We demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.
Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.
References
1
Dahlbäck B.
A new model for coagulation factor V suggesting a unique mechanism of activation. Scand J Clin Lab Invest 1988; 48 (Suppl. 191) 47-61
6
Mazzorana M,
Cornillou B,
Baffet G,
Hubert N,
Eloy R,
Guguen-Guilluozo C.
Biosynthesis of factor V by normal adult rat hepatocytes. Thromb Res 1989; 54: 655-675
7
Wilson DB,
Salem HH,
Mruk JS,
Maruyama I,
Majerus PW.
Biosynthesis of coagulation factor V by a human hepatocellular carcinoma cell line. J Clin Invest 1984; 73: 654-658
9
Tracy PB,
Eide LL,
Mann KG.
Human prothrombinase complex assembly and function on isolated peripheral blood cell populations. J Biol Chem 1985; 260: 2119-2124
11
Annamalai AE,
Stewart GJ,
Hansel B,
Memoli M,
Chiu HC,
Manuel DW,
Doshi K,
Colman RW.
Expression of factor V on human umbilical vein endothelial cells is modulated by cell injury. Arteriosclerosis 1986; 6: 196-202
12
Annamalai AE,
Rao AK,
Chiu HC,
Wang D,
Dutta-Roy AK,
Walsh PN,
Colman RW.
Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium. Blood 1987; 70: 139-146
13
Kane WH,
Davie E.
Cloning of cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplamin. Proc Natl Acad Sci USA 1986; 83: 6800-6804
14
Feinberg AP,
Vogelstein B.
A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984; 137: 266-267
15
Rappolee DA,
Mark D,
Banda MJ,
Werb Z.
Wound macrophages express TGF-alpha and other growth factors in vivo: Analysis by mRNA phenotyping. Science 1988; 241: 708-712
17
Fuqua SA,
Fitzgerald SD,
McGuire WL.
A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. Biotechniques 1990; 9: 206-211
19
Niemetz F,
Morrison DC.
Lipid A as the biologically active moiety in bacterial endotoxin (LPS) initiated generation of procoagulant activity in peripheral blood leukocytes. Blood 1977; 49: 947-956
20
Carlesen H,
Prydz H.
Enhancement of procoagulant activity in stimulated mononuclear blood cells and monocytes by cyclosporine. Transplantation 1981; 43: 543-548
21
Osterud B,
Rapaport SI.
Activation of factor IX by the reaction product of tissue factor and factor VII: Additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-5264
26
Schwartz BS,
Levy GA,
Fair DS,
Edgington TS.
Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase. J Exp Med 1982; 155: 1464-1479
30
Tracy PB,
Mann KG.
Prothrombinase complex assembly on the platelet surface is mediated through the 74,000 dalton component of factor Va. Proc Natl Acad Sci USA 1983; 80: 2380-2384
31
Kane WH,
Ichinose A,
Hagen FS,
Davie EW.
Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats. Biochemistry 1987; 26: 6508-6514
34
Chelly J,
Concordet JP,
Kaplan JC,
Kahn A.
Illegitimate transcription: Transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-2621
35
Brenner CA,
Tam AW,
Nelson PA,
Engelman EG,
Suzuki N,
Frey KE,
Larrick JW.
Message amplification phenotyping (MAPPing), a technique to simultaneously measure multiple mRNAs from small number of cells. BioTechniques 1989; 7: 1096-1103
36
Gregory SA,
Morrissey JH,
Edgington TS.
Regulation of the tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989; 9: 2752-2755
38
Adany R,
Kappelmayer J,
Berenyi E,
Szegedi A,
Fabian E,
Muszbek L.
Factors of the extrinsic pathway of blood coagulation in tumour associated macrophages. Thromb Haemostas 1989; 62: 850-855
39
Osterud B,
Bogwald J,
Lindahl U,
Seljelid R.
Production of blood coagulation factor V and tissue thromboplastin by macrophages in vitro. FEBS Lett 1981; 127: 154-156
