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Biomedical subjects

D F Mosher

Publications and source records attributed to D F Mosher.

At least 145 records · Page 8Linked to original sources
PubMed1985

Incorporation of thrombospondin into fibrin clots.

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.

Blood Coagulation↗
PubMed1985

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.

Binding Sites↗
PubMed1985

Role of fibronectin in human monocyte and macrophage bactericidal activity.

Fibronectin is a high-molecular-weight glycoprotein found as a soluble dimer in plasma and as an insoluble multimer in tissues. It has been proposed that plasma fibronectin facilitates phagocytic removal of lysed cells and damaged tissues. Fibronectin binds avidly to several species of gram-positive bacteria and enhances staphylococcal and streptococcal attachment to cultured cells. Determination of whether fibronectin will enhance the bactericidal activity of monocytes and macrophages has not been reported. The bactericidal activity of freshly isolated monocytes, cultured monocytes, or lymphokine-activated macrophages was tested in the presence of either dimeric or multimeric fibronectin. Freshly isolated monocytes and lymphokine-activated macrophages killed Staphylococcus aureus effectively in the absence of fibronectin or whole serum. In contrast, monocytes cultured for 7 to 10 days had diminished staphylocidal capacity. When the monocytes were cultured with either dimeric or multimeric fibronectin, however, bactericidal capacity was maintained. Thus, although fibronectin did not enhance the bactericidal activity of mononuclear phagocytes, both multimeric and dimeric fibronectin were effective at maintaining the bactericidal capacity.

Blood Bactericidal Activity↗
PubMed1985

Localization of thrombospondin in clots formed in situ.

Thrombospondin is a principal glycoprotein secreted by thrombin-stimulated platelets and has known affinities for fibrinogen and fibrin. We studied the distribution of thrombospondin in clots formed in situ on Formvar-coated coverslips at 37 degrees C for intervals up to 17 hours. The distributions of three other major platelet granular proteins--fibrinogen, fibronectin, and von Willebrand factor (vWF)--were also determined. The portions of the clots adhering to the coverslips after stripping, washing, and fixation with formaldehyde were stained for the four proteins by the peroxidase-antiperoxidase technique. Monoclonal antibodies were used to localize thrombospondin, fibronectin, and vWF; affinity-purified polyclonal antibodies were used to localize fibrinogen. Platelets stained positively for all four proteins. Thrombospondin was maximally present in the fibrin meshwork from 1 1/2 to 2 hours, after which the intensity of staining decreased until only trace amounts of thrombospondin were detectable between four and 17 hours. Antifibrinogen and, to a lesser extent, antifibronectin stained the fibrin meshwork at all time points. The vWF was not detectable in the fibrin meshwork at any time point. Staining of polymorphonuclear leukocytes (PMNLs) in a fine granular pattern was found with antithrombospondin. The fraction of PMNLs staining positively was 6% to 14% at 1/2 to 4 hours and increased at eight hours to 27%. At 17 hours, 52% of the PMNLs stained for thrombospondin. More than 48% of the PMNLs stained with antifibrinogen at all time points. PMNLs did not stain for either fibronectin or vWF. These studies indicate that thrombospondin is a transient component of the temporary fibrin meshwork and has a unique spatial and temporal distribution in the hemostatic plug.

Antibodies, Monoclonal↗
PubMed1984

Effect of anti-thrombospondin antibodies on the hemagglutination activities of the endogenous platelet lectin and thrombospondin.

The proposal that thrombospondin is the endogenous platelet lectin was evaluated using antisera and monoclonal antibodies to thrombospondin. The platelet-bound hemagglutinin activity of human platelets stimulated with A23187 was inhibited by rabbit anti-thrombospondin sera and by a monoclonal anti-thrombospondin IgG. A second monoclonal IgG did not inhibit platelet-bound agglutinin activity. Preparations of purified platelet thrombospondin differed in their hemagglutination activities. The hemagglutination activity of an active preparation of thrombospondin was inhibited by the monoclonal antibody that inhibited platelet-bound lectin activity. The hemagglutination activity of an almost inactive preparation of thrombospondin was enhanced by the anti-thrombospondin monoclonal antibody that did not block platelet-bound lectin activity. The results demonstrate that expression of the platelet-bound form of the endogenous lectin is thrombospondin-dependent and suggest that thrombospondin must become part of a larger complex, either by binding to the platelet surface or by becoming aggregated in solution, before hemagglutination activity can be expressed.

Antibodies, Monoclonal↗
PubMed1984

Plasma fibronectin concentration in patients with acquired consumptive coagulopathies.

Plasma fibronectin was assayed in 179 hospitalized patients referred for workup of possible acquired coagulopathy. Based on laboratory results and chart review, these patients were classified as having no coagulopathy (N = 36), defibrination syndrome (N = 31), compensated defibrination syndrome (N = 100), microangiopathic thrombocytopenia (N = 7), and primary fibrinolysis (N = 5). Compared to patients with no coagulopathy, fibronectin concentration was reduced in patients with defibrination syndrome (p less than 0.005) and compensated defibrination syndrome (p less than 0.10). Fibronectin concentration was not reduced in patients with microangiopathic thrombocytopenia and primary fibrinolysis. In patients with defibrination syndrome, the reduction of fibronectin was correlated to the degree of liver impairment. This finding is consistent with the liver being the primary site of synthesis of plasma fibronectin. Fibronectin was significantly correlated to plasminogen and antithrombin III. The sensitivity of fibronectin for the diagnosis of coagulopathy is low except for patients with defibrination syndrome.

Antithrombin III↗
PubMed1984

Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.

The free sulfhydryl groups of purified plasma fibronectin were blocked by alkylation with iodoacetamide in 3 M guanidine hydrochloride at pH 7.0 (to form fCam-fibronectin). 125I-fCam-fibronectin bound to cell layers of cultured human fibroblasts with kinetics similar to binding of 125I-labeled underivatized fibronectin. Once bound to the cell surface, 125I-fCam-fibronectin, like 125I-fibronectin, became incorporated into a detergent-insoluble extracellular matrix. Matrix-bound 125I-fCam-fibronectin, like matrix-bound 125I-fibronectin, was present as disulfide-bonded multimers as well as disulfide-bonded dimers. These results indicate that the mechanism of formation of disulfide-bonded fibronectin multimers does not involve oxidation of free sulfhydryls. Catheptic D digests of matrix-bound fibronectin were analyzed by gel electrophoresis without and with reduction. In non-reduced digests, the disulfide-rich 70-kDa amino-terminal fragment was found in a large (greater than 300 kDa) complex. Thus, fibronectin multimer formation probably occurs by disulfide exchange in the amino-terminal portion of the molecule.

Cathepsin D↗
PubMed1984

Cross-linking of fibronectin to collagenous proteins.

Attempts were made to cross-link several collagenous proteins to fibronectin with Factor XIIIa (plasma transglutaminase). Cross-linking was demonstrated with type I collagen, type II collagen, type III collagen, type V or AB collagen, and alpha 1(I)-CB7 and alpha 1(I)-CB8 cyanogen bromide fragments of type I collagen. Cross-linking was not demonstrated with type IV collagen, Clq, and cyanogen bromide fragment alpha 1(I)-CB6. The pH optimum for cross-linking of alpha 1(I)-CB7 to fibronectin was 8.5 to 9.6. Cross-linking of alpha 1(I)-CB7 to fibronectin was somewhat enhanced at lower than physiological ionic strength.

Acyltransferases↗
PubMed1984

Binding and degradation of platelet thrombospondin by cultured fibroblasts.

Thrombospondin was purified from human platelets and labeled with 125I, and its metabolism was quantified in cell cultures of human embryonic lung fibroblasts. 125I-Thrombospondin bound to the cell layer. The binding reached an apparent steady state within 45 min. Trichloroacetic acid-soluble radioactivity was detected in the medium after 30 min of incubation; the rate of degradation of 125I-thrombospondin was linear for several hours thereafter. Degradation of 125I-thrombospondin was saturable. The apparent Km and Vmax for degradation at 37 degrees C were 6 X 10(-8) M and 1.4 X 10(5) molecules per cell per minute, respectively. Degradation was inhibited by chloroquine or by lowering the temperature to 4 degrees C. Experiments in which cultures were incubated with thrombospondin for 45 min and then incubated in medium containing no thrombospondin revealed two fractions of bound thrombospondin. One fraction was localized by indirect immunofluorescence to punctate structures; these structures were lost coincident with the rapid degradation of 50-80% of bound 125I-thrombospondin. The second fraction was localized to a trypsin-sensitive, fibrillar, extracellular matrix. 125I-Thrombospondin in the matrix was slowly degraded over a period of hours. Binding of 125I-thrombospondin to the extracellular matrix was not saturable and indeed was enhanced at thrombospondin concentrations greater than 3 X 10(-8) M. The ability of 125I-thrombospondin to bind to extracellular matrix was diminished tenfold by limited proteolytic cleavage with trypsin. Degradation of trypsinized 125I-thrombospondin was also diminished, although to a lesser extent than matrix binding. Heparin inhibited both degradation and matrix binding. These results suggest that thrombospondin may play a transitory role in matrix formation and/or organization and that specific receptors on the cell surface are responsible for the selective removal of thrombospondin from the extracellular fluid and matrix.

Amines↗
PubMed1984

Physiology of fibronectin.

Fibronectin is a major protein of blood and tissues. It has been intensively studied because of its many interactions with cells and other macromolecules. Fibronectin is a principal component of the extracellular matrix, and its most important function seems to be in tissue remodelling during embryogenesis and wound healing. It may influence a number of other physiological processes, including phagocytosis. The concentration of plasma fibronectin and the distribution of tissue fibronectin are altered in disease states, although diseases specifically caused by quantitative or qualitative abnormalities of fibronectin have not been identified.

Binding Sites↗
PubMed1984

Detection of autoantibodies and glomerular injury in rabbits immunized with denatured human fibronectin monomer.

Seven rabbits were studied after immunization with human plasma fibronectin which had been purified by preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis run after reduction. Light- and electron-microscopic examination of kidneys revealed proliferative mesangial and capillary alterations in all of the rabbits immunized with fibronectin, but not in the rabbits immunized with fibrinogen or saline. In addition, one of the rabbits (Rabbit 4) also demonstrated dense deposits in a unique distribution within the glomerular basement membrane. Granular staining for rabbit IgG was present in the mesangium and along the basement membranes of the capillary loops of glomeruli from Rabbit 4 as detected by immunohistochemical methods. Sera from all of the rabbits immunized with human fibronectin contained IgG antibodies that reacted with rabbit fibronectin when tested by the Western blotting method. Preimmune sera and sera from rabbits immunized with fibrinogen or saline recognized neither human nor rabbit fibronectin. Although antibodies from several of the rabbits reacted with the 27,000-dalton, aminoterminal fragments of human fibronectin by the Western blotting method, only antibodies from Rabbit 4 recognized the 27,000-dalton fragment of rabbit fibronectin. These studies indicate that antibodies which recognize fibronectin of the host species and which are involved in the pathogenesis of glomerular injury can be induced by immunization with denatured heterologous fibronectin monomer.

Animals↗
PubMed1984

Fibronectin-induced agglutination of Staphylococcus aureus correlates with invasiveness.

Studies on the interactions of staphylococci with fibrinogen, fibrin split products, and prothrombin have formed the basis for the clumping tests for coagulase and fibrin degradation products. We investigated the role of another circulating protein, fibronectin, in clumping Staphylococcus aureus. Fibronectin is a dimeric glycoprotein with high molecular weight that is present in both blood and tissue and is involved in opsonization, clotting, healing of wounds, cell-to-cell attachment, and differentiation. Each fibronectin molecule has two S. aureus binding sites, thus allowing lattice formation. We defined conditions under which fibronectin will cause agglutination of S. aureus. Strains of S. aureus that were most easily clumped had the largest number of fibronectin receptors. Trypsinization or gentle sonication removed the fibronectin binding and agglutinating receptors from S. aureus. These treatments did not alter viability, which suggests that binding is a superficial component of the organisms. Invasive fibronectin-binding strains were from a wide variety of phage types. Twenty two S. aureus isolates from patients with invasive disease were more readily agglutinated and had a greater number of fibronectin binding sites than 19 noninvasive strains (p less than 2.5 X 10(-4)). This suggests that the pathogenicity of S. aureus invasion may be enhanced by binding of bacteria to tissue fibronectin or by agglutination of bacteria by plasma fibronectin. Thus, the fibronectin receptors on S. aureus that mediate agglutination might also permit invasion of host tissues.

Fibrinogen↗
PubMed1983

Specificity of fibronectin--fibrin cross-linking.

Our experiments indicate that (1) non-covalent binding of fibronectin to fibrin is mediated by sites in a 27 kd NH2-terminal region and a 31 kd COOH-terminal region of fibronectin; (2) the 31 kd region is probably present in both chains of the fibronectin dimer; and (3) covalent (factor XIIIa-mediated) cross-linking of fibronectin and fibrin is between a glutaminyl residue in the 27 kd region of fibronectin and a lysyl residue in the COOH-terminal two-thirds of the fibrin alpha chain.

Amino Acid Sequence↗
PubMed1983

In vitro formation of disulfide-bonded fibronectin multimers.

Fibronectin purified from a plasma protein side fraction in the absence of denaturant contained 1.5 to 1.9 cryptic free sulfhydryl groups per 200- to 250-kDa subunit. Exposure of sulfhydryl groups in physiologic salt solutions required at least 1 M guanidine, and 3 M guanidine was required for optimal exposure. The sulfhydryl groups were not exposed by collagen, a fibronectin-binding collagen fragment, fibrinogen, heparin, hyaluronic acid, calcium ion, EDTA, deoxycholate, or methylamine. One- and two-dimensional gel electrophoresis indicated that a molecule of 40-60 kDa was disulfide-bonded to a minor portion of the fibronectin in whole human plasma and in preparations of purified fibronectin. In addition, traces of disulfide-bonded multimers were present in preparations of purified fibronectin. The proportion of fibronectin in disulfide-bonded multimers increased in guanidine-containing solutions. Compared to dimeric fibronectin, these multimers had limited solubility in physiologic buffers, could be readily cross-linked by Factor XIIIa, and exhibited altered tryptic susceptibility. In free sulfhydryl groups were blocked by prior alkylation with N-ethylmaleimide or iodoacetamide, fibronectin did not form disulfide-bonded multimers in guanidine-containing solutions. The patterns of altered tryptic susceptibility and cyanide cleavage suggested that multimer formation is mediated by both sulfhydryls of fibronectin. The transition from dimeric to multimeric fibronectin can serve as a model for the formation of disulfide-bonded fibronectin multimers in the extracellular matrix.

Disulfides↗
PubMed1983

Fibronectin concentration in plasma of patients with breast cancer, colon cancer, and acute leukemia.

Plasma fibronectin was measured in patients with breast cancer, colon cancer, and acute leukemia. In the patients with solid tumors, mean levels were significantly elevated above the mean level of age- and sex-matched normals whether the disease was thought to be metastatic or not (P less than 0.001). It did not make a difference whether the determinations were done prior to or during chemotherapy. Fibronectin was measured serially in eight hospitalized patients with leukemia during intensive induction chemotherapy. Normal concentrations were found prior to therapy. However, fibronectin concentration fell on the day following chemotherapy in nine of 12 episodes (P less than 0.05), and during sepsis in 13 of 13 episodes (P less than 0.001). Thus, the concentration was influenced by at least two factors: recent chemotherapy and sepsis. Because fibronectin concentration is sensitive to clinical events other than the status of the malignancy, it seems unsuitable as a tumor marker, at least as a single isolated measurement.

Adult↗
PubMed1983

The effect of the dimeric and multimeric forms of fibronectin on the adhesion and growth of primary glomerular cells.

Primary glomerular cells placed in a chemically defined medium containing Waymouth's medium MB 752/1 supplemented with insulin, transferrin, fibroblast growth factor, nonessential amino acids, sodium pyruvate, and antibiotics showed rapid outgrowth of cells which morphologically resembled well differentiated visceral epithelial cells followed by outgrowth of poorly differentiated cells; morphologic evidence suggests these latter cells are precursor cells of the epithelial cell lineage. Whereas the well differentiated glomerular epithelial cells were never observed to divide by sequential phase microscopic observations, a chemically defined medium was developed for optimal growth of the poorly differentiated cell type. This serum-free medium contained Waymouth's medium MB 752/1 supplemented with insulin, transferrin, selenium, and fibronectin (plus non-essential amino acids, sodium pyruvate, and antibiotics). Using this chemically defined medium, we have compared the effects of dimeric and multimeric fibronectin (high molecular weight disulfide-bonded fibronectin produced by incubation of dimeric fibronectin with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11) on the adhesion and growth of the poorly differentiated primary glomerular cell type. Dimeric fibronectin (FN) was twice as effective as multimeric FN in promoting glomerular cell adhesion, although both forms of FN promoted cell adhesion better than an uncoated substratum. In contrast, cell growth studies demonstrated that multimeric FN was a more potent growth stimulant than dimeric FN. The differential effects of dimeric and multimeric forms of FN in vitro suggests that these molecules may have different functions in vivo.

Animals↗