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Biomedical subjects

D F Mosher

Publications and source records attributed to D F Mosher.

At least 127 records · Page 7Linked to original sources
PubMed1988

Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea.

A difference in recognition of the adhesive glycoprotein vitronectin (also called S-protein, serum spreading factor, and epibolin) by monoclonal antibody 8E6 (Hayman EG, et al, Proc Natl Acad Sci USA 80:4003, 1983) was investigated using a competitive enzyme-immunosorbent assay and immunoaffinity chromatography. Recognition of vitronectin in serum was approximately 50-fold greater than recognition of vitronectin in plasma. Recognition of vitronectin incubated with heparin, thrombin-antithrombin III complex, or heparin and thrombin-antithrombin III complex together was 2.5-, 7-, or 32-fold greater, respectively, than recognition of vitronectin alone. Thrombin or antithrombin III by itself did not induce the antigenic change. Factor Xa-antithrombin III was less effective than thrombin-antithrombin III in induction of the change. Dextran sulfate and fucoidan were more potent than heparin in induction of the antigenic change, whereas dermatan sulfate, hyaluronic acid, heparan sulfate, chondroitin sulfate, or keratan sulfate were less effective. Immunoblotting analysis of serum and of vitronectin incubated with thrombin and antithrombin III demonstrated the presence of complexes composed of vitronectin and thrombin-antithrombin III that could only be dissociated with reducing agent. N-ethylmaleimide completely blocked the formation of the presumably disulfide-bonded complexes and partially blocked the antigenic change. Both non-disulfide-bonded and disulfide-bonded vitronectin bound to antibody-Sepharose from a mixture of vitronectin and thrombin-antithrombin III. Treatment of vitronectin with 8 mol/L urea resulted in enhanced recognition by the monoclonal antibody. Thus, the 8E6 antibody reacts with an epitope that is preferentially expressed by noncovalently and covalently linked vitronectin/thrombin-antithrombin III complexes and by urea-treated vitronectin. The change in vitronectin induced by thrombin-antithrombin III, therefore, is a physiological correlate of urea treatment and of adsorption of vitronectin onto tissue culture plastic (as is done in cell adhesion assays). The change may be important for expression of vitronectin activity.

Antithrombin III↗
PubMed1987

Matrix assembly sites for exogenous fibronectin are decreased on human fibroblasts after treatment with agents which increase intracellular cAMP.

Studies with cultured fibroblasts have shown that plasma as well as cellular fibronectin can be organized into fibrillar structures and that this organization is mediated by sites at the cell surface. Treatment of human skin fibroblasts with cholera toxin resulted in a prompt decrease in the number of binding sites for 125I-labeled plasma fibronectin and a 125I-labeled 70-kDa amino-terminal fragment of fibronectin. This decrease was accompanied by less incorporation of labeled fibronectin into deoxycholate-insoluble extracellular matrix. Binding of 125I-fibronectin was also decreased in cultures treated with epinephrine, isoproterenol, or forskolin. These results, therefore, indicate that G proteins and the adenylate cyclase system are involved in regulation of fibronectin matrix assembly sites may be one mechanism whereby hormones or growth factors can modify extracellular matrix characteristics.

Cells, Cultured↗
PubMed1987

The accumulation of a platelet protein, thrombospondin, at the site of arterial thrombus formation: preliminary report.

There are many methods available for the detection of thrombosis, none of which are noninvasive, rapid and accurate. Thrombospondin is a platelet protein that is present in the developing thrombus and may be an effective substance to use for imaging thrombosis. Vascular stenosis and thrombosis were produced in coronary, carotid and femoral arteries in eleven adult mongrel dogs. 131I labeled thrombospondin was administered to each animal to determine whether the radiotracer accumulated at the site of thrombus formation. The radioactivity per gram of vessels with thrombi was significantly different from the control vessels or whole blood (p = 0.0037 and p = 0.0015, respectively, paired t-test). This preliminary work suggests that iodinated thrombospondin accumulates at the site of thrombus formation. Labeled thrombospondin may be a rapid, safe and accurate method of detecting arterial thrombosis.

Animals↗
PubMed1987

Localization of cell surface sites involved in fibronectin fibrillogenesis.

Fibronectin binding sites on cultured human fibroblasts were localized by high voltage electron microscopy using either 5- or 18-nm colloidal gold beads (Au5 or Au18) bound to intact fibronectin, the 70-kD amino-terminal fragment of fibronectin that blocks incorporation of exogenous fibronectin into extracellular matrix, or 160-180-kD fragments of fibronectin with cell adhesion and heparin-binding activities. Binding sites for Au18-fibronectin on the cell surface were localized to specific regions along the edge of the fibroblast and on retraction fibers. Au18-fibronectin complexes at these sites were initially localized in clusters that co-aligned with intracellular microfilament bundles. With longer incubations, Au18-fibronectin complexes were arranged into long fibrillar networks on the cell surface and in the extracellular space. The appearance of Au18-fibronectin in these fibrillar networks and disappearance of clusters of Au18-fibronectin suggest that Au18-fibronectin complexes are arranged into matrix at specific regions of the cell surface. Au18-70-kD fragment complexes initially had a similar distribution to Au18-fibronectin complexes. With longer incubations, Au18-70-kD fragment complexes were found in long linear arrangements on the cell surface. Double labeling experiments using Au18-70-kD fragment and Au5-160-180-kD fragments showed that the 70-kD fragment and the 160-180-kD fragments bind to different regions of the cell.

Cell Membrane↗
PubMed1987

Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation.

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.

Animals↗
PubMed1987

Ex vivo platelet deposition on fibronectin-preadsorbed surfaces.

Temporal platelet deposition profiles of canine plasma fibronectin (CPFN) adsorbed to different polymers ex vivo and the in vitro characteristics of CPFN adsorption were studied in an attempt to correlate the two. The maximum platelet deposition (gamma pltmax) obtained at a protein preadsorption time of 30 min was greater than that obtained using an adsorption time of 120 min for all surfaces studied. At 30 min of preadsorption, gamma pltmax was 520,560 and 1230 platelets/1000 micron2 on Biomer, polyethylene (PE) and oxidized PE (OXPE), respectively. In contrast, the platelet deposition at 120 min. of fibronectin preadsorption was about 60 approximately 90 platelets/1000 micron2 on all polymers studied. The surface concentrations of adsorbed CPFN measured using 125I-CPFN, were in the order PE greater than OXPE greater than Biomer. The adsorbed protein concentration increased with increasing adsorption time. The surface distribution of adsorbed CPFN was visualized with antibody-labelled colloidal gold and scanning electron microscopy. The extent of staining was lowest on PE, greater on Biomer, and highest on OXPE, roughly similar to the order of platelet deposition. Platelet deposition ex vivo appears to correlate with the immunogold-stainable-adsorbed protein rather than with the total amount of adsorbed protein.

Adsorption↗
PubMed1987

Association of congenital deficiency of multiple vitamin K-dependent coagulation factors and the phenotype of the warfarin embryopathy: clues to the mechanism of teratogenicity of coumarin derivatives.

We have evaluated a boy who had excessive bleeding and bruising from birth and showed markedly prolonged prothrombin times, partially correctable by oral vitamin K administration. Additional laboratory studies demonstrated decreased activities of plasma factors II, VII, IX, and X; near normal levels of immunologically detected and calcium binding-independent prothrombin; undercarboxylation of prothrombin; excess circulating vitamin K epoxide; decreased excretion of carboxylated glutamic acid residues; and abnormal circulating osteocalcin. These results all are consistent with effects resulting from decreased posttranslational carboxylation secondary to an inborn deficiency of vitamin K epoxide reductase. This individual also had nasal hypoplasia, distal digital hypoplasia, and epiphyseal stippling on infant radiographs, all of which are virtually identical to features seen secondary to first-trimester exposure to coumarin derivatives. Therefore, by inference, the warfarin embryopathy is probably secondary to warfarin's primary pharmacologic effect (interference with vitamin K-dependent posttranslational carboxylation of glutamyl residues of various proteins) and may result from undercarboxylation of osteocalcin or other vitamin K-dependent bone proteins.

1-Carboxyglutamic Acid↗
PubMed1987

Assembly and alignment of fibronectin-coated gold beads into fibrils by human skin fibroblasts.

The assembly of fibronectin into fibrils was monitored by high voltage electron microscopy using 18 nm colloidal gold beads bound to fibronectin (Au18-fibronectin) or an amino terminal 70 kd fragment of fibronectin (Au18-70 kd) that blocks the incorporation of fibronectin into disulfide bonded fibrils. Subconfluent cultures of human skin fibroblasts were incubated with the colloidal gold complexes for 0.25, 0.5, 1.5 and 5 h. In fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragments for 0.25 and 0.5 h, the complexes of Au18-fibronectin and Au18-70 kd fragment were observed bound to the cell surface in clusters near bundles of intracellular microfilaments, usually along the lateral edges of the fibroblasts. Fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragment for 1.5 and 5 h showed a more linear arrangement of the Au18-fibronectin and Au18-70 kd fragment along the lateral and retracting edges and on filopodia. This alignment of Au18-fibronectin into linear arrangements on the fibroblast cell surface suggests that the assembly of fibronectin fibrils occurs on specific regions of the fibroblast.

Actin Cytoskeleton↗
PubMed1986

Thrombospondin is a substrate for blood coagulation factor XIIIa.

Thrombospondin (TSP) is released from alpha granules of activated platelets, binds to platelet surfaces, and copolymerizes with fibrin. In the present experiments, we investigated the action of factor XIIIa (plasma transglutaminase) on TSP. Factor XIIIa catalyzed incorporation of [14C]putrescine into soluble TSP and ligation of TSP to itself and to fibrin intermediates. Proteolytic digestion of [14C]putrescine-labeled TSP with trypsin or thrombin yielded a labeled disulfide-bonded core of 90 or 120-130 kilodalton (kDa) subunits, labeled fragments of less than 10 kDa, and an unlabeled 30-kDa heparin-binding fragment, indicating the presence of multiple factor XIIIa reactive glutaminyl residues located in several domains of the molecule. TSP became ligated in fibrin clots formed from amidinated fibrinogen, i.e., fibrin that could not contribute lysyl residues to factor IIIa catalyzed cross-links. The disulfide-bonded core of TSP formed upon thrombin digestion copolymerized with fibrin as efficiently as intact TSP. However, a lower proportion of the disulfide-bonded core became ligated. These results indicate that TSP, both in clots and in solution, contributes glutaminyl and lysyl residues to factor XIIIa catalyzed ligation. Cross-linking may be important in stabilizing interactions among TSP, fibrinogen, or fibrin and other molecules in hemostatic plugs.

Blood Platelets↗
PubMed1986

Effects of thrombospondin on fibrin polymerization and structure.

Thrombospondin (TSP) is a trace protein in plasma but is released in high concentrations from alpha-granules of activated platelets during hemostasis. It binds to the platelet membrane and becomes incorporated into fibrin clots. A variety of approaches were taken to learn the effects of TSP on fibrin polymerization and structure. 125I-TSP and 125I-fibrinogen were used to study the effect of TSP concentration on the extent of TSP and fibrin incorporation. Turbidity at 600 nm was used to monitor the time course of polymerization. Wavelength dependence of the turbidity was used to calculate the mass to length ratio, fiber diameter, and fiber density of fibrin formed in the presence and absence of TSP. Morphologies of control and TSP-containing clots were examined by electron microscopy following critical point drying. The initial TSP concentration influenced the amount of TSP incorporated but did not alter the extent of fibrin polymerization. TSP, in a concentration-dependent manner, reduced the lag time to turbidity rise and caused formation of more numerous but thinner fibers. Except for their diameter, these fibers were identical to fibers of control fibrin in terms of density and morphology. It is proposed that TSP interacts with fibrin intermediates to accelerate fiber growth, perhaps by serving as a trifunctional branching unit during network formation. The properties of fibrin around aggregating platelets, therefore, may be influenced considerably by secreted TSP.

Blood Platelets↗
PubMed1986

The influence of preadsorbed canine von Willebrand factor, fibronectin and fibrinogen on ex vivo artificial surface-induced thrombosis.

We have examined the effects of preadsorption of several canine plasma proteins on surface-induced thrombogenesis in a canine ex vivo model. Our technique allowed determination of initial deposition and subsequent embolization of 51Cr-labeled platelets and 125I-fibrinogen onto and from polymeric arterio-venous shunts in non-anticoagulated canines. Segments of the tubing were removed at various time points between 2 and 120 minutes of blood contact for examination of the morphology of the thrombus by scanning electron microscopy. Thrombus deposition was measured on uncoated plasticized poly(vinyl chloride) (PVC) and PVC precoated with canine von Willebrand factor (vWF), fibronectin, partially purified fibrinogen (fibrinogen which contained vWF and fibronectin as impurities), or purified fibrinogen (fibrinogen which had been further purified to remove fibronectin and vWF). Preadsorption of all proteins studied enhanced the thrombogenic response relative to that of the uncoated surface. Precoating with vWF or partially purified fibrinogen resulted in the deposition of the greatest number of thrombi, and embolization was slower than on shunts precoated with canine fibronectin or purified fibrinogen. The deposition-embolization profiles for the fibronectin and purified fibrinogen-coated surfaces were similar. The amount and time sequence of initial adhesion and spreading of platelets was related to the extent and time sequence of peak thrombus formation. The partially purified fibrinogen-coated and vWF-coated surfaces had more adhered and spread platelets at the earliest time points and a greater number of larger thrombi at the peak deposition times. The slowest rate of platelet adhesion and spreading was seen on the purified fibrinogen-coated surface. White blood cells were present very early on surfaces precoated with vWF and partially purified fibrinogen, and were present prior to embolization on all surfaces. Major conclusions from this work indicate that, although fibrinogen and fibronectin promote thrombogenesis when adsorbed to a surface, vWF is even more active in promoting platelet deposition and in anchoring thrombi to the surface of biomaterials. Thus, differences in vWF adsorption to biomaterials may be a determinant of surface-induced thrombogenesis.

Adsorption↗
PubMed1986

Diagnostic efficacy of six plasma proteins in evaluating consumptive coagulopathies. Use of receiver operating characteristic curves to compare antithrombin III, plasminogen, alpha 2-plasmin inhibitor, fibronectin, prothrombin, and protein C.

Six coagulation proteins were measured in 79 consecutive patients referred to the coagulation service for suspected disseminated intravascular coagulation. Antithrombin III, plasminogen, and alpha 2-plasmin inhibitor were measured with fluorescent substrate assays. Fibronectin, prothrombin, and protein C were measured with electroimmunoassays. Using history and physical findings and the results of a coagulation screen (prothrombin time, partial thromboplastin time, fibrinogen, fibrin[ogen] degradation products, platelet count, and peripheral smear), the 79 patients were classified into five categories: no disseminated intravascular coagulation (n = 21), elevated fibrin(ogen) degradation products without other evidence of coagulopathy (n = 44), defibrination syndrome (n = 9), microangiopathic thrombocytopenic purpura (n = 4), and primary fibrinolysis (n = 1). Because the sensitivity and specificity of each of the proteins could not easily be compared, receiver operating characteristic (ROC) curves and areas under the ROC curves were calculated for each of the six proteins as well as for the tests of the coagulation screen. The ROC curves indicated that, apart from plasminogen, the other coagulation proteins provided little additional information about the classification of the coagulopathy.

Antithrombin III↗
PubMed1986

Effects of thrombin on the fibronectin-containing extracellular matrix.

The effects of human alpha-thrombin on the incorporation of exogenous human fibronectin into the extracellular matrix of cultured human skin fibroblasts were studied. Thrombin had no effect on the saturable cell-surface receptor for soluble fibronectin. Thrombin, however, did digest fibronectin bound reversibly to the cell surface and fibronectin incorporated irreversibly into the extracellular matrix. The products of thrombin digestion of cell surface and matrix-bound fibronectin were similar to the products of thrombin digestion of soluble fibronectin. These results indicate that fibronectin can potentially modulate some of the effects of thrombin on cells.

Cells, Cultured↗
PubMed1986

Epsilon-aminocaproic acid in the treatment of patients with acute promyelocytic leukemia and acquired alpha-2-plasmin inhibitor deficiency.

Patients with acute promyelocytic leukemia often develop bleeding diatheses during treatment. In seven patients who had this disease, the plasma level of alpha-2-plasmin inhibitor was the best predictor of severity of coagulopathy and bleeding. Clinical bleeding occurred when alpha-2-plasmin inhibitor levels measured less than 30% of normal levels. Patients with acute promyelocytic leukemia who had acquired deficiencies of alpha-2-plasmin inhibitor were considered to have deficits similar to those in persons congenitally deficient in alpha-2-plasmin inhibitor, and were assumed to be at increased risk for bleeding. Treatment with the fibrinolytic inhibitor, epsilon-aminocaproic acid, along with heparin resulted in prompt cessation of bleeding, reversal of laboratory evidence of fibrinolysis, and a decreased need for blood product support. The only thrombotic complication--thrombosis around a central venous catheter--resolved when treatment with epsilon-aminocaproic acid was discontinued. Epsilon-aminocaproic acid is a safe and effective therapy for those patients with acute promyelocytic leukemia who develop coagulopathy associated with low levels of alpha-2-plasmin inhibitor.

Aminocaproates↗
PubMed1986

On the identity of vitronectin and S-protein: immunological crossreactivity and functional studies.

Vitronectin (serum spreading factor), a major serum cell adhesion molecule, was compared with S-protein, the inhibitor of the C5-9 membrane attack complex. Data from the literature indicate that S-protein and vitronectin are alpha globulins with the same aminoterminal residues, amino acid compositions, and concentrations in normal plasma (150 to 250 micrograms/mL). Both proteins have been reported to interact with the thrombin-antithrombin complex. The cDNA sequences of vitronectin and S-protein were recently determined and found to be almost identical. In the present studies, rabbit-anti-S-protein and a monoclonal antibody to vitronectin both recognized 65,000- and 75,000-molecular weight (mol wt) polypeptides when plasma or serum proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. The 65,000 and 75,000-mol wt polypeptides bound more avidly from serum than plasma to monoclonal anti-vitronectin or heparin coupled to agarose. The presence or absence of the polypeptides constituted a major difference between the heparin-binding proteins of serum and plasma. When complement-activated serum and unactivated serum were separated by gel filtration, vitronectin coeluted with C9 in high-mol-wt fractions of activated serum but not unactivated serum. Purified S-protein was recognized by the monoclonal antibody to vitronectin and promoted spreading of human skin fibroblasts. Both vitronectin and S-protein were degraded by thrombin. On the basis of immunological and functional, as well as biochemical, properties, therefore, S-protein and vitronectin are the same.

Glycoproteins↗
PubMed1986

Serologic and pathologic studies of mice immunized with homologous fibronectin.

It is demonstrated that BALB/c/DUB mice receiving chronic intraperitoneal injection of low doses of murine native dimeric or denatured multimeric or monomeric serum fibronectin in the absence of adjuvant produce a humoral autoimmune response to mouse fibronectin and have significant levels of circulating immune complexes. With the use of the Western immunoblotting technique, sera of mice immunized with native or sodium dodecyl sulfate (SDS)-treated fibronectin reacted with native, reduced and alkylated, and heat-and SDS-treated fibronectin. Sera from mice injected with reduced and alkylated fibronectin, however, reacted preferentially with reduced and alkylated fibronectin. In a competitive inhibition ELISA, the reactivity of immune sera from mice immunized with reduced and alkylated fibronectin to insolubilized reduced and alkylated fibronectin could be inhibited (66%) by preincubation with soluble reduced and alkylated fibronectin, but not by preincubation with native or SDS-treated fibronectin (less than 25%). Glomerular mesangial electron-dense deposits were observed in animals given injections of all three forms of fibronectin, but not in saline-injected animals. Indirect immunoperoxidase studies suggested that these dense deposits were at least partially immune in nature, because the authors were able to demonstrate binding in vivo of mouse IgG.

Animals↗