Factor XIII of blood coagulation in human monocytes

THROMBOSIS RESEARCH 37; 401-410, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

FACTOR XIII OF BLOOD COAGULATION

IN HUMAN MONOCYTES

L. Muszbek, R. Addny, G. Szegedi': J.+Polgar and M. Kavai" Department of Clinical Chemistry and IIIrd Department of Medicine, University School of Medicine, Debrecen H-4012 Hungary (Received 12.9.1984; Accepted in revised from 5.11.1984 by Editor R. Machovich)

ABSTRACT The presence of Factor XIII subunit a was demonstrated in human monocytes by immunoperoxidase staining using specific antisera against Factor XIII and its subunits. This finding was verified bv immunobiochemical techniques, as well. In an immunoblotting system after SDS polyacrylamide gel electrophoresis of denatured monocyte homogenate a protein band comigrating with Factor XIII subunit a showed positive reaction with antibodies against this subunit or whole Factor XIII. In contrast, no subunit b of Factor XIII could be detected by either of these methods in monocytes. Activity measurements were carried out by the dansylcadaverine incorporation assay in the absence and presence of anti-Factor XIII antibody with and without thrombin activation. The expression of transglutaminase activity required thrombin and was completely abolished in presence of anti- Factor XIII antibody, which clearly indicate that practically all the transglutaminase activity measured in monocytes comes from Factor XIII. Factor XIII of monocytes and macrophages might have a role in formation of focal fibrin thrombi as well as in organization of stable, fibrinolysis resistant fibrin clot at the site of inflammation or around tumor cells.

Key words:

Factor XIII, monocytes, immunoblotting

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INTRODUCTION Factor XIII (FXIII) of blood coagulation is a plasma zymoqen (protransqlutaminase) with a tetrameric structure consisting of two types of subunits (22b2). Platelets also contain the a subunit dimer (a 1 but they lack the b subunits. FXIII is converted into an 2 ctive enzyme (plasma-transqlutaminase; FXIIIa) by the ps?teolytic action of thrombin and by the divalent cation Ca . The activated 2 subunits possess transqlutaminase activity, i.e., they catalyze covalent attachment of small molecular weight primary amines to peptide bound glutamine residues or crosslinking of two peptide chains via an 6 -(y-qlutamyl)lysyl bond (isopeptide bond; refs. l-3). FXIIIais to be differentiated from the liver type tissue transqlutaminase. The latter consists of a single polypeptide chain, does not need a proteolytic activation and possesses distinct biochemical and immunological features (1,4,5). Most recently several papers have been published on the existence of tissue transqlutaminase in monocytes and macrophaqes (6-lo), and it has been implicated in receptor mediated endocytosis as well as in phaqocytosis (6,8,11-14). Here we report the identification of 2 but not g subunit of FXIII in human peripheral blood monocytes and show that it exists essentially in the zymoqenic form and can be transformed into active transqlutaminase by thrombin. The low transqlutaminase activity measured in the absence of thrombin is also of FXIII origin. MATERIALS

AND METHODS

Throughout the separation Isolation of human blood monocytes. procedure special effort was taken to eliminate contaminating free and adherent platelets from monocyte preparations. 100-200 ml heparinized venous blood was centrifuged for 10 min at 20°C, platelets were pellet400 q. Platelgt rich plasma was removed, ed (3000 q, 4 C, 10 min) and platelet poor plasma obtained this way was given back to the original red blood cell and buffy coat pellet. Mononuclear cells were separated from this mixture on Ficoll-Hypaque as described by Nakaqawara et al (21). The isolation of monocytes was carried out either by centrifuqation on a continuous ?ercoll gradient (22) or by the adhesion technique of Kumaqai et al(23). Platelets still adherent to the monocytes were removed according to Pawlowski et al (24) with the only modification that for the resuspension and incubation of monocytes 5% bovine serum albumin containing 5 mM EDTA was used instead of EDTA-serum solution. 93-96% of the cells was proved to be monocytes by non-specific esterase staining (25) and the viability of the cells was around 95% by the trypan blue exclusion test. The monocyte platelet ratio in the final preparation was 1OO:l. Phaqocytosis of C3-coated yeast particles by monocytes was carried out as described earlier (26).

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Immunoperoxidase staining of human monocytes for FXIII Monocytes adhered to a glass surface and its subunits. were fixed by 3.5% paraformaldehyde fixative in isoosmolar phosphate buffered saline (PBS) pH 7.2 for 40 min at room temperature. Endogenous peroxidase activity was blocked by 3% H20jt in PBS for 30 min. To prevent aspecific binding of anti F III antibodies preparations were incubated for 30 min in 5% human serum diluted by PBS. For the detection of FXIII and its subunits commercially available rabbit antisera against 5 and b subunits of human FXIII (Behring) as well as rabbit antiserum against highly purified whole human FXIII raised in our laboratory were used. The specificity of the antisera was checked by immunoblotting against human plasma, platelet homogenate, highly purified human and bovine FXIII as well as against guinea pig liver tissue transglutaminase (a gift'of L. Fks&). No reaction with plasma or platelet proteins but the respective FXIII subunits were observed. Antiserum against subunit b showed a slight crossreaction with the a subunit while antiserum against the a subunit possessed an exclusive specificity (see in Fig. 2). None of the antisera crossreacted with tissue transglutaminase. Fixed monocytes pretreated as described above were incubated in 1:200 dilution of antisera for 2 hours. For development biotynilated antirabbit IgG and avidin-biotynilated peroxidase complex (Vectastain ABC kit, Vector Labs.) were used. The reaction was visualized by 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB), 0.01% H202 substrates in 0.1 M Tris HCl buffer pH 7.2. In control normal rabbit serum was replaced for anti FXIII antisera. In another series of experiments anti FXIII antisera exhausted with an excess of highly purified human FXIII were used. Identification of FXIII in monocytes by immunoblottinq. Monocytes separated were sonicated (3x30 set) in denaturing solution and boiled for 5 min. The last supernatant in the preparation procedure was used as control for the neglectable amount of platelets still contaminating the monocyte preparation. This supernatant contained 3 times as many platelets 8s the final monocyte preparation. It was spun down (3000 g, 4 C, 15 min) and treated similarly to monocytes ("platelet control"). Denatured monocyte preparation and "platelet control" as well as human FXIII and molecular weight standard (Sigma, SDS-6H) were submitted to SDS polyacrylamide gel (10%) electrophoresis according to Laemmli (27). Three parallel runs were performed on the same gel, which were separated following electrophoresis. One part was stained by Coomassie blue and destained. The two others were electroblotted to nitrocellulose paper and developed for FXIII subunit a or b respectively by immunoperoxidase technique (BIO BAD IGun-B‘rot Assay Kit with the modification that 0.01% DAB and 0.003% H 0 in Tris buffered saline pH 7.6, were applied as peroxidase su 28 strates). As the source of the first antibody 1:500 dilution of antisera against subunit -a or b of FXIII (Behring) were used.

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Transglutaminase activity-measurement in extract of human monocytes. Monocytes prepared by Percoll gradient centrifugation and depletgd of platelets were extracted by sonication (3x30 set at 4 C)in Tris buffered saline pH 7.6 cgntaining 1% Lubrol. After centrifugation (3000 g, 15 min, 4 C) the supernatant was removed for enzyme activity measurement. A mixture (500 ul final volume) containing monocyte extract (ME; 0.32 mg protein/ml), 8 mM CaCl 4 mM dithiothreitol, 35 mM Tris HCl pH 7.5 (final concen $' rations) with or without 100 ul antiserum against FXIJI subunit 2 (anti FXIII A) was prgincubated for 10 min at 4 C. Then it was warmed up to 37 C and, where indicated, thrombin (50 NIH U/ml) was added. Following another 10 min incubation transglutaminase activity was determined by the dansylcadaverine incorporation method (28). RESULTS AND DISCUSSION Verification of the existence of FXIII in human monocytes was first attempted by immunoperoxidase staining using specific antisera against FXIII or its subunits (Fig. 1). With antisera against FXIII and its subunit a component (Fig. 1A) positive reaction was obtained while no-subunit b could be detected by this technique. The specificity of the reaction was clearly proved by the facts that there was no staining with preimmune or normal rabbit sera and it was abolished by the preabsorption of the antisera with human FXIII (Fig. 1B). Subunit a of FXIII of these cells could be well demonstrated in the cytoplasm also following phagocytosis of yeast particles coated with C3b complement-component (Fig. 1C). No other cellular elements of peripheral blood but platelets and monocytes showed positive reaction, indicating that lymphocytes, granulocytes and red blood cells are devoid of FXIII subunits. The presence of FXIII subunit a in monocytes is further supported by biochemical experiments using immunoblotting technique (Fig.2). The only protein band stained with the respective antiserum in sodium dodecyl sulfate (SDS) extract of monocytes comigrated with subunit a on SDS polyacrylamide gel and no reaction could be demonstratsd with antiserum against b subunit. The same results were obtained when antiserum against whole FXIII was used in separate experiments. During the preparation procedure special effort was made to eliminate contaminating platelets and in the final Fmnocyte pre¶tionthe number of these cells was extremely low. As in control experiments platelets even in concentration 3 times higher than in monocyte preparation showed no detectable reaction by immunoblotting and lymphocytes present in a few percent were not stained in morphological experiments, FXIII detected by this technique is exclusively of monocyte origin. Transglutaminase activity measurements in Lubrol extract of monocytes provide not only a further proof for the existence of FXIII in these cells but also demonstrate that practically all the transglutaminase activity measured in monocytes comes from FXIII (Table 1). Without thrombin activation a rather low activity was obtained that was completely inhibited by antiserum against FXIII subunit d, i.e., in all probability it represented a small portion of FXIII tl?at had been activated within the cells or during the extraction procedure. Thrombin caused

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reaction of cytoplasmic Monocytes stained for FXIII subunit 2 showed a highly intensive localization (A) which was abolished by the preabsorption of the antiserum with FXIII (B). in phagocytosing cells in the cytoplasm among yeast FXIII subunit a was also well detected few lymhocytes contaminating the preparation were consequently negative. particles (C) . -The

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FACTOR XIII

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FIG. 2

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Electrophoretic patterns of monocyte extract (120 ug protein) and human FXIII (5 ug) stained by Coomassie blue (tracks 1,2) .Two identical segments of gel were electroblotted to nitrocellulose and developed for FXIII subunit a (tracks 3,4) or subunit 6 (tracks 5,6). At the left side the horizontal lines with the corresponding numbers indicate the electrophoretic mobility and molecular weight of components in the molecular weight calibration kit.a: subunit 2, b: subunit b of FXIII. In the "platelet control" the cell count and consequently the protein content, as well, were so low that no staining whatsoever could be detected either by Coomassie blue or by immunoblotting (not shown on the Figure). Note slight crossreaction of antiserum against subunit b with the a subunit that could be demonstrated only if high amount of FXIII was applied.

-a -b

123456

an almost 40 fold increase of transglutaminase activity clearly indicating that in resting monocytes FXIII exists predominantly in the zymogenic form. The high activity obtained following thrombin activation was practically also completely diminished by immunoinhibition of FXIII subunit a. TABLE I Transglutaminase

activity in human monocytes

Activity -1 -1 pm01 x min x mg ME ME + Anti FXIII A ME + Thrombin ME + Anti FXIII A + Thrombin

24 0 863 28

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Martaugh et al have reported that tissue transglutaminase was present at low level in freshly isolated human monocytes that was highly increased during adherent culture of these cells in autologous serum or plasma (10). This enzyme was also found in macrophages of various origin (6-9, 14). For this reason it is important to emphasize that in our experimental systems a clear distinction could be made between tissue transglutaminase and FXIII. This is clearly supported by the following pieces of evidence: l/ The antisera we used did not show crossreaction with guinea pig liver tissue transglutaminase by immunoblotting technique and in separate morphological experiments (Adany and Muszbek in preparation) negative reaction was obtained with human hepatocytes and red blood cells (rich sources of tissue transglutaminase). 2/ The electrophoretic mobility of tissue transglutaminase could be well distinguished from that of FXIII subunit a in our Laemmli gel. 3/ There is an enormous increase in transglutaminase activity following thrombin treatment, a characteristic feature of FXIII but not of tissue transglutaminase (1). In conclusion, it is clearly established that FXIII exists in monocytes in a rather high concentration and immunoinhibition experiments suggest that even the low transglutaminase activity measured without thrombin is derived from FXIII activated in vitro or in vivo. This result also indicates that tissue transglutaminase is present, if at all, only in undetectable concentration in these cells. In the experiments of Murtaugh et al (10) tissue transglutaminase was verified by immunoblotting using affinity purified antibody against guinea pig liver transglutaminase. However, this antibody reacted with human platelet homogenate, in which no other transglutaminase but FXIII is present and the protein band of monocyte extract that gave a positive reaction comigrated with the platelet protein but not with purified tissue transglutaminase. Thus, on this basis no clear conclusion could be drawn. Our results, obviously, do not exclude the possibility that during culturing of monocytes or following their transformation into macrophages an intensive synthesis of tissue transglutaminase is initiated, perhaps as a result of gene repression. In this case the coexistence of two types of transglutaminase within the same cell type would represent an interesting biological phenomenon. For more than a decade it was taken for granted that plasma FXIII is synthetized in the liver. Changes of FXIII level in various hepatic diseases (2) and preliminary morphological investigations (15, 16) seemed to support this general belief. Most recently, however, no FXIII was found in human hepatocytes by immunoperoxidase morphological technique (17). These observations of Fear et al well agree with our experience (Adany and Muszbek in pretxarationlandclearly disqualify liver as a candidate for the site of synthesis of plasma FXIII. The presence of FXIII in megacaryocytes (17, 18) and in fibroblasts (17) is well established. By our present finding monocyte is identified as an additional cell type that might be involved in the production of subunit a of _nlasma FXIII. In this context it is important to emphasize,-however, that only the presence Of subunit a is proved by the experiments reported here. On this basis one can surmise that monocytes are capable of synthetizing

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FXIII subunit a but there are other alternatives, as well. The isolated pTnocytosis of subunit a from plasma FXIII is one of the possibilities though not of high probability. The phagocytosis of senescent platelets by autologuos monocytes (19) might also contribute to the FXIII content of these cells. A further puzzling question is if FXIII becomes activated within the monocyte during its maturation or as a consequence of various stimuli and if yes what is the mechanism responsible for it. As far as the role of FXIII in monocytes is concerned one can only speculate at the present moment. Suggestions on the involvement of tissue transglutaminase in receptor mediated endocytosis and phagocytosis of macrophages (11-14) were based on circumstantial insufficient evidence and it is not sure if the same hypothesis could stand for activated PXIII, an enzyme with more restricted substrate specificity. Activated monocytes, as demonstrated earlier by Shelley and Juhlin (20), can induce the formation of focal fibrin thrombi, what in all probability has important physiopathologic implications. FXIII present in inflammatory macrophages and macrophages invading malignant tumors might have a role in the formation of stable, fibrinolysis resistant fibrin clot at the site of inflammation or around tumor cells. ACKNOWLEDGEMENT These studies were conducted in part pursuant to a contract with the National Foundation for Cancer Research, Bethesda, MD. and were also supported by a grant from the Hungarian Ministry of Health. The skillful technical assistance of Mr. R. Herendi is acknowledged. REFERENCES 1. FOLK, J.E. Transglutaminases. 1980.

Ann. Rev. Biochem.

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2. LORAND, L., LOSOWSKY, M.S. and MILOSZEWSKI, K.J.M. Human factor XIII: fibrin-stabilizing factor. Progr. Haemostas. Thrombos. -5,245-290,198o. 3. MUSZBEK, L. and LAKI, K. Interaction of thrombin with proteins other than fibrinogen (thrombin susceptible bonds). Activation of FXIII. In: The Thrombin. R. Machovich (Ed.) Boca Raton: CRC Press, 1984, pp. 321-342. 4. CHUNG, S,I. Comparative studies on tissue transglutaminase and factor XIII. Ann. N.Y. Acad. Sci. 202,240-255, 1972. 5. BOHN, H. Comparative studies on the fibrin-stabilizing factors from human plasma, platelets and placentas. Ann. N.Y. Acad. Sci. 202,256-272, 1972.

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6.

SCHROFF,G., NEUMANN, C. and SORG, C. Transglutaminase as a marker of subsets of murine macrophages. Eur. J. Immunol. 11,637-642, 1981. -

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KANNAGI, R., TESHIGAWARA, K., NORO, N. and MASUDA, T. Transglutaminase activity during the differentiation of macrophaqes. Biochem. Biophys. Res. Comm. 105,164-171, 1982.

8.

LEU, R.W., HERRIOTT, M.J., MOORE, P.E., ORR, G.R. and BIRCKBICHLER, P.J. Enhanced transqlutaminase activity associated with macrophage activation. Exp. Cell Res, 141, 191-199, 1982.

9.

MURTAUGH, M.P., MEHTA, K., JOHNSON, J., MEYERS, M., JULIANO, R.L. and DAVIES, P.J.A. Induction of tissue transglutaminase in mouse peritoneal macrophages. 2. Biol. Chem. 258,11074-11081, 1983.

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MURTAUGH, M.P., AREND, W.P. and DAVIES, P.J.A. Induction of tissue transglutaminase in human peripheral blood monocytes. J. Exp. Med. 159,114-125, 1984.

11.

MAXFIELD, F.R., WILLINGHAM, M.C., DAVIES, P.J.A. and PASTAN,I. Amines inhibit the clustering of a -macroqlobulin and EGF on the fibroblast cell surface. Nagure. 277,661-663, 1979.

12.

DAVIES, P.J.A., DAVIES, D.R., LIVITZKI, A., MAXFIELD, F.R., MILHAUD, P., WILLINGHAM, M.C. and PASTAN, I. Transglutaminase is essential in receptor-mediated endocytosis of a2 macroglobulin and polypeptide hormones. --Nature. 283,162-167, 1980.

13.

LEVITZKI, A., WILLINGHAM, M.C. and PASTAN, I. Evidence for participation of transglutaminase in receptor-mediated endocytosis. Proc. Xatl. Acad. Sci.ilSAn7r 2706-2710, 1980.

14.

FESUS, L., SANDOR, M., HORVATH, L.I., BAGYINKA, C., ERDEI, A. and GERGELY, J. Immune complex-induced transglutaminase activation: role in the F -receptor-mediated transmembrane effect on peritoneal macrgphages. Mol. Immunol. s,633-638, 1981.

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LEE, S.Y. and CHUNC, S.I. Biosynthesis and degradation of plasma protransglutaminase (factor XIII). Fed. Proc. %,1486a, 1976.

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IKEIMATSU, S. An approach to the metabolism Acta Haematol. Jpn. -44r1499-1505, 1981

17.

FEAR, J.D., JACKSON, P., GRAY, C., MILOSZEWSKY, K.J.A. and LOSOWSKY, M.S. Localisation of factor XIII in human tissues using an immunoperoxidase technique. J. Clin. Pathol. -37, 560-563, 1984.

18.

KISSELBACH,T.H. and WAGNER, R.H. Demonstration of factor XIII in human megacaryocytes by a fluorescent antibody technique. Ann. N.Y. Acad. Sci. 202,318-328, 1972.

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19. KHANSARI, N. and FUDENBERG, H.H. Immune elimination of aging platelets by autologous monocytes: role of membranespecific autoantibody. Eur. J. Immunol. 13, 990-994, 1983. 20. SHELLEY, W.B. and JUHLIN, L. Induction of fibrin thrombi by monocytes. Nature 270, 343-344, 1977. 21. NAKAGAWARA, A., NATHAN, C.F. and COHN, Z.A. Hydrogen peroxide metabolism in human monocytes during differentiation in vitro. J. Clin. Invest. 68, 1243-1252, 1981. 22. GMELING-MEYLING, F. and WALDMANN, R.A. Separation of human blood monocvtes and lymphocytes on a continuous Percoll gradient. J: Immunol.-Meth.-33, l-9, 1980. 23. KUMAGAI, K., ITOH, K., MINUMA, S. and TADA, M. Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation. 2. Immunol. Meth. 2, 17-26, 1979. 24. PAWLOWSKI, N.A., KAPLAN, G., HAMILL, A.L., COHN, Z.A. and SCOTT, W.A. Arachidonic acid metabolism by human monocytes. Studies with platelet-depleted cultures. 2. Exp. Med. 158, 393-412, 1983. 25. YAM, L.T., LI, C.Y. and CROSBY, W.H. Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283-290, 1971. 26. KAVAI, M., SANDOR, M., SZEGEDI, G., F&ST, G. and GERGELY, J. Effect of soluble immunocomplexes on Fc and C receptordependent phagocytosis by human monocytes. I&unolosv 44, 599-606, 1981. 27. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680, 1970. 28. LORAND,

deKIEWIET, J.W.C. and NOSSEL, H.L. L., URAYAMA, T., Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation. J. Clin. Invest. 48, 1054-1064, 1969.