Fibrinmonomer binding to macrophages mediated by fibrin-binding fibronectin fragments

THROMBOSIS RESEARCH 38; 183-194, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

FIBRINMONOMER BINDING TO MACROPHAGES MEDIATED FIBRIN-BINDING FIBRONECTIN FRAGMENTS

Helmut

Hormann,

Max-Planck-Institut

Hartmut

Richter

and Viktorija

fiir Biochemie, Martinsried German Federal Republic

BY

Jelinic bei

Munchen

(Received 21.12.1984; Accepted in revised form 27.3.1985 by Editor R. Gollwitzer)

ABSTRACT

Binding of 125-I-fibrinmonomer to peritoneal macrophages was investigated in dependence of plasma fibronectin and of its thrombinor plasmin-derived fragments. P1asm.a fibronectin failed to enhance cell binding of 125-I-fibrinmonomer. In contrast, 30kD-fragments derived from the N-termini of the fibronectin subunits improved binding considerably. The association with the cell surface was completely inhibited by EDTA, 2-5 mM putrescine and to about 40 per cent by 0.1 mM dansyl cadaverine suggesting that a transamidase-catalyzed cross-linking reaction was involved. Thrombin-derived 200kD-remnants of the fibronectin subunit chains failed to mediate cell binding of 125-I-fibrinmonomer provided they had been deprived of residual thrombin activity. Otherwise they were active and their activity was inhibited by the thrombin inhibitor hirudin. Plasminderived 200 kD-fragments were inactive as well. INTRODUCTION

Plasma fibronectin, previously termed cold-insoluble globulin, consists of two nearly identical disulfide-linked subunits of molecular weight ca. 250.000 composed of several domains with affinities to a number of substrates including gelatin, heparin and fibrin (l-3). In addition to non-covalent binding also coKey Words: Abbreviations:

Fibronectin; Fibrinmonomer; Macrophage; Cell Binding; Transamidase EDTA, ethylenediamine tetraacetate; PPACK, nylalanyl-L-prolyl-L-arginyl-chloro-methylketone 183

D-phe,

184

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valent cross-linking with fibrin, collagen or cell walls of reaction ’ various bacteria by a transamidase-catalyzed observed (4-6). As fibronectin also contains a cell-bindi;; attachment of substrates the site, it is capable of mediating So, it functions as an opsonin for mentioned to suitable cells. cells of the phagocytosis of gelatin-containing substances by binding of the reticula-endothelial system (7,8) and mediates some bacteria to neutrophils (9). that plasma fibronectin is evidences suggest Indirect involved in the removal of fibrinmonomer from circulating blood reticula-endothelial system. A decreased fibronectin by the level was found in plasma of patients suffering from severe disseminated intravascular coagulation (10). Furthermore, in vivo, the decay of radioactively labelled fibronectin in blood is considerably promoted by fibrinmonomer and adapts to the rate of fibrinmonomer clearance (11). In spite of those indirect evidences experiments failed to demonstrate a direct role of plasma fibronectin in the binding and internalization of fibrinmonomer by cells reticuloof the endothelial system or to related macrophages. Therefore, the idea rose that plasma fibronectin might be an inactive precursor forming an active mediator after modification. The present paper reports that limited proteolysis of plasma fibronectin with thrombin or plasmin generates fragments capable of mediating fibrinmonomer binding to peritoneal macrophages. MATERIALS

AND METHODS

Fibrinogen was purified from fibrinogen KABI (ca. 500 mg in 50 ml 0.1 M NaCl, 0.025 M e-aminocaproic acid, 0.05 M Tris/HCl, pH 7.4) by passing over gelatin-Sepharose (column 15x5 cm) for removal of fibronectin followed by gelfiltration over Sephacryl S-300 (150x5 cm) in the cold. The main peak contained material which, as checked by sodium dodecylsulfate gel electrophoresis of the reduced sample, consisted of complete A=-, BPand r labelled with chains. Fibrinogen was 125-I by the Iodogen method described by Nieuwenhuizen et a1.(12). Activity generally was in the range of 200.000 cpmlug. 125-I-fibrinogen (1 mg/ml in 0.05 M Tris/HCl, pH 7.4, 0.1 M NaCI, 5 mM EDTA) was coagulated with 10 NIH U/ml thrombin (Thrombinum purum, the Behring) and isolated coagulum dissolved in 1 M KBr, 0.05 M Tris/HCl, pH 5.3, 0.025 M &-aminocaproic acid in presence of 5 NIH U/ml hirudin (Sigma) to yield a concentration of ca. 1 mg/ml. Fibronectin was isolated from titrated human plasma by adsorption onto gelatin-Sepharose according to Miekka et a1.(13). A sample (50 mg) dialyzed against 0.05 M Tris/HCl, pH 7.4, 0.1 M NaCl and adjusted to 2 mg6ml was degraded with 750 NIH U thrombin for 24 hours at 37 C. Proteolysis was terminated by the specific thrombin inhibitor (14) PPACK (Calbiochem-Behring, 0.2 ml 0.1 % solution in isopropanol) unless otherwise indicated and the digest was passed over gelatin-Sepharose (column 10x2,4 cm). Retained material was eluted with 1 M KBr, 0.05 M Tris/HCl, pH 5.3, 0.025 M &-aminocaproic acid and 0.1 M dialyzed against

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The non-bound fraction was NaCl, 0.05 M Tris/HCl, pH 7.4. 30 kD-fragment adsorbed onto heoarin-Seoharose (1.6x15 cm) and pH 7.4, 1 mM EDTA. eluted with 0.25'M NaCl 'in 0.05.M Tris/HCl, It The fragment was purified by gelfiltration over AcA44 (LKB . a 30 showed, when checked by dodecylsulfate gel electrophoresis kD-band and a minor 28 kD-fragment. (80 mg in 80 ml 0.1 M Plasmin digestion of fibronectin NaCl, 0.05 M Tris/HCl, pH 7.8) was performed wit8 12 U p asmin (KABI-Forschungsplasmin) for 24 hours at 37 c. Fol t owing termination by phenylmethane sulfonylfluoride (0.1 M solution in isopropanol - final concentration 1 mM) the digest was processed as described for the thrombin-derived one. cavity of Macrophages were harvested from the peritoneal injection of female guinea pigs 4-5 days after intraperitoneal 20 ml mineral oil (Paraffinum liquidum, DAB 7, Merck). The washed cells were incubated in Hank's balanced salt solution (IO7 cells/ml) with 0.1 mg/ml trypsin (Seromed) followed by washing twice. Binding exp riments were done in plastic tubes (63x11 mm, Sarstedt). balanced 5x10 % cells were suspended in 0.1-0.2 ml salt solution containing 0.5 % bovine serum albumin (Behring). Fibronectin or fibronectin fragments as well as 0.1 ml solution of fibrinogen (20 ug or 2 ug, respectively) and 200 ng 125-1fibrinmonomer in the same buffer were added and the volume was completed to 0.5 ml. Following incubation over night at room once temperature cells were spun down (15 min., 25Oxg), washed and resuspended in 0.45 ml solvent. Cell-bound radioactivity was counted by a Beckman counter To each experiment Gamma 4000. controls without cells were run and radioactivity unspecifically precipitated or adsorbed to vessel walls was determined. Unless otherwise indicated controls were subtracted from the experimental data. Enzymatic activity of thrombin was determined with the synthetic substrate Chromozym TH (Boehringer, Mannheim). Indicated amounts of thrombin 0.1 M or fibronectin fragments in 0.9 ml pH 7.2 were thermostated in cuvettes to Nagl, 0.05 M Tris/HCl, 37 c. After addition of 0.1 ml Chromozym TH solution (1 mg/ml) the time-course of the reaction was recorded at 405 nm. nE/min values measured in the linear range were calculated. RESULTS In order to evaluate any involvement of plasma fibronectin in the removal of fibrinmonomer by phagocytic cells, binding of soluble 125-I-fibrinmonomer to peritoneal macrophages was studied in presence of variable amounts of fibronectin and fibronectin fragments generated by thrombin or plasmin. Those enzymes were selected as they are activated in the process of fibrin formation. In the binding experiments a surplus of fibrinogen was added in order to improve the solubility of fibrin and to render the system more related to natural conditions.

_-• *-. r-=Vol. 39, No. 2

FIBRIN BINDING TO MACROPHAGES

186

0

5

*Cells

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..____---.

*.--

___.-______*______C_----

-Cells

i

8

IS

32

$4

li8

256

5;2

Fibronectin (Pg/mll FIG.1

Binding of 125-I-fibrinmonomer (400 ng/ml) to macrophages in deof plasma fibronectin at 40 pg/ml fibrinogen (solid pendence 125-I-fibrinmonomer deposiline). Dashed line records unspecific tion on vessel walls in the absence of cells (revised result of l.c. 15).

FIG.2 Enhancement of 125-I-fibrinmonomer binding to macrophages by thrombin-derived 30 kD-fibronectin fragment (solid had been line). 30kD-fragment isolated from a digest terminated by PPACK. Dashed line in absence records controls of cells. 400 ng 125-I-fibrin onomer, 40 ug fibrinogen, 10Y cells per ml.

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7'6

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$0

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30k-Fragmentlpg/ml)

Fig.1 shows that plasma fibronectin itself appeared unable to influence fibrinmonomer binding to phagocytes. A small amount of 125-I-fibrin was bound by cells independently on the the presence of plasma fibronectin. In addition to the entire molecule fibronectin fragments were tested on their capability to mediate cell binding of 125-1fibrinmonomer. First, thrombin-generated fragments were applied fibronectin with as this enzyme cleaves rather selectively little further processing of the main fragments (16). Thrombin cleaves the fibronectin dimer close to the C-terminal interchain disulfide link forming single subunits. In addition, it removes an N-terminal fibrin-binding 30 kD-domain from each of the two long chains leaving a group of remnants ranging in the size of

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some undigested 200 kD. These 200 kD-fragments together with fibronectin were separated from the digest by adsorption on gelatin-Sepharose and 30kD-fragment in the non-absorbed fraction purified by affinity chromatography on heparin-Sepharose was followed by gelfiltration over AcA 44. Among the thrombin-derived fragments the N-terminal 30 kD-piece considerably enhanced binding of 125-I-fibrinmonomer to more than 70 macrophages as shown in Fig.2. In this experiment per cent binding was observed 100 ug/ml 30 kDin presence of fragment compared to ca. 10 per cent in its absence. Half-maximal improvement was achieved at 15 lg/ml. For technical reasons binding was generally measured after an overnight incubation at room temperature. At 37 C a significant binding, about half of that recorded under the above conditions, was found already after three hours. Binding of 125-I-fibrinogen to macrophages was elevated by 30 kD-fragment (60 ug/ml) from 3 to about 30 per cent. However, as here only traces of the fibrin precursor were applied, one cannot exclude a conversion to 125-I-fibrinmonomer by a limited coagulant activity of the cells (see discussion). TABiE

1

Inhibition of 30 kD-fragment-mediated monomer to macrophages. Inhibitor

binding

of

125-I-fibrin-

Fibrinogen (tig/ml)

125-I-Fibrin bound (%)

Inhibition (%)

40 4":

43.9 14.6 0.3

66.7 99.4

putrescine putrescine

1 4

67.9 5.2 2.5

9215 96.4

1.0 mM

EDTA

4 4

61.3 4.5

9217

0.1

mM

dansylcadaverine

1

72.9 41.8

42:7

400

ng

125-I-fibrinmonomer,

2.5 5.0

mM putrescine mM putrescine

2.5 5.0

mM mM

50 ug 30kD-fragment

,

lO-/cells per ml.

The promoting effect of the thrombin-derived 30 kD-fragment on fibrinmonomer cell-binding was effectively suppressed by 1 mM EDTA and by 2-5 mM putrescine which was investigated at two different fibrinogen concentrations (Tab.1). Partial inhibition was also achieved by 0.1 mM dansylcadaverine. In this case application of higher concentrations was hampered by the low

188

FIBRIN BINDING TO MACROPHAGES

FIG.3

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80’

o 200k

Experimental

Binding of 125-I-fibrinmonomer (400 ng/ml) to macrophages at 4 ug/ml fibrinogen. Influence of thrombin-derived 200 kDfibronectin remnants (solid line) and remnants previously treated with PPACK (dashed line, upper part). Lower part records controls measured in the absence of cells showing that increased experimental data in the upper range of PPACK-treated 200 kD-fragment are due to unspecific insolubilization of 125-I-fibrinmonomer.

70.

_*

TABLE Residual thrombin by the chromogenic

8

16

32 6L 200 k Fragment

__.’

Thrombinum purum Fibronectin fragments 200 kD 200 kD/PPACK; 30 kD/PPACK

Quantity

,’

,/

200k ” PPACK

a

128 fpg/ml)

200 k

256

2

activity of fibronectin fragments substrate Chromozyme TH.

Sample

.

-=

_*.--

0

200k PPACK

nE405/min

Units

determined Units/mg

0.66

JJg

0.046

0.2

300

0.25 0.25 0.25

mg mg mg

0.057 0.00 0.00

0.25
0.99 (0.04 <0.04

* Fragments isolated from a thrombin fibronectin terminated by PPACK.

digest

of plasma

solubility of the agent. The inhibition studies suggest that a transamidase-catalyzed cross-linking reaction on the cell surface is involved in the binding process. The effective EDTA-concentration corresponded to the calcium content of the medium. Putrescine and dansylcadaverine were active in a range which is inhibitory to other transamidases (17,18). Dansylcadaverine is the strongest specific transamidase inhibitor known (19). In contrast to the 30 kD-fragment the gelatin-binding 200 kD fibronectin remnants failed to mediate binding of 125-I-fibrinmonomer to macrophages. This result was obtained

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with fragments isolated from a thrombin digest terminated by the specific thrombin inhibitor PPACK. If inactivation of thrombin isolated by adsorption on was omitted, the 200 kD-fragments gelatin-Sepharose still contained residual thrombin activity as (Tab.2). In revealed by the chromogenic substrate Chromozym TH considerably the 200 kD-fragments enhanced cell this case was, binding of 125-I-fibrinmonomer (Fig.3). The activity indicating hirudin that the however, completely abolished by to adherent mediator function is thrombin to be attributed Actually, binding rather than to the 200 kD-fragments (Tab.3). of 125-I-fibrinmonomer to macrophages could be considerably improved by small amounts of thrombin. The effects of thrombin 125-I-fibrinmonomer or thrombin-containing 200 kD-fragments on shown). In cell binding were not influenced by putrescine (not the experiments with thrombin or thrombin-containing fragments unspecific fibrin deposition was prevented by using low fibrinogen concentrations in the assay system. TABLE

3

Inhibition by hirudin of 125-I-fibrinmonomer cell binding mediated by thrombin-derived 200 kD-fibronectin fragments containing residual thrombin activity. Hirudin (pg/ml) 0°D3 0:12 400 ng ments,

Inhibition (%)

125-I-Fibrin bound (%) ;; 3

125-I-fibrinmonomer, 107 cells per ml.

96

4 ug fibrinogen,

TABLE

130 ug 200

kD-frag-

4

Enhancement of 125-I-fibrinmonomer binding to macrophages thrombinand plasmin-derived fibronectin fragments. Fragment 30 kD 200

kD

Digest

ug/ml

thrombin plasmin

::

thrombin* plasmin

260 260

125-I-Fibrin

bound

by (%)

61.4 42.5 o.o** 3.2

* digest terminated with PPACK ** camp. Fig.3 400 ng 125-I-fibrinmonomer, 4 ug fibrinogen, lo7 cells

per ml.

The 200 kD-fragments negligeably influenced the promoting effect of the 30 kD-peptide (60 ug/ml) on fibrinmonomer cell binding up to a concentration of 60 ug/ml. At higher

190

concentrations precipitation. more detailed

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technical difficulties arose due to unspecific This multicomponent system, however, deserves a investigation.

Fragments of similar size originating from the same regions as those produced by thrombin are also generated by plasmin (20,21). So, an early plasmin digest was as well separated into gelatin-binding high molecular weight core peptides and nonbinding 30 kD-fragments. Again the latter were active in enhancing 125-I-fibrinmonomer binding to macrophages and the 200 kDremnants showed little effect (Tab.4). In case of the 30 kD-fragment the mediator function was inherent to the peptide and not dependent on any residual thrombin activity. The fragment used in the experiment shown in Fig.2 was isolated from a thrombin digest terminated by PPACK and was shown to be free of thrombin activity (Tab.2). DISCUSSION Removal of soluble fibrinmonomer complexes from circulating blood is an important function of the body to prevent deposition of fibrin within the vessels giving rise to stenosis or occlusion. Clearance of fibrinmonomer takes place by cells of the reticula-endothelial system which remove it far more rapidly than circulating fibrinogen (22). Blockade of this system can occur in severe cases of disseminated intravascular coagulation or other events when large amounts of soluble fibrin emerge. It is suggested that the failure accounts for the consumption of a plasma factor involved in the clearing reaction. Generally, it is assumed that fibrinmonomer first associates with a plasma factor and that the complex is recognized by the reticula-endothelial system which consists of macrophage-like Kupffer cells. Several evidences (10,ll) point in the direction that plasma fibronectin might be such a factor, although direct proof failed to demonstrate an involvement. Now, the presented data show that processing of fibronectin by thrombin or plasmin produces fragments active in mediating cell binding of fibrinmonomer. Evidently, the plasma factor mediating fibrinmonomer clearance has to be liberated from the plasmatic fibronectin precursor by proteases generated in the course of fibrin formation and processing. The active fragments represent the N-terminal 30 kD-domain consisting of five homologous peptide sequences each containing two intrachain disulfide bonds (21,23). The domain the bears main fibrin-binding site of fibronectin (24,25) but is devoid of a cell-binding site. Close to the N-terminus of peptide its chain a sensitive glutamine residue is located capable of forming cross-links to fibrin and some other substrates by a transamidase-catalyzed reaction (26). This site appears to be important for the mediator function of the fragment in fibrinmonomer cell binding as the binding is inhibited by 2 - 5 mM putrescine and to about 40 per cent by 0.1 mM dansylcadaverine,

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two competitive inhibitors of transamidase-catalyzed cross-linkIt is possible that covalent binding of the 30 kD-fragment ing. for cell binding of a with a cell-surface receptor is essential complex consisting of fibrinmonomer and the 30 kD-fragment. is the formation of an effective cellAnother possibility binding site on the fibrin M-chains after covalent linking of cell-binding site the 30 kD fibronectin fragment. A potential was already postulated in that part of the fibrin molecule (27). The remaining fibronectin remnants represented by a group of peptides of ca. 200 kD size still contained fibrin-binding domains (25,28-30) and even transamidase-reactive cross-linking sites (31). These remnants were ineffective in mediating binding of their of fibrinmonomer However, in case to phagocytes. generation by limited proteolysis with thrombin followed by incomplete separation from the protease, the isolated fragments promoted cell binding of fibrinmonomer. The active principle of this fraction was inhibited by hirudin and, therefore, was due to residual thrombin. Whether these fragments contribute to fibrinmonomer clearance by complexing thrombin and transporting it from the area of blood clotting to the reticula-endothelial system or whether they play any other role in the multicomponent system acting on the surface of those cells must be clarified by further experiments. Fibronectin 200 kO-remnants produced by plasmin digestion had no influence on fibrinmonomer binding to macrophages. The binding studies had been performed at a surplus of fibrinogen which served as a solubilizing agent for fibrinmonomer. In addition, it was important to demonstrate that 30 binding if fibrinogen kD-fragment promoted fibrinmonomer cell was present. As considerable amounts of 125-I-fibrinmonomer were bound by the macrophages, fibrinoa significant competition by gen appears unlikely. fibrinogen, On the other hand, labelled applied without cold fibrinogen, was remarkably bound by the cells in presence of 30 kD-fragment. These controversal findings become understandable if one considers a limited conversion of fibrinogen to fibrin by the macrophages as already shown for monocytes (32). Its extent appears to depend on the cell prepar-. ation. In the experiment of Fig.2 fibrinmonomer generation clearly was insufficient to compete effectively for cell binding of labelled fibrinmonomer. However, with macrophages from another animal a reduced cell binding was observed at a higher fibrinogen concentration Therefore, in (camp. Table 1). later experiments a fibrinogen content of only 4 ug/ml was applied (Table 2 and 4). The interaction of macrophages with fibrinogen deserves a more critical investigation. ACKNOWLEDGEMENTS We are indebted to Mrs. C. Wendt The investigation was supported by schaft, Sonderforschungsbereich 0207.

for technical assistance. Deutsche Forschungsgemein-

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