels than those without lesions detectable by quantitative coronary angiography (n=30) (1.47+/-0.33 ng/ml versus 0.60+/-0.06 ng/ml, p<0.02). A strong correlation between the percent of stenosis of left anterior descending coronary artery and serum cathepsin L levels in patients with stenosis (R=0.542, p<0.0001), also suggests involvement of cathepsin L in these vascular diseases.     

Functional domain structure of fibronectin.

Structural domains of fibronectin (FN) and their ability to associate with cell surface components have been systematically investigated. Plasma FN was cleaved into three structural domains (Mr 150,000-140,000, 40,000, and 32,000) by sequential digestion with trypsin and thermolysin. A single digestion with thermolysin alone generated Mr 150,000-140,000, 40,000, and smaller fragments. With the inclusion of thermolysin, but not with other proteases, one can, with a high yield dissect FN simultaneously into three clearly distinctive functional domains. Of three major fragments only the Mr 40,000 fragment bound to a gelatin column; this fragment contained essentially all of the carbohydrates present in the original FN. In contrast, heparin-binding sites were localized on both the Mr 150,000-140,000 and 32,000 fragments but not on the Mr 40,000 fragment. Only the Mr 150,000-140,000 fragments and intact FN promoted cell spreading, whereas the Mr 40,000 and 32,000 fragments could induce cell attachment but failed to promote cell spreading. These results indicate that FN is composed of (at least) three structural domains that are functionally distinct from each other.