©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin Receptor-mediated Endocytosis of Vitronectin Is Protein Kinase C-dependent (*)

(Received for publication, May 12, 1995)

Tracee Scalise Panetti (§) Sarah A. Wilcox Carol Horzempa Paula J. McKeown-Longo (¶)

From the Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Previous studies have demonstrated that the alpha(v)beta(5) integrin receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J.(1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J.(1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the beta(5) integrin or exogenous heparin, suggesting that both integrin receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha(v)beta(5) integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-alpha(v)beta(5) antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the alpha(v)beta(5) integrin following activation of protein kinase C.


INTRODUCTION

Vitronectin is a conformationally labile plasma protein, which is found in both plasma and the extracellular matrix. A variety of functions have been described for vitronectin including mediating cell adhesion, regulating the activity of both thrombin and plasminogen activator, as well as modulating the membrane attack complex of complement(3, 4) . The Arg-Gly-Asp (RGD) sequence in vitronectin can interact with either the alpha(v)beta(3) or the alpha(v)beta(5) integrin receptor on the cell surface to mediate cell adhesion(5, 6) . Vitronectin is also the primary binding site for plasminogen activator inhibitor, Type I (PAI-1) (^1)in the extracellular matrix(7, 8, 9) . PAI-1 bound to vitronectin can inhibit both plasminogen activator (10, 11) as well as thrombin(12, 13, 14) , suggesting that matrix vitronectin may be important in the regulation of hemostasis.

Vitronectin has been identified in at least two conformations(15) . Native vitronectin is found in the plasma in a monomeric form that binds weakly to heparin(16, 17) . Treatment of vitronectin with denaturants, such as urea, results in a multimeric form of vitronectin with increased affinity for heparin and with an exposed epitope for the 8E6 monoclonal antibody(17, 18) . Such multimeric forms of vitronectin have been identified in platelet releasate(18) . Native vitronectin forms complexes with components of the coagulation and complement cascades. The interaction of vitronectin with thrombin-serpin complexes, as well as with the terminal complement complex C5b-9, exposes both the heparin binding domain and the 8E6 epitope in vitronectin(15, 19, 20) . Thus, the interaction of vitronectin with these complexes triggers the conformational changes seen in the multimeric form of vitronectin.

The binding of multimeric vitronectin as well as the ternary complex of vitronectin-thrombin-antithrombin to the surface of vascular endothelial cells has been shown to be dependent on the heparin binding domain of vitronectin(21, 22) . The binding of vitronectin to the endothelial cell surface is sensitive to heparatinase and colocalizes with proteoglycans(21, 22) , suggesting that vitronectin-containing complexes bind to heparan sulfate proteoglycans on the cell surface. In fibroblast cell layers, multimeric and native vitronectin bind to the same site in the cell layer, and localize to extracellular fibrils (23) . Fibroblasts internalize the multimeric vitronectin and direct it to lysosomes for degradation(1) . This endocytic process, but not initial binding to the matrix, is inhibited by exogenous heparin and dependent on the interaction of vitronectin with the alpha(v)beta(5) integrin receptor(1, 2) , suggesting that endocytosis of vitronectin requires the interaction of vitronectin with both the alpha(v)beta(5) integrin and a species of heparan sulfate proteoglycan. Binding to the extracellular matrix is a prerequisite for internalization as multimeric vitronectin does not appear to be degraded from the fluid phase(1, 23) . Native vitronectin is not internalized and degraded but remains bound in the native form (23) to the extracellular matrix(1, 23) . Addition of -thrombin, which is known to alter the conformation of native vitronectin, triggers the degradation of native vitronectin by the cells(1) . The fact that degradation requires the exposure of vitronectin's heparin binding domain and that degradation is inhibited by exogenous heparin is consistent with the hypothesis that heparan sulfate proteoglycans are involved in the endocytosis of vitronectin.

The present studies were conducted to determine the intracellular signaling pathways involved in the endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha(v)beta(5) integrin in the activation of these signaling pathways. Our data suggest that vitronectin internalization is mediated by the alpha(v)beta(5) integrin receptor, following a proteoglycan-dependent activation of protein kinase C.


MATERIALS AND METHODS

Cell Culture

Human foreskin fibroblasts were a gift from Dr. Lynn Allen-Hoffmann (University of Wisconsin, Madison, WI). The cells were cultured in Ham's F-12 nutrient medium (F-12) (Life Technologies) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (100 units/ml), and streptomycin (100 µg/ml). Fibroblasts were plated in T-75 flasks (Becton Dickinson Laboratories, Lincoln Park, NJ) at 5 10^5 cells/flask and reached confluence in 5-7 days. Experiments were done on cells between passages 4 and 12. For experiments, fibroblasts were grown to confluence in 12- or 24-well plates. After the cells achieved confluence, fresh ascorbic acid (50 µg/ml) was added to the medium daily for three to four days prior to use in experiments. The addition of ascorbate was found to increase the binding of vitronectin to the cell layer.

Purification and Iodination of Vitronectin

Conformationally altered vitronectin was purified from human plasma by heparin affinity chromatography according to the method of Yatohgo et al.(24) as described previously(1) . Conformationally altered vitronectin (400 µg) was labeled with NaI (DuPont NEN) using either chloramine T or lactoperoxidase and glucose oxidase (Sigma) as described previously(1, 2) . Iodinated vitronectin was stabilized with 1% bovine serum albumin (BSA) and 0.1 mM phenylmethylsulfonyl fluoride, dialyzed against phosphate-buffered saline (PBS), and frozen at -80 °C until use. Integrity of the labeled protein was assessed by gel electrophoresis and autoradiography.

Binding of Vitronectin to Fibroblast Monolayers

Monolayers of human skin fibroblasts were incubated at 37 °C with F-12 containing 0.2% BSA and 620 ng/ml I-vitronectin. To measure bound vitronectin, medium containing labeled protein was removed, cultures were rinsed three times with PBS, and the monolayer was solubilized in 1 N NaOH. Radioactivity was determined by scintillation counting.

Degradation of Vitronectin by Fibroblasts

Cells were incubated at 37 °C with F-12 containing 0.2% BSA and 620 ng/ml I-vitronectin. Degradation of I-vitronectin by the cultures was monitored by the appearance of radioactivity in the medium that was soluble in 10% trichloroacetic acid. To estimate background (i.e. cell-independent) degradation, blank culture dishes were preincubated with complete medium (F-12, 10% fetal bovine serum) for 1 h at 37 °C, rinsed, and subjected to the same procedure as above. Vitronectin degradation in the absence of cells was less than 1% over the time course of the experiment. Background degradation was subtracted from total trichloroacetic acid-soluble radioactivity. Radioactivity was determined in a counter.

To examine the role of integrins in the regulation of vitronectin degradation, turnover of I-vitronectin by the cell layers was measured in the presence of RGDS or RGES peptides (Sigma) or a monoclonal antibody against the alpha(v)beta(5) integrin receptor, P1F6 (Life Technologies, Inc.). To determine the role of glycosaminoglycans in vitronectin metabolism, degradation of I-vitronectin was examined in the presence of soluble heparin (Sigma). In other experiments, heparan sulfate was removed from the cell surface by pretreatment with 5 units/ml heparinase I (heparinase) (Sigma), for 1 h. These enzyme amounts were similar to those used earlier to demonstrate the binding of vitronectin-thrombin-anti-thrombin III complexes to endothelial cell surface proteoglycans(21) . Fresh enzyme was added with the radiolabeled vitronectin and remained in the culture medium for the duration of the experiment. The integrity of I-vitronectin in the culture medium in the presence of heparinase was verified by SDS-polyacrylamide gel electrophoresis and autoradiography. All experiments were done at least three times. Data presented are from one representative experiment done in triplicate.

Modulators of Protein Kinase

The protein kinase C inhibitors calphostin C and staurosporine as well as the protein kinase A inhibitor KT5720 (LC Laboratories, Woburn, MA) were solubilized in dimethyl sulfoxide (Me(2)SO). The stock solution of inhibitors was diluted in F-12 with 0.2% BSA and incubated with the fibroblast monolayers for 1 h before the addition of PMA or I-vitronectin. Calphostin C was light-activated according to the protocol of Bruns et al.(25) . The protein kinase C activator, PMA, was solubilized in Me(2)SO, and diluted to a working concentration of between 50 and 500 nM in F-12 with 0.2% BSA.

Indirect Immunofluorescence

Vitronectin was localized within cell monolayers by indirect immunofluorescence. Cell layers were pretreated with PMA, calphostin C, or staurosporine 1 h prior to addition of exogenous vitronectin (12.5 µg/ml). The monolayers were rinsed with PBS and fixed with 3% paraformaldehyde. Intracellular vitronectin was visualized by treating fixed cells with -20 °C acetone for 10 min. Cell layers were incubated with hybridoma medium (diluted 1:10 in PBS), which contained a monoclonal antibody (8E6) directed against human vitronectin (generous gift from Dr. Deane F. Mosher, University of Wisconsin, Madison, WI). The secondary antibody was rhodamine isothiocyanate-conjugated goat anti-mouse IgG (Cappel, Organon Teknika Corp., Durham, NC). Control studies using no primary antibody were negative. Samples were viewed using a Nikon Microphot microscope equipped with epifluorescence.


RESULTS

Vitronectin Degradation Is Modulated by Effectors of Protein Kinase C

Previously we have demonstrated that conformationally altered, multimeric vitronectin was internalized from the extracellular matrix by receptor-mediated endocytosis and subsequently degraded in lysosomes (1) . In these studies soluble heparin, RGD peptides, and antibodies against the beta(5) integrin were all shown to block the internalization and degradation of vitronectin, without affecting the initial binding of vitronectin to the extracellular matrix(1, 2) . Because previous studies have shown that the beta(5) subunit of the vitronectin receptor may be a substrate for protein kinase C (26, 27) , we asked whether protein kinase C may be part of the intracellular signaling pathways involved in the regulation of vitronectin endocytosis. As shown in Fig. 1, vitronectin degradation was up-regulated 2.5-fold by a 1-h pretreatment of cells with the activator of protein kinase C, PMA. PMA concentrations between 50 and 500 nm gave identical results (data not shown). Baseline levels of vitronectin degradation were decreased about 60% in the presence of 5 mM staurosporin, an inhibitor of protein kinase C.


Figure 1: Effect of PMA and staurosporin on vitronectin (Vn) degradation. Human fibroblasts were pretreated with 500 nM PMA, Me(2)SO control (C), or 5 mM staurosporine (Staurosp'n) for 1 h. F-12 containing I-vitronectin and 0.2% BSA was added to the cells for 5 h. Degradation of vitronectin was determined by measuring the trichloroacetic acid-soluble radioactivity in the culture medium. Data are expressed as mean ± S.E.



To examine further the role of protein kinase C in vitronectin degradation, confluent fibroblast monolayers were pretreated with increasing doses of the protein kinase C inhibitors staurosporine or calphostin C for 1 h. Degradation of I-vitronectin was measured over 5 h in the presence of increasing concentrations of the drugs. Calphostin C inhibited vitronectin degradation in a dose-dependent manner, with half-maximal inhibition occurring at 0.05 µM (Fig. 2A). Staurosporine also inhibited vitronectin degradation (Fig. 2B). Neither staurosporine nor calphostin C inhibited vitronectin binding to the cell layer. The protein kinase A inhibitor, KT5720, had no effect on vitronectin degradation (data not shown). These data demonstrate that vitronectin degradation is protein kinase C-dependent.


Figure 2: Effect of calphostin C (A) and staurosporine (B) on I-vitronectin binding and degradation. Confluent fibroblasts were incubated with increasing concentrations of calphostin C, staurosporine, or vehicle (Me(2)SO) for 1 h. F-12 containing I-vitronectin, 0.2% BSA, and increasing concentrations of protein kinase C inhibitors was added to the cells. After 5 h, the trichloroacetic acid-soluble radioactivity in the culture medium was determined as an index of degradation. To measure vitronectin binding, the cell layers were rinsed and solubilized in 1 N NaOH. Radioactivity was determined by scintillation. Data are expressed as percent of control, where binding and degradation in the absence of drugs were set at 100%.



PMA Overcomes the Inhibition of Vitronectin Degradation by Heparin and Heparinase

Our earlier studies have shown that degradation of vitronectin is blocked by exogenous heparin, suggesting a role for cell surface proteoglycans in the internalization of vitronectin. These earlier observations, coupled with the ability of PKC effectors to regulate vitronectin degradation, suggest that one possible mechanism by which proteoglycans and alpha(v)beta(5) integrin could interact to mediate internalization of vitronectin might be through a proteoglycan-dependent activation of protein kinase C. We tested whether the inhibition of vitronectin degradation by exogenous heparin might be overcome with the addition of PMA. To block vitronectin binding to cell surface proteoglycans, cell monolayers were either incubated with exogenous heparin or glycosaminoglycans were digested with heparinase as described under ``Materials and Methods.'' Heparinase-treated cell layers and non-digested cell layers were pretreated with PMA for 1 h. The monolayers were then incubated with I-vitronectin for 5 h in the presence of heparinase or soluble heparin, and degradation was determined. As demonstrated previously(1) , heparin inhibited the degradation of vitronectin to trichloroacetic acid-soluble radioactivity (Fig. 3) but did not affect the binding of vitronectin to cell layers (data not shown). Heparinase also blocked the degradation of vitronectin (Fig. 3) but did not affect binding to the cell layers (data not shown), confirming the role of proteoglycans in the degradation of vitronectin. PMA was able to reverse the inhibition of vitronectin degradation seen in the presence of heparin or in heparinase-treated cells (Fig. 3). The PMA-dependent increase in vitronectin degradation on heparinase-treated cells could be blocked using staurosporine (data not shown). The ability of staurosporine to block the PMA reversal of heparinase treatment demonstrates that a protein kinase C signaling event generated by PMA triggers vitronectin degradation.


Figure 3: PMA overcomes the inhibition of vitronectin degradation by heparin and heparinase (Hep'ase). Confluent fibroblasts were pretreated with 5 units/ml heparinase for 1 h prior to receiving PMA (500 nM) or vehicle (Me(2)SO). After a 1-h treatment with PMA, F-12 containing I-vitronectin and 0.2% BSA was added to monolayers in the presence of heparin (100 µg/ml) or heparinase (5 units/ml). After 5 h, the trichloroacetic acid-soluble radioactivity in the culture medium was measured as an index of degradation. Data are expressed as mean ± S.E.



Protein Kinase C Regulates Vitronectin Internalization

Indirect immunofluorescence was used to localize vitronectin in cell layers treated with various effectors of protein kinase C. In control cells, vitronectin was localized to intracellular vesicles (Fig. 4A), and localization within vesicles was blocked by exogenous heparin (Fig. 4B). Cells treated with calphostin C showed little vesicular staining (Fig. 4C), consistent with data showing that staurosporine blocked vitronectin degradation (Fig. 2). The addition of PMA to heparin-treated cells resulted in a reappearance of vitronectin within vesicular structures (Fig. 4D), indicating that the PMA-dependent increase in vitronectin degradation seen in heparin-treated cells (Fig. 3) results from the ability of PMA to trigger vitronectin internalization. The PMA-induced increase in vitronectin internalization was blocked by calphostin C (Fig. 4E). Identical results were obtained when staurosporine was used to inhibit protein kinase C (data not shown). These data indicate that effectors of protein kinase C modulate vitronectin degradation by regulating the internalization of vitronectin. These studies demonstrate that under conditions where protein kinase C activity is increased, vitronectin degradation does not require interaction of vitronectin with cell surface heparan sulfate proteoglycans.


Figure 4: Effect of PMA and calphostin C on vitronectin internalization. Human fibroblasts were pretreated with either 500 nM PMA or 1 µM calphostin for 1 h and then incubated with vitronectin (12.5 µg/ml) for 5 h in F-12 containing 0.2% BSA in the presence or absence of 1 mg/ml heparin. After the cells were fixed and permeabilized, vitronectin was visualized by indirect immunofluorescence using the 8E6 monoclonal antibody. A, control monolayers; B, monolayers incubated with heparin; C, monolayers pretreated with calphostin; D, monolayers pretreated with PMA and incubated with heparin; E, monolayers pretreated with calphostin and PMA and then incubated with heparin. Bar = 20 µM.



PMA Has No Effect on Inhibition of Vitronectin Degradation by RGDS Peptides or Antibodies to the alpha(v)betaIntegrin Receptor

To demonstrate that vitronectin binding to the alpha(v)beta(5) vitronectin receptor was required for vitronectin degradation, we examined the effect of PMA on inhibition of vitronectin degradation by peptides and antibodies to the vitronectin receptor. Confluent fibroblasts were pretreated with the phorbol ester PMA for 1 h, and then vitronectin degradation was examined in the presence of RGDS peptides or antibodies to the alpha(v)beta(5) integrin receptor. RGDS peptides and antibodies to the alpha(v)beta(5) vitronectin receptor inhibited vitronectin degradation by 80-85% (Fig. 5). The control peptide RGES had no effect on vitronectin degradation (data not shown). PMA treatment had no effect on the inhibition of vitronectin degradation by the antibodies or peptides. The inability of PMA to overcome the inhibition of vitronectin degradation by RGDS and anti-alpha(v)beta(5) antibodies suggests that the binding of vitronectin to the alpha(v)beta(5) integrin receptor is required for internalization and degradation.


Figure 5: PMA has no effect on inhibition of vitronectin degradation by RGDS peptides or antibodies to the alpha(v)beta(5) integrin receptor. Human fibroblasts were pretreated with PMA (500 nM) or vehicle (Me(2)SO) for 1 h. F-12 containing I-vitronectin and 0.2% BSA was added to the cells for 3 h in the presence of RGDS peptides or antibodies to the alpha(v)beta(5) integrin receptor (P1F6). Degradation of vitronectin was determined by measuring the trichloroacetic acid-soluble radioactivity in the culture medium. Data are expressed as mean ± S.E.




DISCUSSION

Previously we have shown that conformationally altered vitronectin was cleared from the extracellular matrix by receptor-mediated endocytosis and subsequently degraded in lysosomes(1) . The fact that degradation was blocked by pretreating cell layers with heparinase or by antibodies against the alpha(v)beta(5) integrin indicated that the binding of vitronectin to cell surface heparan sulfate proteoglycans as well as the alpha(v)beta(5) integrin was required for vitronectin's degradation. Localization of vitronectin by indirect immunofluorescence indicated that soluble heparin blocked vitronectin degradation by preventing the internalization of vitronectin into intracellular vesicles. These data suggest that the binding of vitronectin to both heparan sulfate proteoglycans as well as the alpha(v)beta(5) integrin trigger vitronectin's internalization by the cell.

Binding of vitronectin to cell surface proteoglycans has been demonstrated on endothelial cells(21, 22) . Binding of vitronectin multimers to proteoglycans on the surface of endothelial cells has been proposed to mediate the internalization and transcytosis of plasma vitronectin to the subendothelium(22) . Our studies suggest that, in fibroblast cells, the actual internalization event may only require the alpha(v)beta(5) integrin. The addition of PMA to the cells could substitute for the proteoglycan binding event, but not the integrin binding event. These findings are consistent with a mechanism by which the proteoglycan acts as a signaling molecule triggering the alpha(v)beta(5)-mediated internalization of vitronectin. Recent studies have suggested that the alpha(v)beta(3) and alpha(v)beta(5) integrin receptors may function in the internalization of adenovirus (28, 29) and fibrinogen(30) .

Degradation of vitronectin was sensitive to inhibitors of protein kinase C. The doses of calphostin C used to block vitronectin degradation were within the range of doses used in earlier studies on fibroblasts, which have documented a role for protein kinase C in cell spreading (31, 32) and matrix assembly(33) . Indirect immunofluorescence showed that both calphostin C and staurosporine blocked degradation by preventing internalization of vitronectin. The initial binding of vitronectin to the cell layers was not inhibited by either drug. PMA was able to increase base-line degradation of vitronectin. PMA was also able to overcome the inhibition of vitronectin degradation in the presence of heparin and heparinase. PMA, staurosporine, and calphostin C appear to prevent vitronectin degradation by regulating vitronectin internalization, rather than by regulating the flow of vesicular traffic within the cells. The absence of vitronectin-containing endocytic vesicles in the presence of calphostin C and staurosporine and the return of endocytic vesicles with PMA in heparin-blocked cells is consistent with the idea that the protein kinase C signal regulates internalization.

The ability of PMA to activate the alpha(v)beta(5)-dependent internalization of vitronectin suggests that alpha(v)beta(5) function can be regulated by protein kinase C. Whether PMA is triggering the internalization of vitronectin bound to alpha(v)beta(5) or whether PMA is affecting the affinity of the alpha(v)beta(5) for vitronectin has not yet been determined. Protein kinase C activation has been shown to increase the rate of internalization of the transferrin receptor (34) and to increase the formation of clathrin coated vesicles in Schizosaccharomyces pombe(35) , suggesting that protein kinase C is involved in the up-regulation of the endocytic process. Alternatively, PMA has been shown to alter the binding properties of several integrins for their ligands, suggesting that protein kinase C may be a component of the ``inside-out'' signaling pathways which affect the interaction of integrins with their ligands (reviewed in (36) and (37) ). PMA-dependent affinity modulation of the alpha(5)beta(1)(38) , alpha(6)beta(1)(39) , and alpha(2)beta(1)(40) has been described. The specific physiological mediators that activate protein kinase C-dependent integrin modulation are not well understood. Thrombin (41) and ligation of T-cell receptor (42) can activate integrin receptors on platelets and leukocytes. Activation of both the alphabeta(3) and the alpha(L)beta(2) can also be stimulated by phorbol esters(43, 44) , indicating that protein kinase C is part of the signaling pathway between transmembrane signaling receptors(45, 46) . Recent studies have suggested that the alpha(v)beta(3) integrin receptor may regulate the phagocytic function of the alpha(5)beta(1) receptor via a protein kinase C-dependent pathway(47) .

Our study presents the first evidence that signaling pathways from cell surface proteoglycans may regulate integrin function. The inhibition of alpha(v)beta(5)-dependent internalization of vitronectin by both exogenous heparin as well as heparinase treatment suggests that internalization requires the interaction of vitronectin with heparan sulfate proteoglycans on the cell surface. Previous studies have suggested that heparan sulfate proteoglycans may transduce signals from the extracellular matrix that promote the formation of focal contacts during cell adhesion to fibronectin (48, 49, 50) . The fact that PMA can subserve the vitronectin-proteoglycan binding suggests that the interaction of vitronectin with heparan sulfate proteoglycans may contribute to an increase in protein kinase C activity necessary to trigger alpha(v)beta(5)-dependent endocytosis. Regulation of vitronectin endocytosis may be important in the control of hemostasis. Native vitronectin bound to the extracellular matrix in a non-wound environment may mediate cell adhesion through interaction with cell surface integrin receptors. During tissue injury, the interaction of native vitronectin with PAI-1 and thrombin would be expected to result in a ternary complex that is multivalent for both integrin and proteoglycan binding. The interaction of such a multivalent ligand with cell surface proteoglycans as well as the alpha(v)beta(5) integrin may result in the clearance of vitronectin thrombin-serpin complexes from the pericellular space. Further studies are needed to delineate the role of heparan sulfate proteoglycans in receptor-mediated endocytosis of vitronectin.


FOOTNOTES

*
This work was supported by Grant CA-58626 from the National Institutes of Health, Grant AHA-93013270 from the American Heart Association, and Predoctoral Training Grant HL-07194 from the NHLBI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Depts. of Medicine and Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706.

To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-5666; Fax: 518-262-5669.

^1
The abbreviations used are: PAI-1, plasminogen activator inhibitor-type 1; F-12, Ham's F-12 nutrient medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate.


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