t of Arteries, Veins, Capillaries, and Lymphatics, and on Syncytiotrophoblast of Human Placenta

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodutin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain. Endothelium has the important function of maintaining blood fluidity. This is accomplished in part by the production of the icosanoid mediator PGI2 in response to endothelial injury (2), thrombogenic stimuli such as thrombin (24), or inflammatory mediators such as histamine (l) and bradykinin (17). PGI2 acts as a vasodilator and inhibits platelet activation (3). Recently, other endothelial factors that inhibit coagulation reactions have been described. These include heparinlike molecules on the cell surface that act as a cofactor for anti-thrombin III in inhibiting several coagulation factor proteases (13), and the cell surface protein, thrombomodulin, that acts as a cofactor for thrombin-catalyzed activation of protein C. Activated protein C inhibits coagulation by inactivating factors Va and VIIIa. Thrombomodulin was first isolated from detergent extracts of rabbit lung by affinity chromatography on thrombin-Sepharose (5-8). The protein was postulated to be of endothelial origin since perfusion of thrombin and protein C through rabbit vessels led to accelerated rates of protein C activation (6). A similar protein was thought to be present in humans since accelerated rates of thrombin-catalyzed protein C activation could also be demonstrated in the presence of intact human umbilical vein endothelial cells in culture (6), and antibodies directed against rabbit thrombomodulin partially inhibited protein C activaTHE JOURNAL OF CELt BIOLOGY . VOLUME 101 AUGUST 1985 363-371 © The Rockefeller University Press • 0021-9525]85[08]0363]09 $1.00 Lion on human cells (15). The existence of thrombomodulin in human tissues has been recently established by the isolation of the protein from extracts of human placenta. Human thrombomodulin is similar but not identical to the rabbit protein (14, 20). Thus, human thrombomodulin activity is stimulated by coagulation factor Va and its isolated light chain. We have recently prepared antibodies against human thrombomodulin. Using these antibodies, we have studied the tissue distribution of thrombomodulin. Using an avidinbiotin immunoperoxidase method, we now report that thrombomodulin is found in most human endothelium including that of arteries, veins, capillaries, and lymphatics. Another cell type found to contain thrombomodulin is the placental syncytiotrophoblast. This cell has also been found to contain yon Willebrand factor antigen, another marker of endothelium. MATERIALS AND METHODS Except when indicated, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Human thrombin (16), human protein C (22), and antithrombin IIl (18) were isolated from human plasma as described. Anti-human von Willebrand factor rabbit lgG was purchased from Dako Corp. (Santa Barbara, CA). Nitrocellulose sheets were purchased from Bio-Rad Laboratories (Richmond, CA). 363 on Jauary 8, 2018 jcb.rress.org D ow nladed fom Preparation of Anti-thrombomodulin IgG: Two male rabbits were immunized intradermally at 300 sites with 60 #g each of thrombomodulin in complete Freund's adjuvant by the method of Vaitukaitis (23). After 2 mo, potent antiserum was obtained. Anti-thrombomodulin IgG or control IgG was isolated using protein A-Sepharose (Pharmacia Fine Chemicals, Piscataway, N J) as follows: 5 ml of the anti-thrombomodulin serum or control serum was applied on a protein A-Sepharose column (1 x 5 cm) equilibrated with phosphate-buffered saline (PBS; l0 mM phosphate, 0.15 M NaCl, pH 7.4). After the column was washed with 50 ml of PBS, lgG was eluted with 0. l M glycine, pH 3, and immediately dialyzed against the buffer with 20 mM TrisHCI, 0.15 M NaCI, pH 7.4. 0.05 pmol human thrombomodulin isolated from placenta was incubated with 0-10 pg of anti-thromhomodulin IgG for 15 min at 37"C, and then cofactor activity of the thrombomodulin was assayed in reaction mixtures containing 1.0 pM protein C and 40 nM thrombin in a total volume of 30 pl 20 mM Tris-HCI, pH 7.4, 0.15 M NaC1, l mM CaCl2, and 5 mg/ml bovine serum albumin (BSA). Protein C activation was terminated by addition of 40 U/ml hirudin and 350 pg/ml anti-thrombin III. The amount of activated protein C was assayed by measuring the rate of hydrolysis of 0.2 mM o-Phe-pipecolyl-Arg-p-nitroanilide ($2238, Kabi Diagnostica, Sweden). Immunoblot of Thrombomodulin: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed by the method of Laemmli (l l) using 4% acrylamide in the stacking and 10% acrylamide in the running gel. Samples of placental thrombomodulin were electrophoresed with 2-mereaptoethanol. Immunoblotting was performed by the method of Burnett~ (4). Anti-thrombomodulin serum was used at a 1:250 dilution, and detection was made using ~2~I-protein A and autoradiography. Cell Cultures and the Functional Assay of Thrombomodulin Activity: Primary cultures of human umbilical vein endothelial cells were prepared as previously described (10). One of the human choriocarcinoma cell lines, JAr, was provided by Dr. Irving Boime, Washington University, and the other, BeWo, was from American Type Culture Collection. These cells were cultured in plastic 30-100-mm petri dishes or 6-16-mm multi-well plates. Protein C activation was examined on these cells as previously described (15). The cultured cells were also used for immunochemical study. Tissue Preparation: Tissues (excluding placenta) fixed in 10% formalin buffered with 0.1 M calcium acetate, pH 7.4, for a minimum of 24 h, were obtained from the autopsy pathology service. Placenta fixed in 10% formalin buffered with 0.15 M sodium acetate, pH 7.0, for a minimum of 24 h, was obtained from the surgical pathology service. Tissues were embedded in paraffin, and 6-/am-thick sections were cut and placed on microscope slides that had been cleaned with 10% potassium dichromate, 1% sulfuric acid solution, and coated with 0.25% gelatin containing 0.05% chrome alum as an adhesive (19). Sections were then dried at 60"C and stored at room temperature. Immunocytochemistry: Sections were de-waxed in 100% xylene for l0 min with one change, and rehydrated in 100% isopropyl alcohol for l0 min with one change, 70% isopropyl alcohol for 5 min, and PBS for 15 min with two changes. Rabbit anti-thrombomodulin serum and control rabbit serum were diluted l:l,000; rabbit anti-thrombomodulin IgG and normal control rabbit IgG were diluted to l0 pg/ml; and rabbit anti-von Willebrand factor lgG and its control rabbit IgG were diluted to 20 pg/ml. All dilutions were made in 10% normal human serum in PBS. Diluted reagents were centrifuged at 20,000 g for 20 rain to remove any large aggregates. Normal human serum provided optimal blocking of nonspecific staining compared with normal goat serum or no serum, with no impairment of specific staining. For anti-von Willebrand factor staining, sections were preincubated with 0.025% trypsin (KC Biological Inc., Lenexa, KS) in 0.1% CaCl2, 0.05 M Tris-HC1,' pH 7.6 for 30 rain at 37"C. This was necessary in order to expose von Willebrand factor antigenic determinants in formalin-fixed tissue. Thrombomodulin staining was unaffected by the trypsin preincubation. These sections were then washed in PBS for 15 min with two changes. All sections were incubated with either antiserum, immune IgG, or control preparations for 16-20 h at 4"C in a humid atmosphere, gently washed in PBS for 15 min with two changes, and then incubated with biotinylated affinity-purified goat anti-rabbit immunoglobulin (Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature. The latter reagent was diluted to the recommended concentration (45 #l in 10 ml) in 10% normal human serum in PBS. Sections were again gently washed in PBS for 15 min with two changes, incubated with avidin-biotinylated horseradish peroxidase complex (ABC reagent, Vector Laboratories, Inc.) at the recommended concentration (90 pl avidin and 90 ul biotinylated horseradish peroxidase in 10 ml PBS, allowed to incubate for 30 min before use) for 45 min, washed in PBS for 5 min and 100 mM Tris-HCl buffer, pH 7.2, for 10 min with one change. After incubation in 0.05% diaminobenzidine tetrahydrochloride, 0.01% H202 in Tris-HCl buffer, pH 7.2, for 5 min, sections were washed in the Tris-HCl buffer for 10 min with one change and in deionized water for 5 min. Sections were then counter-stained in 0.5 % Harris hematoxylin 364 THE JOURNAL OF CELL BIOLOGY . VOLUME 101, 1985 (21) for 2 min, differentiated by quick dipping in acid alcohol (1% HCI, 70% ethanol), and blued in gently running tap water for 5 min. Sections were finally dehydrated in 70% isopropyl alcohol for 5 min, 100% isopropyl alcohol for 10 min with one change, and 100% xylene for l0 min with one change, and mounted in Permount. Cultured cells of monolayers were also fixed and stained as described above. 5pecific Absorption of Antibodies: Thespecificityofanti-thrombomodulin lgG and anti-von Willebrand factor IgG was established by absorption with the respective purified antigens. Anti-thrombomodulin lgG (20 pg/ ml) was incubated with 30 pg thrombomodulin/ml (threefold excess) isolated from human placenta for 24 h at 4"C and then centrifuged at 20,000 g for 30 min and the supernatant collected and used for immunohistochemistry. Antivon Willebrand factor lgG (20 pg/ml) was incubated with 25 t~g yon Willebrand factor/ml as described above. The concentration of von Willebrand factor was five times that required to neutralize the antibody. The protein was homogeneous as determined by SDS PAGE and was a gift from Drs. Evan Sadler (Washington University) and Earl Davie (University of Washington). The respective antisera were also absorbed with human serum albumin as a control. Sections of human placenta were stained using these absorbed lgG solutions as described above.