Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3793-3797, May 1989 Medical Sciences von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets PHILIP G. DE GROOT*t, AUGUSTO B. FEDERICIt, HETTY C. DE BOER*, PATRIZIA D'ALESSIO*, PIER M. MANNUCCIt, AND JAN J. SIXMA* *Department of Haematology, University Hospital Utrecht, Catharijnesingel 101, 3511 GV Utrecht, The Netherlands; and tA. Bianchi Bonomi Hemophilia and Thrombosis Center and Institute of Internal Medicine, University of Milano, Milan, Italy Communicated by Kenneth M. Brinkhaus, December 12, 1988 ABSTRACT Endothelial cells were isolated from the um- vWF is synthesized by endothelial cells and megakaryo- bilical vein of a patient with subtype IIB von Willebrand cytes (5, 6). The vWF protein is synthesized with a signal disease, and the biosynthesis and function of von Willebrand peptide (22 amino acids), a precursor part (vWF antigen II, factor (vWF) synthesized by these cells were compared with 741 amino acids), and the mature protein (2050 amino acids) those of vWF synthesized by endothelial cells from normal (7, 8). There are three possible destinations of newly syn- individuals. The patient's endothelial cells synthesized, stored, thesized vWF: secretion into the culture medium ofthe cells, and secreted vWF indistinguishably from normal endothelial incorporation into the extracellular matrix, and storage inside cells: it was synthesized as a prepolypeptide of Mr 270,000 and the cell in the Weibel-Palade bodies (5, 9, 10). The secretion had a mature form of 220,000; the full spectrum of of vWF from the cells is a continuous process through Mr constitutive release, but there is also regulated release, multimers was found both inside the cells and in the culture induced by stimuli, from the Weibel-Palade bodies (11). medium; it was stored normally, in the Weibel-Palade bodies; Multimer assembly takes place during the processing of and similar amounts of vWF were secreted into the medium vWF in the cell (12). Constitutively released vWF consists of and deposited in the extracellular matrix. In a perfusion set-up, intermediate multimerized forms, whereas vWF present in- the extracellular matrix from JIB cells supported platelet side the Weibel-Palade bodies is highly multimerized (13). A adhesion similarly to the matrix from normal cells. vWF number of steps in the multimerization of vWF have been secreted constitutively by JIB cells into the culture medium characterized (14, 15), and abnormalities in posttranslational bound to platelets at concentrations of ristocetin lower than processing could play a role in the pathogenesis of type II those necessary for vWF from normal cells. vWF stored in the vWD. However, to understand the molecular defects under- Weibel-Palade bodies of type JIB cells was released upon lying the different type II vWD subtypes, culture of mutant stimulation with phorbol ester and bound almost completely to endothelial cells from IIB vWD patients is necessary. Levene platelets even in the absence of ristocetin. Moreover, sponta- et al. (16) isolated endothelial cells from a patient with type neous platelet aggregation was induced by vWF synthesized by IIA vWD and showed that this phenotype is caused by type IIB cells. These data support the hypothesis that the increased proteolytic sensitivity ofthe vWF protein. We have absence of highly multimeric forms of vWF in plasma of type isolated endothelial cells from the umbilical cord ofa type IIB IIB von Willebrand disease patients is due to specific removal vWD patient and studied the biosynthesis of vWF by these of these multimers by platelets. cells. The mutant cells synthesized an abnormal vWF mol- ecule with an increased affinity for platelets, but this protein von Willebrand factor (vWF) is a glycoprotein necessary for was normally synthesized, processed, and multimerized by adhesion of platelets to subendothelium after vascular injury the IIB vWD endothelial cells. These data support previous (1). vWF also plays an important role in the interaction suggestions that the absence of high molecular weight mul- between endothelial cells and their matrix (2). Qualitative and timers in plasma may have been caused by selective removal quantitative deficiencies of vWF are associated with an of these multimers by platelets in vivo. autosomal hemorrhagic disorder, von Willebrand disease (vWD) (3). vWD is a bleeding disorder that is heterogeneous MATERIALS AND METHODS in its genetic transmission, its clinical and laboratory mani- festations, and its underlying pathogenetic mechanisms. Description of the Patient. The mother of the newborn vWF is present in plasma as a set ofmultimers with molecular patient was originally diagnosed as type IIB together with x several other affected members ofa large family (17). She has weights of 2-20 106. Analysis of the multimeric structure a severe bleeding disorder characterized by a markedly of vWF with high-resolution agarose gel electrophoretic prolonged bleeding time [25 min; Simplate (General Diag- systems has been used for the differentiation of vWD into nostics, Morris Plains, NJ)], decreased levels ofvWF antigen subtypes. Type II vWD is characterized by the lack of large (45 units/dl) and ristocetin cofactor activity (12 units/dl), vWF multimers in plasma. In a variant form of type II vWD, normal clotting assay (factor VIII procoagulant activity, 65 subtype IIB, there is hyperresponsiveness of platelet-rich units/dl), the absence of high and intermediate vWF multi- plasma to low doses of ristocetin. Patients suffering from mers in plasma, and the presence ofall multimers in platelets. subtype IIB vWD may develop thrombocytopenia, especially Her platelets show an enhanced responsiveness to ristocetin after treatment with 1-desamino-[8-D-arginine]vasopressin. (0.5 mg/ml induced 30% platelet aggregation). In the course In contrast to subtype IIA, in JIB vWD the full spectrum of of her pregnancy the mother developed a thrombocytopenia vWF multimers is present in the platelets of the patients (4). (down to 66,000 platelets per ,l) and spontaneous aggrega- The publication costs of this article were defrayed in part by page charge Abbreviations: vWD, von Willebrand disease; vWF, von Willebrand payment. This article must therefore be hereby marked "advertisement" factor; PMA, phorbol 12-myristate 13-acetate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. Downloaded by guest on September 29, 2021 3793 3794 Medical Sciences: de Groot et al. Proc. Natl. Acad Sci. USA 86 (1989) tion; spontaneous aggregation and thrombocytopenia disap- Perfusion Studies. After isolation of the endothelial cells peared after delivery. Her child, a girl born by caesarean from the umbilical vein, the arteries were prepared from the delivery, also expressed only the low multimer forms of vWF umbilical cord for studies with an annular perfusion chamber (Fig. 1). as described by Baumgartner (25) with some modifications Cell Culture. Human umbilical vein endothelial cells from (26). Perfusions with steady flow were carried out with this normal individuals were isolated and cultured as described annular perfusion chamber. A rectangular perfusion chamber (18). The cells were grown on culture plastics (Nunc), pre- as described by Sakariassen et al. (27) was used for studies coated with fibronectin, in a culture medium consisting of with glass coverslips coated with the endothelial cell extra- RPMI-1640 (GIBCO) supplemented with 10% human serum, cellular matrix. Perfusions were carried out with washed acidic fibroblast growth factor (19) at 150 heparin human platelets resuspended in human albumin solution, to Ag/ml, which washed and packed erythrocytes were added to a (Organon) at 5 units/ml, and antibiotics. To study platelet hematocrit of 0.4 (26). Platelets were washed as described interaction with endothelial matrices, the cells were subcul- (26). The final platelet count was 120,000 per ,ul. This tured on gelatin-coated glass coverslips. When the cells were perfusate (prewarmed for 5 min at 37°C) was recirculated confluent, the cultures were exposed to 0.1 M NH40H for 30 through the perfusion chamber for the indicated period at the min to isolate the extracellular matrix (20). The matrix-coated indicated shear rate. coverslips were stored in phosphate-buffered saline (PBS) at After perfusion, the artery segments were fixed in glutar- 40C and used for experiments the same day. dialdehyde and embedded in Epon for microscopic evalua- Endothelial cells were metabolically labeled with [31S]me- tion (28). Sections =1 ,um thick were stained with methylene thionine (DuPont/NEN). Cell lysate and medium fractions blue and basic fuchsin. The coverslips with matrix were were prepared and, after preclearing of the fractions with washed and fixed with 0.5% glutardialdehyde and directly gelatin coupled to Sepharose to remove fibronectin, vWF stained with May-Grunwald/Giemsa reagent. Platelet adhe- was immunoprecipitated with an anti-vWF monoclonal an- sion was expressed as percentage ofthe surface covered with tibody coupled to Sepharose (21). (The monoclonal antibody platelets. This was evaluated by en face light microscopy. against vWF was a generous gift ofJ. A. van Mourik, CLB, The light microscope was interfaced with an image analyzer Amsterdam.) To isolate vWF stored inside the cells, endo- (AMS, Saffron Walden, U.K.). thelial cells were stimulated for 1 hr with phorbol 12- Binding Studies. Human platelets were washed (26) and myristate 13-acetate (PMA; Sigma) at 20 ng/ml in RPMI- resuspended in Tyrode buffer (137 mM NaCI/2 mM MgCl2/ 1640. This treatment of the cells released all vWF present 0.42 mM NaH2PO4/11.9 mM NaHCO3/2.9 mM KCI/5.5 mM inside the Weibel-Palade bodies (22). glucose/10 mM Hepes, pH 7.35). Endothelial cell culture Assays. vWF antigen was determined by ELISA (21). The medium (0.25 ml) from normal or IIB cells (untreated or amount of vWF and fibronectin in the extracellular matrix PMA-stimulated) or control or IIB plasma was incubated was determined as described (20).