Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes

Abstract Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK−), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.

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Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes

Blood ◽

10.1182/blood.v91.7.2305.2305_2305_2312 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2305-2312 ◽

Cited By ~ 2

Author(s):

Triantafyllos Chavakis ◽

Sandip M. Kanse ◽

Barbara Yutzy ◽

H. Roger Lijnen ◽

Klaus T. Preissner

Keyword(s):

Monoclonal Antibody ◽

Extracellular Matrix ◽

Cell Surface ◽

Plasminogen Activator ◽

Umbilical Vein ◽

Urokinase Receptor ◽

Enzyme Linked Immunosorbent Assay ◽

Tissue Remodelling ◽

Amino Terminal ◽

Plasminogen Activator Activity

Urokinase-type-plasminogen activator (uPA) and its receptor are localized in the vessel wall where they are involved in cellular activation and remodelling processes. Besides the cell surface glycolipid (GPI)-anchored urokinase receptor (uPAR), which binds uPA with high affinity, recent evidence points to the existence of soluble uPAR (suPAR), as well. In the present study, the origin, binding mechanism, and cellular effects of suPAR were examined. Under basal conditions human vascular smooth muscle cells (HVSMC), human umbilical vein endothelial cells (HUVEC), and monocytic cells released 0.1 to 2 ng/mL suPAR, which was increased twofold to fivefold after phorbol ester (PMA) stimulation, as measured by a function-dependent enzyme-linked immunosorbent assay (ELISA). suPAR alone did not bind to HVSMC or HUVEC, but reduced cellular uPA binding by 50% to 70%. However, after removal of GPI-uPAR with phosphatidylinositol-specific phospholipase C, suPAR dose-dependently increased uPA binding by fourfold to fivefold. This increase in binding was completely inhibited by vitronectin (VN) and by a monoclonal antibody against VN, but not by other matrix proteins or antibodies. Thus, VN-mediated uPA binding to cells was regulated by the ratio of soluble to surface-associated uPAR. In a uPAR-deficient cell line (LM-TK−), suPAR increased uPA binding up to 10-fold, whereas the truncated receptor lacking the amino-terminal uPA-binding domain was ineffective. The formation of a ternary uPA/suPAR/VN-complex on the cell surface and the free extracellular matrix could be inhibited by a monoclonal antibody against VN, as well as by plasminogen activator inhibitor-1 (PAI-1). Moreover, VN-mediated binding of the uPA/suPAR-complex led to a fivefold increase in plasminogen activator activity. Through this novel pathway, VN concentrates the uPA/suPAR-complex to cell surfaces and extracellular matrix sites, leading to the accumulation of plasminogen activator activity required for cell migration and tissue remodelling processes.

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Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650304 ◽

1996 ◽

Vol 75 (03) ◽

pp. 497-502 ◽

Cited By ~ 10

Author(s):

Hadewijch L M Pekelharing ◽

Henne A Kleinveld ◽

Pieter F C.C.M Duif ◽

Bonno N Bouma ◽

Herman J M van Rijn

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Plasminogen Activator ◽

Binding Sites ◽

Umbilical Vein ◽

Single Step ◽

Plasminogen Activation ◽

Structural Homology ◽

Tissue Plasminogen ◽

Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

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SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA

10.1055/s-0038-1644425 ◽

1987 ◽

Cited By ~ 1

Author(s):

J Grøndahl-HANSEN ◽

N Agerlin ◽

L S Nielsen ◽

K Danø

Keyword(s):

Plasminogen Activator ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Mean Values ◽

Amino Terminal ◽

Type Plasminogen Activator ◽

Healthy Donors ◽

Urokinase Type Plasminogen Activator ◽

The Mean

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.

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Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4234-4242.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4234-4242

Author(s):

B L Allen-Hoffmann ◽

C L Crankshaw ◽

D F Mosher

Keyword(s):

Extracellular Matrix ◽

Growth Factor ◽

Cell Surface ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Human Fibroblasts ◽

Tgf Beta ◽

Surface Binding ◽

Amino Terminal ◽

Cell Surface Binding

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.

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A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽

10.1182/blood.v75.9.1794.1794 ◽

1990 ◽

Vol 75 (9) ◽

pp. 1794-1800 ◽

Cited By ~ 27

Author(s):

PJ Declerck ◽

HR Lijnen ◽

M Verstreken ◽

H Moreau ◽

D Collen

Keyword(s):

Monoclonal Antibody ◽

Human Plasma ◽

Plasminogen Activator ◽

Fibrin Clot ◽

Clot Lysis ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

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Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

Clinical Chemistry ◽

10.1093/clinchem/39.4.583 ◽

1993 ◽

Vol 39 (4) ◽

pp. 583-591 ◽

Cited By ~ 13

Author(s):

M J Hursting ◽

B T Butman ◽

J P Steiner ◽

B M Moore ◽

M C Plank ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Human Plasma ◽

Sandwich Elisa ◽

Peptide Sequence ◽

Carboxyl Terminus ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombotic Risk ◽

Prothrombin Fragment ◽

Amino Terminal

Abstract Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

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Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis

Blood ◽

10.1182/blood.v80.7.1758.1758 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1758-1764 ◽

Cited By ~ 28

Author(s):

J Kassis ◽

J Hirsh ◽

TJ Podor

Keyword(s):

Endothelial Cell ◽

Plasminogen Activator ◽

Plasma Levels ◽

Umbilical Vein ◽

Interleukin 1 ◽

Plasminogen Activator Inhibitor ◽

Enzyme Linked Immunosorbent Assay ◽

Type I ◽

Activator Inhibitor ◽

Pai 1

Abstract Postoperative fibrinolytic shutdown has been attributed to an increase in plasma levels of type I plasminogen activator inhibitor (PAI-1) activity and may contribute to postoperative venous thrombosis. The purpose of this study was to determine whether the postoperative increase in PAI-1 is contributed to by a plasma mediator(s) that stimulates PAI-1 synthesis and secretion by vascular endothelium. Plasma samples collected from patients (N = 11) before and after surgery for total hip replacement were (1) assayed for endogenous plasma PAI-1 antigen and activity, and (2) incubated with cultured human umbilical vein endothelial cells (HUVECs) and PAI-1 antigen and activity measured in the conditioned medium (CM). Eighteen hours after surgery, endogenous plasma levels of PAI-1 antigen and activity were increased by 225% (P = .003) and 190% (P = .04), respectively over the preoperative values. In addition, compared with preoperative plasma, postoperative plasma increased HUVEC secretion of PAI-1 antigen and activity by 99% (P = .001) and 66% (P = .002), respectively. This increase in HUVEC PAI-1 secretion reflects an increase in PAI-1 mRNA expression and protein biosynthesis as confirmed by metabolic radiolabeling, immunoprecipitation, and Northern blot analysis. Ultra- filtration experiments indicate that the postoperative plasma mediator(s) that stimulates HUVEC PAI-1 biosynthesis is in a molecular weight (MW) range of approximately 30 to 100 Kd. Heat treatment (56 degrees C; 30 minutes) of postoperative plasma abolished the induction of HUVEC PAI-1 production. Enzyme-linked immunosorbent assay and immunoneutralization experiments indicate that tumor necrosis factor- alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) do not contribute to the postoperative plasma effect on HUVEC PAI-1 synthesis. These observations demonstrate that postoperative patient plasma contains a factor(s) that may stimulate endothelial cell PAI-1 biosynthesis in vivo and thus mediate postoperative fibrinolytic shut- down.

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Glycosylphosphatidylinositol Anchored TFPI As Main Regulator of Factor X Activation On the Surface of Endothelial Cells.

Blood ◽

10.1182/blood.v120.21.2210.2210 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2210-2210

Author(s):

Michael Dockal ◽

Robert Pachlinger ◽

Angelina Baldin-Stoyanova ◽

Fabian Knofl ◽

Nadja Ullrich ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Tissue Factor ◽

Conflicts Of Interest ◽

Factor Viia ◽

Umbilical Vein ◽

Surface Proteins ◽

Enzyme Linked Immunosorbent Assay ◽

Factor X ◽

Extrinsic Pathway

Abstract Abstract 2210 Tissue factor pathway inhibitor (TFPI) is a key regulator of factor X (FX) activation in the extrinsic pathway of blood coagulation. TFPI inhibits FXa generation by formation of a quaternary complex consisting of factor VIIa (FVIIa), tissue factor (TF), FXa and TFPI. The main portion (∼80%) of TFPI in humans is reportedly associated with endothelial cells. We used human umbilical vein endothelial cells (HUVECs) as a model to obtain further insight into the function of TFPIα and the glycosylphosphatidylinositol (GPI) anchored TFPI form, which represents TFPIα bound to GPI-anchored surface proteins and/or TFPIβ. In contrast to TFPIα, which consists of 3 Kunitz domains (KD) and a basic C-terminal part, GPI-anchored TFPIβ lacks the third Kunitz domain (KD3) and the basic C–terminal region due to alternative splicing. In TFPIβ these two domains are replaced by a sequence that adds a GPI anchor to the protein linking it to the cell membrane. Treatment of HUVECs with phosphatidylinositol phospholipase C (PI-PLC) that cleaves GPI-anchors and subsequent fluorescence activated cell sorting (FACS) on living cells showed that GPI-anchored TFPI represents about 70–80% of cell surface TFPI. Staining of TFPI on and in fixed and permeabilized cells (total TFPI) demonstrated that GPI-anchored cell surface TFPI contributes to ∼20% of total cellular TFPI. Enzyme-linked immunosorbent assay (ELISA) showed that PI-PLC treatment released a TFPI protein lacking the KD3 and basic C-terminus. These findings strongly suggest that TFPIβ is the predominant GPI-anchored form of TFPI on HUVECs. FX activation assays performed on the cell surface of PI-PLC treated living HUVECs showed the importance of GPI-anchored TFPI on extrinsic Xase complex activity. PI-PLC treatment resulted in increased FX activation. Although GPI-anchored TFPI displays ∼70–80% of cell surface TFPI, overall FXa generation was increased only by ∼50%. In conclusion, HUVEC surface TFPI is predominantly TFPIβ, and GPI-anchored TFPI is the main but not sole regulator of FX activation on the surface of HUVECs. Disclosures: No relevant conflicts of interest to declare.

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Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody

Infection and Immunity ◽

10.1128/iai.68.3.1649-1654.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1649-1654 ◽

Cited By ~ 126

Author(s):

Yongmoon Han ◽

Marcia H. Riesselman ◽

Jim E. Cutler

Keyword(s):

Monoclonal Antibody ◽

Cell Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Survival Times ◽

Vaginal Infection ◽

Igg Antibody ◽

Disseminated Candidiasis

ABSTRACT We previously reported that a liposome-mannan vaccine (L-mann) ofCandida albicans induces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for aC. albicans cell surface β-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6.1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginal Candida CFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.

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Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures.

The Journal of Cell Biology ◽

10.1083/jcb.111.1.249 ◽

1990 ◽

Vol 111 (1) ◽

pp. 249-256 ◽

Cited By ~ 68

Author(s):

D M Peters ◽

L M Portz ◽

J Fullenwider ◽

D F Mosher

Keyword(s):

Monoclonal Antibody ◽

Extracellular Matrix ◽

Cell Surface ◽

Fluorescein Isothiocyanate ◽

Plasma Fibronectin ◽

Cultured Fibroblasts ◽

Human Fibroblast Cultures ◽

Fibroblast Cultures ◽

Extracellular Fibrils ◽

Cellular Fibronectin

Exogenous plasma and endogenous cellular fibronectins on the surface of cultured fibroblasts and in extracellular matrix fibrils were colocalized by fluorescent and high voltage immunoelectron microscopy. Fibroblast cultures grown in the presence or absence of cycloheximide were incubated with exogenous plasma fibronectin labeled with fluorescein isothiocyanate. A monoclonal antibody specific for the EIIIA sequence of cellular fibronectin was used to detect cellular fibronectin. A rabbit antifluorescein antibody identified fluoresceinated plasma fibronectin. In cultures incubated in the presence of cycloheximide, plasma fibronectin was bound to the cell surface and was assembled into extracellular fibrils. In cultures grown in the absence of cycloheximide, plasma and cellular fibronectins were observed in the same matrix fibrils and in the same locations on the cell surface. There was not, however, random admixture of the two proteins.

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