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Endothelial Cell Synthesis of Von Willebrand Antigen II, Von Willebrand Factor, and Von Willebrand Factor/Von Willebrand Antigen II Complex

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Endothelial Cell Synthesis of Von Willebrand Antigen II, Von Willebrand Factor, and Von Willebrand Factor/Von Willebrand Antigen II Complex Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex. D R McCarroll, … , E G Levin, R R Montgomery J Clin Invest. 1985;75(4):1089-1095. https://doi.org/10.1172/JCI111802. Research Article von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules. […] Find the latest version: https://jci.me/111802/pdf Endothelial Cell Synthesis of von Willebrand Antigen ji, von Willebrand Factor, and von Willebrand Factor/von Willebrand Antigen 11 Complex David R. McCarroll, Eugene G. Levin, and Robert R. Montgomery The Blood Center of Southeastern Wisconsin, Inc., and Department of Pediatrics, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233; and Departments of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037 Abstract determinants (4, 5), their levels in plasma are linearly associated (6). vWf and vW AgII are both synthesized by cultured von Willebrand antigen II (vW AgIl) and von Willebrand human endothelial cells and released concomitantly after in factor (vWf) are immunochemically distinct proteins that are vivo stimulation with l-desamino-8-D-arginine-vasopressin deficient in the plasma and platelets of patients with severe (DDAVP) (7). Both proteins are also found in platelets and von Willebrand's disease. Normal human umbilical vein en- are released together after platelet activation (8). dothelial cells were cultured in the presence of V3Slmethionine. vWf is a glycoprotein that circulates in plasma as a series Crossed immunoelectrophoresis of endothelial cell supernates of high molecular weight multimers (1-3) and mediates the and detergent-solubilized endothelial cells demonstrated specific adhesion of platelets to exposed subendothelium (9). These incorporation of the I3Simethionine into vW AgIl. Furthermore, multimers are comprised of 220,000-mol-wt subunits that are when endothelial cells were lysed in the presence of proteolytic held together by disulfide bonds and range in molecular inhibitors, a second, less anodal peak was identified on crossed weights up to 20 X 106. vWf is measured antigenically as von immunoelectrophoresis. This peak represented a complex of Willebrand antigen or Factor-VIII-related antigen (10) and vW AgIl and vWf and demonstrated a reaction of complete functionally as the cofactor that mediates platelet aggregation identity with the vW AgHI immunoprecipitate. When plasma, in the presence of the antibiotic ristocetin (1 1). vWf is synthe- serum, or platelets were evaluated by crossed immunoelectro- sized in endothelial cells (12-14) and megakaryocytes (15). phoresis, this "complex" peak was not present. When antibodies Studies of vWf synthesized in cultured human umbilical vein to vWf, fibronectin, or fibrinogen were present in the first endothelium indicate processing that includes not only glyco- dimension of crossed immunoelectrophoresis, only the antibodies sylation but also cleavage of a 260,000-mol-wt precursor to vWf removed the complex. Radioiodinated polyclonal and molecule into the 220,000-mol-wt vWf subunit (14). Intracel- monoclonal antibodies to vWf also localized vWf to this lularly, vWf appears to be present as a series of multimers complex. Under reducing conditions, sodium dodecyl sulfate- with molecular weights equivalent to those of normal plasma, polyacrylamide gel electrophoresis of 135Sjmethionine-labeled reaching at least 10 X 106 mol wt (16). Most of the protein, immunoprecipitates indicated that the molecular weight of vW however, appears to be present in the dimeric (14) or tetrameric AgHI is 98,000 and that vWf was present as two species of (16) form. vWf has demonstrated in Weibel-Palade bodies 220,000 and 260,000 mol wt, respectively. Immunofluorescent (17), although the significance of this subcellular localization microscopy of endothelial cells demonstrated colocalization of is not well understood. vWf secreted into the culture fluid of vW AgHI and vWf in endothelial cells with intense immuno- endothelial cells possesses the multimeric conformations typical staining of the same subcellular granules. of plasma vWf (14, 16). vW AgII is a second protein described as deficient in von Introduction Willebrand's disease (4). vW AgII is found in plasma and platelets, with approximately two-thirds circulating in platelets (8). It is released from platelets during activation by thrombin, von Willebrand's disease is characterized by a deficiency or ADP, or collagen and is present at increased levels in the structural abnormality in von Willebrand factor (vWf)' (1-3). plasma of individuals with syndromes associated with platelet A second II protein, von Willebrand antigen (vW AgII), is also activation, such as disseminated intravascular coagulation and deficient or absent in von Willebrand's disease (4). While both hemolytic uremic syndrome (8). vW AgIl shows an immuno- vWf and vW AgIl have not been shown to share antigenic chemical reaction of nonidentity with vWf (4) by crossed immunoelectrophoresis (5). Thus an enigma is presented: two Dr. Montgomery is an Established Investigator of the American Heart distinct Association. Address reprint requests to Dr. Montgomery, The Blood proteins are both absent in a single genetic disorder, Center of Southeastern Wisconsin, Inc., Milwaukee, WI 53233. and yet they are found in amounts that are linearly correlated Received for publication 5 March 1984 and in revised form 3 in the circulation of normal individuals and of individuals December 1984. with classic von Willebrand's disease. Their potential relation- ship, therefore, may be pertinent to the pathophysiology of 1. Abbreviations used in this paper: DDAVP, l-desamino-8-D-arginine- von Willebrand's disease. This study was undertaken to char- vasopressin; PMSF, phenylmethylsulfonylfluoride; SDS-PAGE, sodium acterize vW AgIl more fully and to determine the association dodecyl sulfate-polyacrylamide gel electrophoresis; vW AgII, von Wil- between vW AgIl and vWf within the endothelial cell. lebrand antigen II; vWf, von Willebrand factor. J. Clin. Invest. Methods © The American Society for Clinical Investigation, Inc. 0021-9738/85/04/1089/07 $ 1.00 Materials. Unless otherwise noted, supplies and reagents used in Volume 75, April 1985, 1089-1095 endothelial cell culture were purchased from Gibco Laboratories, Endothelial vWf/vWAgII Complex 1089 Grand Island, NY. Other materials included Type I collagenase (Wor- agarose containing the appropriate antibodies. Gels were washed in thington Biochemicals Corp., Freehold, NJ), fetal calf serum (Biocell, several changes of 1% NP40, 10 mM benzamidine HCI, 0.15 M NaCI, Carson, CA), bovine pituitaries (Pel-Freeze Biologicals, Rogers, AR), pressed, dried, and autoradiographed at -80'C with Kodak X-Omat [35Slmethionine (800 mCi/mM sp act; Amersham Corp., Arlington RP film (5). Radiolabeling of immunoprecipitates formed on crossed Heights, IL), fluorescein- and rhodamine-conjugated, preabsorbed an- immunoelectrophoresis was done by (a) using endogenously labeled tibodies to mouse and rabbit IgG (Tago Inc., Burlingame, CA), Hemofil endothelial cell proteins (7); (b) forming immunoprecipitates with (Hyland Laboratories, Costa Mesa, CA), protein A Sepharose 4B antibodies with which 125I had been coupled, using the chloramine T (Pharmacia Fine Chemicals, Div. of Pharmacia Inc., Piscataway, NJ), method (21); or (c) using '25I-labeled plasma protein that had been Biogel Al 5 (Bio-Rad Laboratories, Richmond, CA), SeaKem Agarose partially purified on Biogel A15 as described (5). ME (FMC Corporation, Marine Colloids Division, Springfield, NJ), Immunofluorescent labeling of endothelial cells. Intracellular local- and nonionic detergent NP40 (Particle Data, Inc., Elmhurst, IL). All ization of vWf and vW AgIl was determined by double label indirect other reagents were purchased from commercial sources and were of immunofluorescence. Cells were grown on 4 cm2 glass coverslips the best available grade.
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