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J Biol Chem, Vol. 274, Issue 42, 30215-30221, October 15, 1999


Insulin-like Growth Factor-binding Protein-3 Binds Fibrinogen and Fibrin*

Phil G. CampbellDagger , Susan K. Durham§, James D. HayesDagger , Adisak Suwanichkul§, and David R. Powell§

From the Dagger  Institute for Complex Engineered Systems, Carnegie Mellon University, Pittsburgh, Pennsylvania 15212 and the § Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Following tissue injury, a fibrin network formed at the wound site serves as a scaffold supporting the early migration of stromal cells needed for wound healing. Growth factors such as insulin-like growth factor-I (IGF-I) concentrate in wounds to stimulate stromal cell function and proliferation. The ability of IGF-binding proteins (IGFBPs) such as IGFBP-3 to reduce the rate of IGF-I clearance from wounds suggests that IGFBP-3 might bind directly to fibrinogen/fibrin. Studies presented here show that IGFBP-3 does indeed bind to fibrinogen and fibrin immobilized on immunocapture plates, with Kd values = 0.67 and 0.70 nM, respectively, and competitive binding studies suggest that the IGFBP-3 heparin binding domain may participate in this binding. IGF-I does not compete for IGFBP-3 binding; instead, IGF-I binds immobilized IGFBP-3·fibrinogen and IGFBP-3·fibrin complexes with affinity similar to that of IGF-I for the type I IGF receptor. In the presence of plasminogen, most IGFBP-3 binds directly to fibrinogen, although 35-40% of the IGFBP-3 binds to fibrinogen-bound plasminogen. IGFBP-3 also binds specifically to native fibrin clots, and addition of exogenous IGFBP-3 increases IGF-I binding. These studies suggest that IGF-I can concentrate at wound sites by binding to fibrin-immobilized IGFBP-3, and that the lower IGF affinity of fibrin-bound IGFBP-3 allows IGF-I release to type I IGF receptors of stromal cells migrating into the fibrin clot.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factor-binding protein-3 (IGFBP-3),1 one of 6 structurally related IGFBPs that bind IGF peptides with high affinity, is a 29-kDa protein found in body fluids as multiple ~40-50-kDa forms due to differential glycosylation (1-3). IGFBP-3 is the most abundant serum IGFBP during extrauterine life, where it circulates with one IGF peptide and a single acid-labile subunit in a ~150-kDa ternary complex (1, 4, 5). Expression of IGFBP-3 by many tissues suggests that it is also available locally to modulate the autocrine/paracrine actions of the IGF peptides (1).

IGF-I is a 7.6-kDa protein with mitogenic, metabolic, differentiative, chemotactic, and anti-apoptotic effects (1, 6, 7). Since IGFBPs such as IGFBP-3 have higher affinity for IGF-I than does the type I IGF receptor, it is not surprising that IGFBP-3 can inhibit IGF action (1, 8). However, IGFBPs bound to extracellular matrix may have lower IGF affinity (9-11). By binding an 18-amino acid heparin binding domain (HBD), which is highly conserved in IGFBP-3 and the closely related protein IGFBP-5, heparin and certain other glycosaminoglycans apparently change the conformation of these IGFBPs, resulting in significantly lower affinity for IGF-I (9, 12). In addition, IGFBP-3 proteolysis has been described in many in vivo and in vitro situations, resulting in IGFBP-3 fragments with low affinity for IGFs (1, 13-15). In these situations, IGF-I may be released from IGFBP-3 to type I receptors on the cell surface with subsequent induction of IGF-I effects (1, 2, 8, 13-15).

The IGF system plays an important role in wound healing (16, 17), and both IGF-I and IGFBP-3 are present in wound fluid in significant concentrations (16-19). Recent studies show that plasminogen (Glu-Pg) binds IGFBP-3 and the binary IGFBP-3·IGF-I complex with high affinity by interacting directly with the IGFBP-3 HBD (20). This suggests that Glu-Pg, which plays a crucial role in wound healing and binds to the fibrin clot with high affinity (21-23), may localize IGF-I to the wound site by binding directly to both IGFBP-3·IGF-I complexes and the fibrin clot. However, another possibility is that the IGFBP-3·IGF-I complex binds directly to fibrin; this would be reminiscent of the ability of fibrinogen/fibrin to bind with high affinity to basic fibroblast growth factor, another mitogen that plays a role in the wound healing process (24). This paper describes studies which show that IGFBP-3 and IGFBP-3·IGF-I complexes do indeed bind with high affinity to fibrinogen/fibrin and to fibrin clots, and suggest that the IGFBP-3 HBD participates in this binding process.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human IGF-I and LR3-IGF-I were purchased from GroPep, Ltd (Adelaide, Australia). Recombinant human IGFBP-5 was produced in a baculovirus expression system and purified by affinity chromatography and reverse phase-high performance liquid chromatography (25). Most studies used recombinant human IGFBP-3 expressed in Chinese hamster ovary cells and purified as described previously (20, 26). Some experiments used recombinant human IGFBP-3BV and IGFBP-3hbdBP1BV produced in a baculovirus expression system and purified as described previously (20, 26); IGFBP-3hbdBP1BV is identical to native IGFBP-3, except that the heparin binding domain has been replaced by the homologous but non-heparin binding sequence of IGFBP-1 (26). IGFBP-1 was purified from human amniotic fluid by affinity chromatography and reverse phase-high performance liquid chromatography (20); quantitation was by radioimmunoassay using a kit from Diagnostic Systems Laboratories, Inc. (Webster, TX). Fibrinogen was obtained from Enzyme Research Laboratories, Inc. (South Bend, IN). Peptide IGFBP-3hbd (KKGFYKKKQCRPSKGRKR), which encodes the heparin binding domain of IGFBP-3 (20), was synthesized by Genemed Synthesis, Inc. (South San Francisco, CA). Glu-Pg was purified as described previously (20). Plasmin (Pm) was obtained from American Diagnostics (Greenwich, CT). Human citrate plasma was obtained from outdated blood bank supplies or from laboratory personnel.

Solid-phase Plate Binding Assay-- Binding of IGFBP-3 to fibrinogen and fibrin was characterized using an immobilized ligand-based assay system adapted from a similar system used to characterize IGFBP interactions with other proteins (20, 26, 27). Optimal binding conditions, fibrinogen coating concentration, plate type, and incubation parameters were determined and are presented in the following assay protocol. 96-well immunological plates (Polysorb, Nunc, Fisher Scientific, Pittsburgh, PA) were coated with 5 µg/ml fibrinogen, unless otherwise stated, in 0.1 M Na2CO3, pH 9.8, overnight at 4 °C. The plates were rinsed with 200 µl of 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.01% Tween 20, 0.2% bovine serum albumin (BSA), 0.2% NaN3. To convert fibrinogen to fibrin, 100 µl/well of 20 NIH units/ml human thrombin (Calbiochem, San Diego, CA) in 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.01% Tween 20, 0.2% BSA, 0.2% NaN3, 1 mM CaCl2 was added to washed wells and incubated for 1 h at 37 °C. Fibrinogen conversion to fibrin was terminated by rinsing the plates three times with 1 M NaCl, 8 mM CaCl2, 0.01% Tween 20, 0.2% NaN3. An additional rinse was performed with 5 mM sodium phosphate, pH 6.8, 0.01% Tween 20, 0.2% NaN3. Both fibrinogen and fibrin plates were blocked in 10 mM sodium phosphate, pH 6.8, 150 mM NaCl, 0.2% BSA, 10 mM lysine, 0.2% NaN3; tightly sealed in plastic wrap; and stored for up to 1 month.

For an assay, plates were rinsed twice with 200 µl of 30 mM Tris-acetate, pH 7.4, 10 mM sodium phosphate, 0.1% Tween 20, 0.2% NaN3 (assay buffer). IGF-I, and IGFBP-3 expressed in Chinese hamster ovary cells, were each iodinated by the chloramine-T method to a specific activity of ~150 µCi/µg protein (13). 125I-IGFBP-3 (50,000 cpm) or 125I-IGF-I (50,000 cpm) were incubated with various concentrations of IGFBP-3, IGFBP-3BV, IGFBP-3hbdBP1BV, IGFBP-1, IGFBP-5, IGFBP-3hbd, IGF-I, fibrinogen, Pm, Glu-Pg, epsilon -aminocaproic acid (epsilon ACA), arginine, or heparin in 100 µl of assay buffer; unless stated otherwise, incubations were for 1 h at 37 °C. Unbound radioactivity was removed by rinsing the wells twice with 200 µl of ice-cold assay buffer. Bound radioactivity was solubilized with 200 µl of 1 N NaOH, transferred to 12 × 75-mm glass test tubes, and counted for radioactivity.

Fluid-phase Plate Binding Assay-- 96-well immunological plates (MaxiSorb, Nunc) were coated with 5 µg/ml rabbit anti-human fibrinogen antibody (Enzyme Research Laboratories) overnight at 4 °C or for 3 h at 23 °C in 100 µl of 0.1 M Na2CO3, pH 9.8. Plates were rinsed with PBS and blocked as described in the solid-phase plate assay protocol. In a separate, uncoated 96-well test plate (Sarstedt) various concentrations of fibrinogen, IGFBP-3BV, 125I-IGFBP-3, and/or human citrate plasma were incubated in a total volume of 200 µl of assay buffer for 1 h at 37 °C; this allows all reactants to associate in the fluid phase. At the end of the incubation period, 100 µl of reactants were transferred to the blocked and rinsed 96-well plates that were coated with the anti-fibrinogen antibody. After 30 min at 23 °C, unbound radioactivity was removed by rinsing wells twice with assay buffer. Bound radioactivity, representing IGFBP-3·fibrinogen complexes, was released by incubating wells with 200 µl of 1 M acetic acid for 10 min at 23 °C. Acid washes were transferred to 12 × 75-mm glass test tubes and counted for radioactivity. Radioactivity bound in the absence of added fibrinogen served as an estimate of nonspecific binding.

Fibrin Clot Binding Assay-- Fibrin clots were formed in 1.5-ml Eppendorf tubes by mixing citrate plasma with assay buffer containing CaCl2; total reaction volume was 100 µl, final CaCl2 concentration was 5 mM and, unless stated otherwise, 25 µl of citrate plasma were added. After adding ~100,000 cpm of 125I-IGF-I or 125I-IGFBP-3, either with or without IGFBP-3, the mixture was incubated for 90 min at 37 °C. Nonspecific incorporation of trace into fibrin clots was determined in mixtures not containing CaCl2. To terminate incubations, fibrin clots were carefully suspended in 500 µl of ice-cold assay buffer and then pelleted by centrifuging at 14,000 × g for 2 min. After this wash step was repeated, the vial tips were cut off and then counted for radioactivity.

Biotinylation of IGFBP-3-- IGFBP-3BV (13 µg) was diluted in 0.25 M NaHCO3, pH 9.2, to a total volume of 45 µl. After 1.4 mg of NHS-LC-biotin (Pierce ) was dissolved in 40 µl of H2O, 5 µl of this solution were mixed with the IGFBP-3BV solution. The mixture was placed on an orbital shaker for 1 h at 22 °C, after which the reaction was terminated by adding 200 µl of 1 M Tris-HCl, pH 7.4. Unincorporated biotin was separated from biotinylated IGFBP-3BV (bn-IGFBP-3) using a Quick-spin column as instructed by the manufacturer (Roche Molecular Biochemicals). By SDS-PAGE, IGFBP-3BV in bn-IGFBP-3 was the size expected for the intact native protein. Preparations of bn-IGFBP-3 were stored at 4 °C.

Western Ligand Blotting Using bn-IGFBP-3-- 10 µg each of pure fibrinogen, Glu-Pg, and IGFBP-1 were electrophoresed on a 7.5-15% gradient SDS-polyacrylamide gel under nonreducing conditions and then transferred to a nitrocellulose membrane as described previously (20). The membrane was washed for 30 min with 3% Nonidet P-40 in Tris-buffered saline (TBS), blocked for 2 h at 4 °C in 0.5% BSA in TBS, washed for 10 min with 0.1% Tween 20 in TBS (TBST), and then incubated overnight with 32 ng/ml bn-IGFBP-3 in TBST. After washing in TBST buffer, the membrane was incubated in streptavidin-horse radish peroxidase diluted in TBST buffer (1:500) for 45 min at 22 °C. After several washes in TBST, the membrane was incubated with the chemiluminescent reagants provided in the ECL kit (Amersham Pharmacia Biotech) following the recommendations of the manufacturer. The membrane was then exposed to Kodak film for 5-30 min at 22 °C. ECL reagants and streptavidin-horseradish peroxidase were from Amersham Pharmacia Biotech.

Immunohistochemistry-- Normal human citrate plasma was clotted by addition of CaCl2 to a final concentration of 5 mM. After 90 min, the resultant fibrin clot was rinsed and cryosectioned into 5-µm sections. Sequential sections were incubated with normal rabbit serum or with rabbit antisera to plasminogen (Roche Molecular Biochemicals) or IGFBP-3 (Diagnostic Systems Laboratories, Inc.). Immunofluorescent staining was performed using fluorescein isothiocyanate-labeled donkey anti-rabbit IgG as the second antibody (Jackson Immunoresearch Laboratories, West Grove, PA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

125I-IGFBP-3 Binds Immobilized Fibrinogen/Fibrin-- 125I-IGFBP-3 bound specifically to fibrinogen or fibrin immobilized on immunocapture plates. As shown in Fig. 1A, increasing the amount of fibrinogen/fibrin immobilized on the plates resulted in increased binding of 125I-IGFBP-3. IGFBP-5 and IGFBP-3, but not IGFBP-1, efficiently competed with 125I-IGFBP-3 for binding to fibrinogen (Fig. 1B) and fibrin (data not shown). Scatchard analysis revealed that: 1) IGFBP-3 bound comparably to fibrinogen (Kd = 0.67 ± 0.2 nM; mean ± S.E. of three independent experiments) and fibrin (Kd = 0.7 ± 0.2 nM; mean ± S.E. of three independent experiments); 2) 1 mol of fibrinogen bound 0.15 mol of IGFBP-3; and 3) 1 mol of fibrin had 59 ± 15% more 125I-IGFBP-3 binding sites than did 1 mol of fibrinogen. As shown in Fig. 1C, IGFBP-3 binding to immobilized fibrinogen or fibrin was quite stable but was rapidly reversible in the presence of 500 mM arginine.


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  		Fig. 1.   125I-IGFBP-3 binding to fibrinogen/fibrin immobilized on immunocapture plates. Except where noted, plates were coated overnight at 4 °C with 5 µg/ml fibrinogen. In some experiments, fibrinogen was converted to fibrin as described under "Experimental Procedures." Nonspecific binding was determined by adding unlabeled IGFBP-3 or was estimated by determining the binding of IGFBP-3 to wells in which buffer without fibrinogen/fibrin was absorbed; this value was subtracted from total binding to give specific binding. Each data point is the mean of triplicate determinations. A, 125I-IGFBP-3 binds to fibrinogen/fibrin. ~100,000 cpm of 125I-IGFBP-3 was equilibrated with either solid-phase fibrinogen or fibrin for 1 h at 37 °C. B, competition curve for 125I-IGFBP-3 binding to fibrinogen. ~50,000 cpm of 125I-IGFBP-3 was equilibrated with solid-phase fibrinogen for 1 h at 37 °C either without unlabeled IGFBPs or in the presence of increasing concentrations of IGFBP-1, IGFBP-3, or IGFBP-5. C, 125I-IGFBP-3 binding to fibrinogen/fibrin is stable but reversible. ~50,000 cpm of 125I- IGFBP-3 was equilibrated with either solid-phase fibrinogen or fibrin for 1 h at 37 °C. After unbound 125I-IGFBP-3 was removed by rinsing, either buffer alone or buffer containing 0.5 M arginine, pH 7.4, was added and the incubations continued at 37 °C. At the indicated time points, specific wells were rinsed and bound radioactivity was measured.

IGFBP-3 also bound fibrinogen by ligand blot. Fibrinogen, Glu-Pg, and IGFBP-1 were electrophoresed by SDS-PAGE, transferred to a nitrocellulose membrane, and then incubated with bn-IGFBP-3. As shown in Fig. 2, bn-IGFBP-3 bound as expected to the ~86-kDa Glu-Pg protein but not to IGFBP-1. In addition, bn-IGFBP-3 bound to at least two proteins of mass >200 kDa in the fibrinogen lane; the size of these proteins is consistent with the expected size of the common forms of plasma fibrinogen (24, 28).


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  		Fig. 2.   bn-IGFBP-3 binding to fibrinogen by ligand blot. Fibrinogen, Glu-Pg, and IGFBP-1 (10 µg each) were separated by SDS-PAGE on a 7.5-15% gradient gel and then transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4 °C with bn-IGFBP-3, washed, and then visualized as described under "Experimental Procedures." The molecular mass, in Kd, of protein markers is shown on the left.

Role of the IGFBP-3 HBD in Fibrinogen/Fibrin Binding-- Since the IGFBP-3 HBD participates in binding to plasminogen, prekallikrein, and the IGF·acid-labile subunit binary complex (4, 20, 26), it seemed likely that the IGFBP-3 HBD participates in fibrinogen binding. To test this hypothesis, heparin and a synthetic 18-amino acid peptide encoding the IGFBP-3 HBD were evaluated for their ability to compete with 125I-IGFBP-3 for binding to fibrinogen/fibrin. Both IGFBP-3hbd (Fig. 3A) and heparin (data not shown) interfered in a dose-dependent manner with binding of 125I-IGFBP-3 to immobilized fibrinogen or fibrin. In addition, IGFBP-3hbdBP1BV, a full-length IGFBP-3 protein mutated to replace the HBD with the homologous but non-heparin binding region of IGFBP-1, had a ~10-fold lower affinity for fibrin than did native IGFBP-3BV (Fig. 3B).


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  		Fig. 3.   The IGFBP-3 HBD participates in binding to fibrinogen/fibrin. In each study, ~50,000 cpm 125I-IGFBP-3 was incubated with or without competitors in fibrinogen- or fibrin-coated plates. Bo was determined in the absence of any competing ligand. Nonspecific binding was determined in the presence of unlabeled IGFBP-3 or was estimated by determining the binding of IGFBP-3 to wells in which buffer without fibrinogen/fibrin was absorbed. A, increasing amounts of IGFBP-3hbd peptide were coincubated with 125I-IGFBP-3. Each data point is the mean of triplicate determinations. B, increasing amounts of native IGFBP-3BV or IGFBP-3hbdBP1BV were coincubated with 125I-IGFBP-3. Each data point is the mean ± S.E. of triplicate determinations.

IGF-I·IGFBP-3 Complex Binds Immobilized Fibrinogen/Fibrin-- Coincubating 125I-IGF-I and increasing amounts of unlabeled IGFBP-3 in plates coated with fibrinogen or fibrin resulted in a steady increase in 125I-IGF-I binding to these plates (Fig. 4A).


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  		Fig. 4.   IGFBP-3 mediates the binding of 125I-IGF-I to fibrinogen/fibrin. In each study, incubations were performed in fibrinogen- or fibrin-coated plates using ~50,000 cpm 125I-IGF-I. A, 125I-IGF-I was coincubated with increasing amounts of unlabeled IGFBP-3. Bound 125I-IGF-I was measured. Nonspecific binding was determined by measuring the binding of 125I-IGF-I in the absence of IGFBP-3. Each data point is the mean ± S.E. of triplicate determinations. B, IGFBP-3 (100 ng/ml) was bound to solid-phase fibrinogen or fibrin for 1 h at 37 °C. Unbound IGFBP-3 was removed by rinsing. 125I-IGF-I and increasing concentrations of unlabeled IGF-I were equilibrated with bound IGFBP-3 for 1 h at 37 °C. Bound 125I-IGF-I was determined and Scatchard plot calculated. Each data point is the mean of triplicate determinations. C, IGFBP-3 (100 ng/ml) was bound to solid-phase fibrin for at least 1 h at 37 °C. Unbound IGFBP-3 was removed by rinsing. 125I-IGF-I and increasing concentrations of unlabeled IGF-I or LR3-IGF-I were equilibrated with bound IGFBP-3 for at least 1 h at 37 °C. Bound 125I-IGF-I was determined. Each data point is the mean ± S.E. of triplicate determinations. D, ~50,000 cpm of 125I-IGFBP-3 and increasing concentrations of IGF-I were equilibrated with either solid-phase fibrinogen or fibrin for at least 1 h at 37 °C. Specifically bound 125I-IGFBP-3 was determined. Each data point is the mean ± S.E. of two separate experiments performed in duplicate.

Similar results were noted when increasing amounts of unlabeled IGFBP-3 were incubated with immobilized fibrinogen or fibrin prior to the addition of 125I-IGF-I (data not shown). In each of these experiments, very little 125I-IGF-I bound to the fibrinogen- or fibrin-coated plates in the absence of IGFBP-3, and coincubating 125I-IGF-I with 100 ng/ml IGFBP-3 did not result in specific 125I-IGF-I binding if plates were not coated with fibrinogen or fibrin; these results suggest that 125I-IGF-I was binding to fibrinogen- or fibrin-bound IGFBP-3.

To determine the affinity of IGF for IGFBP-3 bound to immobilized fibrinogen/fibrin, increasing amounts of unlabeled IGF-I were coincubated with 125I-IGF-I in the presence of IGFBP-3·fibrinogen complexes (70 fmol/well) or IGFBP-3·fibrin complexes (115 fmol/well). As shown in Fig. 4B, IGF-I bound the immobilized IGFBP-3·fibrinogen complex with Kd = 2.9 ± 0.6 nM (mean ± S.E. of three independent experiments) and bound the immobilized IGFBP-3·fibrin complex with Kd = 2.3 ± 0.3 nM (mean ± S.E. of three independent experiments); in addition, there was a 63 ± 10% increase in binding of 125I-IGF-I to IGFBP-3·fibrin compared with IGFBP-3·fibrinogen (data not shown). Further studies (Fig. 4C) showed that 125I-IGF-I binding to the IGFBP-3·fibrin complex was efficiently competed by IGF-I but not the IGF-I mutant LR3-IGF-I, which has a decreased affinity for IGFBP-3 (29). Finally, coincubating unlabeled IGF-I with 125I-IGFBP-3 did not affect the ability of 125I-IGFBP-3 to bind immobilized fibrinogen or fibrin (Fig. 4D); this suggests that fibrinogen and fibrin bind equally well to IGFBP-3 and the IGFBP-3·IGF-I complex.

125I-IGFBP-3 Binds Fibrinogen in the Fluid Phase-- As shown in Fig. 5A, wells coated with anti-fibrinogen antibodies were able to bind 125I-IGFBP-3 that had been preincubated with purified fibrinogen in the fluid phase; in these studies, the binding of 125I-IGFBP-3 to fibrinogen was dose-dependent and saturable. As shown in Fig. 5B, soluble 125I-IGFBP-3·fibrinogen complexes were also detected when citrate plasma was used as a source for native fibrinogen; binding specificity was demonstrated by the ability of excess unlabeled IGFBP-3 to completely inhibit 125I-IGFBP-3 binding (data not shown). Nevertheless, as shown in Fig. 5C, fluid-phase fibrinogen competed only minimally (~30%) for 125I-IGFBP-3 binding to immobilized fibrinogen, suggesting that IGFBP-3 binding to a fibrin clot can occur in the presence of physiologic concentrations (~3-4 mg/ml) of plasma fibrinogen (30).


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  		Fig. 5.   125I-IGFBP-3 binds to fluid-phase fibrinogen. ~50,000 cpm of 125I-IGFBP-3 were incubated with increasing concentrations of fluid-phase fibrinogen or citrate plasma for at least 1 h at 37 °C. Reactants were then added to wells precoated with 5 µg/ml anti-fibrin ogen antibodies. After 1 h at 37 °C, unbound reactants were removed. Bound radioactivity was released from wells using 1 M acetic acid for 10 min at 23 °C. Nonspecific binding (125I-IGFBP-3 binding in the absence of fibrinogen) was subtracted. A, 125I-IGFBP-3 binding to pure fibrinogen. Each data point represents the mean ± S.E. of duplicate determinations. B, 125I-IGFBP-3 binding to fibrinogen in citrate plasma. Each data point represents the mean ± S.E. of duplicate determinations. C, 125I-IGFBP-3 binding to solid-phase fibrinogen in the presence of increasing concentrations of fluid-phase fibrinogen. Each data point represents the mean ± S.E. of duplicate determinations.

IGFBP-3 Increases IGF-I Binding in Fibrin Clots-- Native fibrin clots, induced using calcium saturation of citrate plasma, specifically bound 125I-IGFBP-3 (10,600 ± 130 cpm specifically bound/100,000 cpm 125I-IGFBP-3 added; mean ± S.E. of three experiments performed in triplicate). This suggests that IGFBP-3 can be bound during clot formation. Furthermore, the amount of 125I-IGF-I specifically bound within these fibrin clots is directly related to the amount of plasma used to generate the clot (data not shown). As IGFBP-3 is the primary circulating IGFBP, it is likely that IGFBP-3 is primarily responsible for immobilizing IGF-I in the fibrin clot. When forming fibrin clots using 25 µl of citrate plasma, 125I-IGF-I was specifically bound (6,200 ± 140 cpm specifically bound/100,000 cpm 125I-IGF-I added), and 125I-IGF-I specific binding increased by 62 ± 11% upon the further addition of 100 ng of IGFBP-3 (mean ± S.E. of two experiments performed in quadruplicate). These data suggest that IGF-I is immobilized in fibrin clots by associating with fibrin-bound IGFBP-3.

Fibrin Clots Contain Both IGFBP-3 and Glu-Pg-- Immunofluorescent staining was also used to determine whether IGFBP-3 was incorporated into native fibrin clots. As shown in Fig. 6, specific antibodies to IGFBP-3 and Glu-Pg did indeed recognize each protein in cross-sections of fibrin clots. This is consistent with the ability of Glu-Pg to bind fibrin with high affinity (22, 23). However, both fibrin and Glu-Pg bind with high affinity to IGFBP-3, and Fig. 6 does not discriminate which protein binds IGFBP-3 in fibrin clots.


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  		Fig. 6.   Both Glu-Pg and IGFBP-3 are detectable in fibrin clots. Normal human citrate plasma was clotted by addition of CaCl2 to a final concentration of 5 mM. After 90 min, the resultant fibrin clot was rinsed and cryosectioned into 5-µm sections. Sequential sections were incubated with normal rabbit serum (NRS) or with rabbit antisera to plasminogen or IGFBP-3. Immunofluorescent staining was recorded using fluorescein isothiocyanate-labeled donkey anti-rabbit IgG as the second antibody.

IGFBP-3 Binds Fibrinogen Both Directly and via Glu-Pg-- As shown in Fig. 7A, partial proteolysis of immobilized fibrinogen by 5 ng/ml Pm completely inhibits IGFBP-3 binding, presumably by destroying IGFBP-3 binding sites. However, coincubation of IGFBP-3 with Glu-Pg allows IGFBP-3 binding to fibrinogen partially proteolyzed by 5 ng/ml Pm. This increase is likely due to IGFBP-3 binding to fibrin-bound Glu-Pg, since partial digestion of fibrinogen may actually increase Glu-Pg binding by uncovering previously hidden Glu-Pg binding sites (22).


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  		Fig. 7.   IGFBP-3 binds to fibrinogen both directly and via Glu-Pg. A, fibrinogen immobilized on immunocapture plates was pretreated with increasing concentrations of Pm for 1 h at 37 °C. Pm was removed by rinsing wells extensively with 200 mM epsilon ACA followed by buffer alone. ~50,000 cpm 125I-IGFBP-3 were equilibrated with digested fibrinogen for at least 1 h at 37 °C with and without 10 µg/ml Glu-Pg. Unbound 125I-IGFBP-3 was removed by rinsing. Bound radioactivity was determined by addition of 1 N NaOH to well contents for 10 min at 23 °C. After nonspecific binding was subtracted out, specific binding was normalized to % control values (i.e. binding of IGFBP-3 to solid-phase fibrinogen not exposed to Pm). Each data point is the mean ± S.E. of quadruplicate determinations. B, ~50,000 cpm 125I-IGFBP-3 were equilibrated with solid-phase fibrinogen and with increasing concentrations of either arginine or epsilon ACA for at least 1 h at 37 °C. Unbound 125I-IGFBP-3 was removed by rinsing. Bound radioactivity was determined by addition of 1 N NaOH to well contents for 10 min at 23 °C. Each data point is the mean ± S.E. of duplicate determinations. C, ~50,000 cpm 125I-IGFBP-3 were equilibrated with solid-phase fibrinogen and either with or without 10 µg of Glu-Pg along with 0, 10, or 25 mM epsilon ACA for at least 1 h at 37 °C. Unbound 125I-IGFBP-3 was removed by rinsing. Bound radioactivity was determined by addition of 1 N NaOH to well contents for 10 min at 23 °C. After nonspecific binding was subtracted out, specific binding was normalized to percentage of control values (i.e. 125I-IGFBP-3 alone or 125I-IGFBP-3 + Glu-Pg). Each data point is the mean ± S.E. of quadruplicate determinations.

Previous studies found that 10 mM epsilon ACA completely blocks Glu-Pg binding to fibrinogen (31). However, as shown in Fig. 7B, 25 mM epsilon ACA only weakly inhibits fibrinogen binding to IGFBP-3; as expected, arginine is a potent inhibitor of fibrinogen binding to IGFBP-3. This suggests that epsilon ACA can be used to discriminate between IGFBP-3 binding to fibrinogen and to fibrinogen-bound Glu-Pg. Indeed, as shown in Fig. 7C, when solid-phase fibrinogen is pre-equilibrated with Glu-Pg, IGFBP-3 binding is inhibited 35-40% in the presence of 10 or 25 mM epsilon ACA, suggesting that 35-40% of IGFBP-3 binds to fibrinogen-bound Glu-Pg.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IGF-I participates in wound healing by acting as a chemotactic agent supporting the early migration of stromal cells into the fibrin clot at the wound site, and by stimulating proliferation of fibroblasts and endothelial cells (16, 17). Potential sources for IGF-I found at the wound site include plasma, platelets (32), monocytes (33), and macrophages (34). As with IGFs in other body fluids, IGF-I in wound fluid is complexed with IGF-binding proteins, primarily IGFBP-3, which is abundant in serum and in platelets (18, 19, 32). Studies presented in this report, which show that IGFBP-3·IGF-I binary complexes bind with high affinity to fibrinogen/fibrin, suggest that direct binding of IGFBP-3·IGF-I complexes to the fibrin clot is an important mechanism by which IGF-I levels may be maintained at the wound site.

Complex formation between IGFBP-3 and fibrinogen or fibrin was shown by solid- and fluid-phase plate binding assays and by Western ligand blotting using biotinylated IGFBP-3. Similar techniques have been used to identify other proteins which bind IGFBP-3 (20, 26). The binding affinity of IGFBP-3 for immobilized fibrinogen and fibrin was Kd = 0.67 and 0.7 nM, respectively. These values suggest that fibrinogen/fibrin affinity for IGFBP-3 is comparable to or stronger than fibrinogen/fibrin affinity for Glu-Pg, tissue plasminogen activator, plasminogen activator inhibitor, apolipoprotein(a), thrombin, factor Xa, factor XIII, fibronectin, thrombospondin and basic fibroblast growth factor (22-24, 35-41). The binding affinities of IGFBP-3 for fibrinogen/fibrin are roughly comparable to the affinity of the IGFBP-3·IGF-I binary complex for acid labile subunit (5), and to the affinity of IGFBP-3 for Glu-Pg and prekallikrein (20, 26).

Fibrinogen and fibrin appear to interact with a specific HBD of IGFBP-3, which is rich in lysine groups and which participates in IGFBP-3 binding to acid-labile subunit, Glu-Pg, and prekallikrein (4, 20, 26). The sequence and location of the IGFBP-3 HBD is conserved in IGFBP-5 (12). Studies presented here show that IGFBP-5 efficiently competes with IGFBP-3 binding to fibrinogen or fibrin, suggesting that IGFBP-5 also binds fibrinogen and fibrin with high affinity. In contrast, the IGFBP-3 HBD sequence is not conserved in IGFBP-1 (12), IGFBP-1 does not bind appreciably to fibrinogen, and replacing the IGFBP-3 HBD sequence with homologous sequence from IGFBP-1 results in a 10-fold loss of affinity for fibrin. The residual ability of IGFBP-3hbdBP1BV to compete with native IGFBP-3 for binding to fibrin suggests that, in addition to the HBD, other IGFBP-3 regions may participate in binding to fibrinogen/fibrin.

Polymerization of fibrinogen into fibrin did not alter affinity for IGFBP-3, but it did increase the number of 125I-IGFBP-3 binding sites by 59% and the number of 125I-IGF-I·IGFBP-3 binding sites by 63%. These data suggest that cleavage of fibrinogen exposes new binding sites for IGFBP-3, just as it exposes new binding sites for Glu-Pg (22). These data also suggest that formation of binary complexes between IGFBP-3 and immobilized fibrin does not prevent the further binding of IGF-I to IGFBP-3, thus forming a ternary complex. Additional studies confirmed that IGF-I binds to IGFBP-3 complexed with immobilized fibrin (Kd = 2.3 nM), and also found that IGF-I binds to IGFBP-3 complexed with immobilized fibrinogen (Kd = 2.9 nM). These Kd values are higher than the range of values (0.03-0.5 nM) reported previously for IGF-I binding to IGFBP-3 (42), and are comparable to the affinity of IGF-I for the type I IGF receptor (43). This suggests that type I IGF receptors on fibroblasts and other cells that are migrating into the fibrin clot can remove IGF-I from fibrin-bound IGFBP-3, resulting in proliferation of these cells at the wound site.

In addition to the direct binding of IGFBP-3 to fibrinogen, studies presented here suggest that IGFBP-3 also binds indirectly, to fibrinogen-bound Glu-Pg. It is well known that Glu-Pg binds with high affinity to fibrinogen/fibrin and that, through this interaction, Glu-Pg plays an important role in the wound healing process (21-23). It is also known that IGFBP-3 and IGF-I·IGFBP-3 complexes bind Glu-Pg with high affinity, and that IGFBP-3 binds to multiple sites on Glu-Pg (20). Thus, a complex of fibrin·Glu-Pg·IGFBP-3·IGF-I is likely to form; in one possible arrangement, the kringle 1-3 regions of Glu-Pg bind to lysines in fibrinogen/fibrin, while the kringle 5/catalytic region of Glu-Pg binds to the HBD of IGFBP-3. If formed, this complex would provide another mechanism for releasing IGF-I at the wound site, since tissue plasminogen activator can activate immobilized Glu-Pg that is complexed with IGFBP-3·IGF-I, resulting in IGFBP-3 proteolysis and IGF-I release (20). Indeed, in tissue culture, activation of Glu-Pg to Pm results in IGFBP-3 proteolysis and release of bound IGF-I to cultured cells where mitogenic and metabolic pathways are stimulated (44, 45); thus, activation of Glu-Pg that is complexed both with fibrinogen and with IGFBP-3·IGF-I should ultimately make IGF-I available to stromal cells present at the wound site. In support of this hypothesis, additional studies show that plasminogen activators can induce proteolysis of IGFBP-3 complexed with fibrin-bound Glu-Pg, resulting in release of IGF-I (manuscript in preparation).

The observations that 125I-IGFBP-3 and 125I-IGF-I localize to fibrin clots generated from plasma in vitro, and that addition of exogenous IGFBP-3 greatly increases the incorporation of 125I-IGF-I into these clots, suggest that IGF-I·IGFBP-3 complexes bind to fibrin clots in vivo at wound sites. This possibility is supported by immunoblot detection of IGFBP-3 in these fibrin clots. The further detection of Glu-Pg in these fibrin clots by immunoblotting suggests that IGFBP-3 may bind these clots directly through fibrin and indirectly through fibrin-bound Glu-Pg. Thus, data obtained from the study of these fibrin clots support the hypothesis that IGFBP-3·IGF-I complexes bind to fibrin clots formed at wound sites in vivo, and are consistent with the possibility that at least two mechanisms exist for the ready release of bound IGF-I to fibroblasts and endothelial cells migrating into the wound site.

    ACKNOWLEDGEMENTS

We thank Heide Eash and Richard Ting for their assistance in conducting the solid- and fluid-phase plate binding assays.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Award RO1 DK-38773 (to D. R. P.) and by a grant from the Beta Sigma Phi Research Fund, Houston City Council (to D. R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Texas Children's Hospital, Feigin Center, MC 3-2482, 6621 Fannin, Houston, TX 77030. Tel.: 713-770-3800; Fax: 713-770-3889; E-mail: dpowell@bcm.tmc.edu.

    ABBREVIATIONS

The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; bn-IGFBP, biotinylated insulin-like growth factor-binding protein; Glu-Pg, plasminogen; PAGE, polyacrylamide gel electrophoresis; Pm, plasmin; IGF, insulin-like growth factor; epsilon ACA, epsilon -aminocaproic acid; HBD, heparin binding domain; BSA, bovine serum albumin; TBS, Tris-buffered saline; TBST, TBS with Tween 20.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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