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Clotting Factors Secreted by Monocytes and Macrophages Clotting factors secreted by monocytes and macrophages Citation for published version (APA): van Dam-Mieras, M. C. E., Muller, A. D., van Deijk, W. D., & Hemker, H. C. (1985). Clotting factors secreted by monocytes and macrophages: Analytical considrations. Thrombosis Research, 37(1), 9-19. https://doi.org/10.1016/0049-3848(85)90028-3 Document status and date: Published: 01/01/1985 DOI: 10.1016/0049-3848(85)90028-3 Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.umlib.nl/taverne-license Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim. Download date: 07 Oct. 2021 '1985 THR0MB0SISRESEARCH 37; 9-19, 0049-3848/8553.99 + .00 Printedin the USA. Copyright(c) 19S5Pergamon Press Ltd. A11rights reserved. CLOTTING FACTORS SECRETED BY MONOCYTES AND },IACROPHAGES: ANALYTICAL CONSIDEMTIONS. lvlaria C.E. van Dam-Mieras, Annemarie D. Muller, Wijgert A. van Deijk, Il. Coenraad l{emker, Department of Biochemistry, Limburg University, Maastricht, The Netherlands. (Received8.11.1983; Accepted in revisedform 19.9.1984 by Editor De Gaetano) (Receivedin fina1 form by ExecutiveEditorial 0ffice 2.'10..|984) ABSTMCT The secretlon of clotting factors by raE spleen macrophages and human peripheral blood monocytes has been studied. Ttre results show that the amount of clotting factors measured depends critically upon the characterist,ics of the assay system used. Ihe presence of warfarin, sa1lcylic acid or thrombin in the eulture mediun is shown to decrease the vitamln K dependent clotting factor actlvlty in the supernatant after in vitro culture of rat spleen macrophages and human perlpheral blood monocytes. INTRODUCTION The course of life of ce1ls belonging to the monocyte-macrophage series is given schematically below: bone marrow nultipotential stem cell vI rnonoblast J promonocyte monocyte ixed cel1 of the retlculo- endothellal system nflammatory ce11 recruited into tissues by chemotactlc signals Key words: monocytes, macrophages, clotting factors, clotting assays. 9 '10 PHAGOCYTES:PROCOAGULANT ACTIVITY Vo'|.37,No.1 Ce11s of the monocyte-macrophage series can be triggered by a variety of stimuli and the responses observed are determined by the stinuli used. Acrong the responses observed are: phagocytosis, bactericidal activity, turnoricidal activity and secretion of biologically active products into the extracellular surroundings. Among the secreted products are enzymes that can activate the plasma proteins of the coagulation-, fibrinolytic- and complement pat.hways. These enrynes forn a link between the hemostatic and immune systems (l-3). Ce1ls of the monocyte-macrophage series can also expose procoagulant activity on their surface after exposure to a variety of stinuli including bacterial endotoxins, antigen/antibody complexes, mitogenic lectins and the proteolytic producls of complement (2r4-10). Different types of ce11ular procoagulant activity have been described: l. tissue factor-like activity (2,4-7'10,11) 2. prothrombin activating enzyme(s) (2,6'L2,I6) 3. actlvated clotting factor VII (i3,14,15) 0sterud et a1. (17,18) and Lindahl et al. (19) have shown that un- stimulated murine peritoneal macrophages cultured in serum free medium secrete the coagulation factors IITVTVII,IX and X into the culture medium and do not expose tissue factor on their surface under these conditions. If, however, the macrophages have been stimulated during their isolation by endotoxin (in the presence of T-lynphocytes), thromboplastin is generated on the ce1lu1ar surface and the coagulation factors secreted ioto the mediun occur in their activat.ed forms. The thrombin level in the medium is reduced under these conditions. The possibility, that a combination of the capacity of uonocytes and macrophages to generate and expose tissue factor complex components (aPopro- tein and phospholipid) on the cellular surface and their capacity to syn- thesize and secrete clotting factors into the surrounding rnedium might expla|n the different procoagulant phenotypes that have been describedr has not received much attention in the literature as far as we know. As the ttcellular procoagulant activityrt of monocytes and macrophages forrns a 'rce1lu1ar link between immunology and coagulation, the possible role of the procoagulant activityrr in the defense against infection and rnalignancy and the possibility to use it as a parameter to fo1low pathological processes is very interesting. We therefore deeided to carefully characterize the dlf- ferent procoagulant factors secreted by rat spleen macrophages and hr:man peripheral blood monocytes and to set up species specific test systems. We further investigated if compounds like warfarin and aspirin, known to inhibit the carboxylation of vitamin K-dependent coagulation factors in the liver, also inhibited the secretion of the analogous cloLt.ing facEors by monocytes and macrophages. MATERIALS AND METIIODS Only analytical grade chemicals were used; all solutions were prepared in destilled water. Michaelis buffer: Na-veronal' 0.15 M; Na-citrate, 0.109 M; NaCl, 0.15 M; plt 7.35. I{uman thronbin (Serva Feinbiochemica, Heidelberg). Rat spleen macrophages were isolated from Brown Norway rats according to Bdyun (20) and cultured in serum free medium as described by Lindah..l et a1 (19). The macrophage cootent of the preparation was at least 967.. ce11 viability was 97%. Iluman peripheral blood monocytes were isolated and cultured as described by Bdyun (20) using ficoll paque (Pharmacia). The monocyte content of the preparation was at Least 957"; cel1 viability was 96%. Vol.37,No.l PHAGOCYTES:PROCOAGULANT ACTIVITY il Analysis of ce1l composi.tion was perforned on cytocentrifuge preparations stained with Giensa or non specific esterase (21). At least 200 cells were counted per s1ide. Viability was assessed by trypan blue exclusion. Culture medium: RPMI 1640 nediun (F1ow laboratories), peniciline (Gibco, fj-nal concentration 100 IU/m1), streptomycine (Gibco, final concentration 100 IU/n1), transferrine (Behringwerke, final concentration LOll g/^t), albumin (Behringwerke, final eoncentration 0.4 ng/n1). In several experiments this culture medium was supplemented wit.h additional compounds; the exact composition of the eulture medium will be specified in the description of these experinents. Culture medium and reagents were free from endotoxin contamination as determined by a chrornogenic substrate endotoxin assay (Kabivitrun Diagnostica ) (ZZ) . The in vltro effect of phospholl,pase C on the procoagulant activity generated by monocyt.es or macrophages was tested as follows (23124): a mixq_ure of 0.1 ml ci.trat.ed plasrna, 0.1 ur1 sample and 0.1 rn1 phospholipase C (I g/nL) was -C, lf incubated for 3 min at 37 than the clotting was initiated by the addition of 0.1 nl CaCl, (0.33 M). The clotting time observed was compared to that obtained when carrying out the same test with 0.1 n1 Michaelis buffer instead of phospholipase C. Rat standard plasma was obtained by pooling equal amounts of platelet free plasma fron 30 Brown Norway rats (I5 rnales and 15 fenales) and was stored in smal1 port,ions at -20 "C until use (25). Rat braln thromboplastin (Bror.m Norway rats) was prepared as described before (25). nat artificial factor II reagent was prepared according to Koller et al. (26) using oxalated rat plasna and rat serum. Human standard plasma was obtaj-ned by pooling equal amounts of platelet free plasma from at least 30 healtly individuals (15 nales aqd 15 females, average age 30 years) and was stored in smal1 portions at -20 "C prior to use (25). Iluman brain thromboplastin was prepared as described before (25). Iluman artificial factor II reagent T^rasprepared according to Koller et a1. (26) using human oxalated plasma and human serum. For the determination of the clotting factors VII, IX and X human congenital factor deficient reagenta were used. APTT-reagent r{as obtalned from Dade. thronboplastin tirne: plasma sanple 0.1 n1 t.hronboplastin 0.1 nl oC^ 30 sec incubatlon at 37 in a glass tube CaCl" (0.033 M) 0.1 n1 regi6tration of the clotti.ng tine APTT plasma sample 0.1 nl APTT reagent 0.1 n1 oC- 120 sec incubation at 37 in a glass tube CaC1, (0.033 M) 0.1 n1 regiEtrati-on of the clotti.ng tine One stage factor II, VII and X determination: sample, diluted with Michaelis buffer 0.1 m1 faetor-deficient plasma (IITVII or X) 0.1 ml thromboplastin 0.1 ml oC 30 sec incubation at 37 in a glass tube Cac1, (0.033 M) 0.1 nl regiStration of the clotting time No.l 12 PHAGOCYTES:PROCOAGULANT ACTIVITY Vo].37, One stage factor IX deternilqtigni - fer 0.
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