The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4/~M heparin and 0.9/tM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3/~M. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaC! as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.
Introduction
Fibronectin, glycosaminoglycans and various collagens are major components of extracellular matrices, and they are present on the surface of different types of cells (Refs. 1-5, and references therein).
The dynamic interactions of such components may be involved in attachment or migration of various cells [6-9], and may participate in the formation of extracellular matrices. In addition, the enzymatic degradation of such structures may play a role in angiogenesis, inflammation and the metastasis of malignant cells [10-20].
Correspondence (present address): R. Machovich, Mayo Clinic, Hematology Research, Rochester, MN 55905, U.S.A.
[21]. The cooperative nature of heparin and fibronectin binding to collagen has also been re- ported [22]. The dissociation of gelatin-fibronectin complexes requires a higher urea concentration if the complex is formed in the presence of heparin [22]. The enhancement of binding of fibronectin to both native and denatured collagen by heparin, and the precipitation of collagen type III and fibronectin in the presence of heparin have been reported as well [23,24].
However, the mechanism of heparin-fibronec- tin-collagen interaction is not well understood. To obtain additional data concerning the associa- tion/dissociation of such a complex, we de- termined the dependence of the rate of complex formation on the absolute and relative concentra- tion of the individual components. We also in- vestigated the effect of NaC1 and CaC1 z, as well as the heparin-antagonist polybrene [25] or sper- min, which are known to inhibit fibronectin-gela-
tin binding [26]. In addition, the possible effect of plasmin on this complex was also investigated. We have found that the rate of ternary complex for- mation is dependent on the concentration of the components, is inhibited by high NaC1 concentra- tions, by calcium ions and by spermin or poly- brene. Heparin incorporated into the insoluble complex can be released by plasmin in active anticoagulant form.
Materials and Methods
Sepharose 4B was from Pharmacia. Cyanogen bromide and lactoperoxidase were purchased from Sigma. Cloramine T was from Merck. The chro- mogenic substrate for plasmin, D-Val-Leu-Lys p- nitroanilide dihydrochloride (S-2251), was ob- tained from KABI, and used according to the instructions of the manufacturer.
Na125I (carrier free) and NaB3H4 were the products of the Isotope Institute of the Hungarian Academy of Sciences. Other chemicals were purchased from Reanal Fine Chemicals, Hungary. Heparin (from porcine intestine, 179 U/rag) was the product of G. Richter Pharmaceuticals, Hungary.
Collagen type III (from calf skin, acid-soluble, as Sigma type I collagen) was from Sigma. Prior to experimentation, the substance was dissolved at 4°C in 50 mM acetic acid to a concentration of 5 mg/ml.
Fibronectin was purified from outdated human plasma by the method of Vuento and Vaheri [27].
Plasminogen was isolated from human plasma by lysine-Sepharose 4B affinity chromatography and activated by streptokinase by established methods [28,29].
Plasmin activity was measured photometrically with S-2251 tripeptide chromogenic substrate as described earlier [29].
Human thrombin was isolated from Cohn frac- tion III [30] and further purified to obtain c~- thrombin [31]. Its activity was 3200 NIH units/mg protein as measured in a fibrinogen clotting test system [32].
Antithrombin III was isolated from human plasma by heparin-Sepharose affinity chromatog- raphy [33].
Collagen and fibronectin were 125I-labeled by the chloramine T method [34] or the lactoperoxi-
dase method [35], respectively. Their specific activ- ity was 680000 dpm/#g and 32000 dpm//.tg, respectively. Prior to experimentation, labeled proteins were diluted 100-fold with their unlabeled counterparts. Heparin was tritiated by the method of Hatton et al. [36] to a specific activity of 12400 dpm//~g.
Protein concentrations were determined by the method of Lowry et al. [37], with bovine albumin as standard. For calculations of molar concentra- tions, molecular weight standards of M r 11000, 283000, 440000, 36000 and 81 500 were used for heparin [38], collagen [1], fibronectin [21] throm- bin [30] and plasmin [39], respectively.
For turbidimetric assays, components and buffer solution were pipetted into a plastic cuvette, then repipetted immediately several times to en- sure complete mixing, and the absorbance at 600 nm was recorded by a Beckman model 25 spectro- photometer at 37 °C. The rate of precipitate for- mation was calculated from the slope of the fastest, linear part of the curve.
Incorporation of the components into the precipitate The components and buffer solution were
pipetted into a 1 cm diameter flat-bottomed vial, mixed thoroughly, then closed and incubated at 37°C overnight. After centrifugation at 800 × g for 10 rain at room temperature, the precipitate was washed three times with 4 ml of 50 mM sodium phosphate (pH 7.4) at room temperature for 30 min. To determine radioactivity incorpo- rated into the complex, it was solubilized in a toluene scintillation cocktail containing Triton X- 100 and the radioactivity measured by a Beckman LS 7800 liquid scintillation counter or a Gamma NK314 gamma-counter for 3H or 125I, respec- tively.
For the determination of proteolytic digestion, the complex, prepared as outlined above, was in- cubated with plasmin or urokinase in 50 mM Na-sodium phosphate buffer (pH 7.4) at room temperature. As a control, human serum albumin or phenylmethylsulphonyl fluoride (PMSF)-treated proteinases were used. At various intervals aliquots were taken and counted for radioactivity.
The biological activity of the heparin released from the insoluble complex was also determined by a clotting assay. 3 NIH units of thrombin (10
/zl) were mixed with an equimolar amount of antithrombin III (50 ~1) and with 240 /~1 of 50 mM sodium phosphate buffer (pH 7.4). After incubation at room temperature for 1 or 3 min, 100/.tl were withdrawn, added to 200 ~1 (250 t~g) fibrinogen, and the clotting time was determined [291.
Results
Kinetics of the insoluble complex formation Heparin, fibronectin and collagen incubated to-
gether at physiological ionic strength and temper- ature formed a precipitate, which was measured by the change of turbidity of the solution. When the components were incubated alone or in pairs, they remained in solution (Fig. 1). The pattern of the turbidimetric curves was dependent on the sequence of the addition of the components. If fibronectin was added last, there was a latency period of approximately 10 min. This phenome- non was not seen if the components were added in any other sequence.
At fixed fibronectin and collagen concentra-
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E 0.15 E
O O
0.10
/ /
/ /
/ /
/ /
/ /
/ o.o5 / /
10 20 30 60
Incubnfion time (rain)
Fig. 1. Time dependence of precipitate formation by heparin, fibronectin and collagen: Effects of preincubation. Collagen (0.6 ~M), fibronectin (1 ~M) and heparin (1 ~M) were in- cubated at 37 o C in 100 mM Tris-HC1 buffer (pH 7.5) contain- ing 150 mM NaCI, and the change of absorbance was de- termined at 600 nm. Collagen preincubated with heparin for 1 min, thereafter, fibronectin added ( ). Fibronectin prein- cubated with either heparin or collagen for 1 min, thereafter collagen or heparin, respectively, added ( - - - - - ) . Compo- nents incubated alone or in pairs at any combination ( . . . . . ).
243
25
E v
5
J / - •
/ ' ' I / 3 J
c~ // ~
/
Incub~ation time (rain;
i i L I ~ i I i
2 /* 18 20
[Heparin] (#M)
Fig. 2. The rate of ternary complex formation by heparin, fibronectin and collagen at various heparin concentrations. The slope (dA/dt) of the linear parts of the turbidimetric curves is plotted versus heparin concentration in the presence of fibronectin (1 ~M) and collagen (0.6 ~tM). Inset: Precipitate formation at various heparin concentrations. Heparin was pre- incubated with collagen (0.6 #M) for 1 min, thereafter fibronectin (1 ~M) was added and the absorbance at 600 nm registered. Final heparin concentrations are shown on the
figure.
tions, the rate of precipitate formation was pro- portional to heparin concentration up to 4 ~M, and at higher heparin concentrations the rate of complex formation did not increase (Fig. 2, and inset).
When the fibronectin concentration was varied at fixed heparin and collagen concentrations, the rate of precipitate formation was linearly related to the fibronectin concentration, even at 3 ~M, a concentration which is 5-fold higher than its plasma level (Fig. 3).
When collagen concentration varied at fixed heparin and fibronectin concentrations, the rate of precipitate formation followed similar kinetics as in the case of change in heparin concentration, and the maximal rate of complex formation was reached at a collagen concentration of 0.9 ~M (Fig. 3B).
The rate of insoluble complex formation de- creased with increasing NaCl concentration (Fig. 4). At 0.1-0.2 M NaC1 a sharp decrease of pre- cipitate formation was measured, and at over 0.25 M NaC1 precipitate formation was not detectable. At low NaCl concentration the complex formation
244
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E
o 0 o 8 X
-o 4
/ /
/
I I
1 2
[Fibr0nectin] (.uH)
A 10
, r i ¢-
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o 0 • -- 5 X
"0
B j o o f
./ /
I, l I I I I l I I I
3 0.1 0.3 0.5 0.7 0.9 [C011agenI (,uM)
Fig. 3. The rate of ternary complex formation by heparin, fibronectin and collagen. (A) At various fibronectin concentrations (1 #M heparin, 0.6 #M collagen). (B) At various collagen concentrations (1/.tM fibronectin, 1/xM heparin).
was fast, but macroscopic aggregates were formed. Ca 2 + also inhibited the formation of the insoluble complex (Fig. 5); 1 mM Ca 2+ caused a more than
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\
\ , \
I & , o--o
0.10 020
[ N'aC[ ] (H) \ \
\ 10 •
o _ _
. , ,
0.1 0.2 0.3 0.8
INo[I] (H) Fig. 4. Effect of ionic strength on complex formation. Heparin (1 ~M), fibronectin (1 #M) and collagen (0.6 ~M) were in- cubated at various NaC1 concentrations in 100 mM Tris/HC1 buffer (pH 7.5) at 37°C and the rate of ternary complex formation determined. Inset: The amount of 3H-labeled heparin precipitation at various NaCI concentrations in 100 mM Tris- HCI buffer (pH 7.5). Initial concentrations: 1 /~M heparin, 1
t~M fibronectin, 0.6 t.tM collagen.
80% decrease in the rate of precipitation, and 5 mM Ca 2+ completely inhibited the complex for- mation. The inhibitory effect of Ca 2÷ was antagonized by EDTA (Fig. 5, inset).
Polybrene (18 /.tg/ml) and spermin (50 /.tM) also inhibited complex formation (data not shown).
Composition of the insoluble complex The a m o u n t of h e p a r i n i n c o r p o r a t e d i n to the
6
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o z, x
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I/,\ ,4 6 O0 I, P
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005 ~- ÷EDTA /
i
5 10 15 men
~ O I n I I I / 1 i ~ -
0.5 1.0 1.5 2.0 25 5.0 [[aC[ z] CraM)
Fig. 5. The effect of Ca 2+ on ternary complex formation. The rate of complex formation among heparin (1/tM), fibronectin (1 #M) and collagen (0.6 /tM) was determined at various CaC12 concentrations in 150 mM NaC1/100 mM Tris-HC1 (pH 7.5) at 37°C. Inset: Effect of EDTA (5 mM) on the
inhibitory effect of 5 mM CaC12.
\ \
complex was proportional to its initial concentra- tion and reached a plateau at about 8 ~tM, as determined with tritiated glycosaminoglycan (Fig. 6). Raising the NaC1 concentration decreased the amount of heparin incorporated into the complex, while at 50 mM NaC1 more than 25% of heparin entered the complex. No insolubilization was found over 200 mM NaCI (Fig. 4, inset). Control experiments indicated that specific precipitation of heparin occurred only in the presence of both proteins, since if tritiated heparin was used in their absence the radioactivity found in an insolu- ble form was only 15% of the amount precipitated in the presence of fibronectin and collagen.
Complex formation was also measured using uSI-labeled proteins. The molar amounts of the
245
precipitated molecules for both fibronectin and collagen type III were similar, suggesting that they are equimolar in the complex (Fig. 6, inset).
Proteolysis of the ternary complex When the insoluble complex, prepared with
3H-labeled heparin, was incubated with 0.8 ~tM plasmin, radioactivity was found in the fluid phase. Urokinase (400 U/ml), albumin or plasmin blocked in the active center by PMSF had no such effect (Fig. 7).
Similar experiments were also carried out with radiolabeled fibronectin and collagen to determine whether they are also released by plasmin. The results obtained indicate that radioactivity of the ternary complex prepared with either 125I-labeled
100
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g.
l - -
t- O
o~
50
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/ 0 ~ z0
1 2 3 ~ 5 ~ 7 ~
[] fibronectin • cottogen
I I I
8 10
I I I I I I
2 4 6
[ H e p o r i n ] ( p H I
Fig. 6. Incorporation of heparin into the insoluble complex. The amount of 3H-labeled heparin precipitated at various initial concentrations of the labeled glycosaminoglycan with constant amounts of fibronectin (1/~M) and collagen (0.6 ~M) was determined in 100 mM Tris-HCl buffer (pH 7.5) contain- ing 150 mM NaCI in a final volume of 500 ~1. Inset: The amount of insolubilized fibronectin (empty columns) or col- lagen (filled columns), in the presence and absence of heparin. The amount of the labeled molecules was determined alone (columns 1, 2), in the presence of the other protein (columns 3, 4) or heparin (columns 5, 6). Columns 7 and 8 represent the amount of precipitated fibronectin or collagen, respectively, in the presence of all three components. Initial concentrations were 1/~M for heparin or fibronectin and 0.6/~M for collagen
fibronectin or 125I-labeled collagen is releasable by plasmin digestion (data not shown). Furthermore, the heparin released by proteolysis was active as an anticoagulent in the thrombin-antithrombin III reaction; an acceleration of the rate of thrombin inactivation by antithrombin III was observed in the presence of the released heparin (Fig. 7, inset).
Discussion
Our results demonstrate that when heparin, fibronectin and collagen type III are combined at physiological ionic strength and temperature, a precipitate is formed which contains heparin and equimolar amounts of the two proteins.
This process likely requires the binding of heparin to fibronectin via its calcium-sensitive heparin-binding domain [40]. The binding of either heparin or collagen to fibronectin facilitates the subsequent binding of the other molecule, as re- vealed by the lack of a lag period in the turbidi- metric curve. Such a cooperative model for the complex formation is in agreement with the find- ing that heparin strengthens the interaction of gelatin and collagen with fibronectin [22-24].
Another set of experiments shows that heparin released from the ternary complex by plasmin digestion is in a biologically active form. Since fibronectin may serve as a substrate for plasmin [41], these data suggest that heparin is bound in the complex via fibronectin.
These results also draw attention to an unex- pected biological function of proteinases; plasmin attacks a protein substrate, fibronectin, which in turn results in the release of a glycosaminoglycan with various biological consequences. Such an ac- tion of plasmin on an extracellular matrix may play a role in tumor invasion, metastasis forma- tion and/or tumor angiogenesis by liberating molecular components from an insoluble state, unmasking their bioregulatory properties [42].
Acknowledgements
We wish to thank Dr. Mihfily Nrmeth-Cs6ka for helpful discussions and Mrs. Agnes Himer and Mrs. Ida Horvfith for their excellent assistance. This work was supported by the Hungarian Ministry of Health and the Hungarian Academy
of Sciences, grant No. 20.057/85 and OKKFT Tt 'Biological Basic Research', and in part by the Pharmaceutical Works of G. Richter, Ltd., Hungary.
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