Von Willebrand Factor Synthesized by Endothelial Cells from a Patient With

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 molecule 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). The multimeric composi- with 5 x 104 platelets per ,l (final concentration) without or tion of vWF in plasma and culture medium was analyzed by with various concentrations of ristocetin. After incubation SDS/agarose gel electrophoresis with detection by 125i- without stirring for 30 min, the mixture was layered on top of labeled rabbit antibodies monospecific for vWF (23). Immu- 1 ml of 20% (wt/vol) sucrose in Tyrode buffer and then nofluorescence studies were carried out with cells grown on centrifuged for 2 min at 10,000 x g. The supernatant was glass coverslips (22). assayed for vWF by ELISA. For immuno electron microscopy, ultrathin cryosections Aggregation Studies. Ristocetin cofactor activity was mea- of cultured cells were incubated with immunopurified rabbit sured with formalin-fixed platelets as described (29). Fixed anti-vWF IgG (50 ,ug/ml in phosphate buffer), washed, and platelets (3 x 107) in 50 Al of 0.02 M Tris HCI pH 7.3/0.15 M incubated with porcine anti-rabbit IgG (dilution 1:2000 in NaCI were added to 450 ,l of culture medium containing phosphate buffer; Nordic, Tilburg, The Netherlands). Incu- vWF antigen at 300 ng/ml. Platelet aggregation was assayed bation of the sections with colloidal gold-labeled protein A spectrophotometrically at 37°C with constant stirring (900 (9-nm gold particles) visualized the bound IgG (24). rpm). RESULTS Endothelial cells isolated from the newborn patient with type IIB vWD were morphologically indistinguishable from normal endothelial cells. The IIB vWD endothelial cells exhib- ited no abberrant features detectable by phase-contrast light microscopy or transmission electron microscopy. Immuno- fluorescence analysis of the IIB cells with antibodies against vWF showed the well-known rod-shaped structures charac- teristic of the Weibel-Palade bodies (Fig. 2A). Immuno ., electron microscopy with vWF-monospecific rabbit antibodies labeled with colloidal gold showed that, just as in normal endothelial cells, the vWF in IIB cells is stored in the Weibel- Palade bodies (Fig. 2B). After stimulation ofthe IIB cells with the phorbol ester PMA, the rod-shaped structures disap- 1 2 3 4 5 peared, indicating that the stimulus-induced release of vWF in IIB cells behaves in the same way as in normal cells (data FIG. 1. vWF multimers separated by SDS/agarose gel electro- not shown). phoresis (1% low-geling-temperature agarose; low resolution power). Arrow indicates the interface between stacking and running To examine the synthesis ofvWF by the IIB cells, cultures gel. Lane 1, normal endothelial cell lysate (106 cells); lane 2, ofthe patient and control endothelial cells were metabolically endothelial cell lysate from the newborn type IIB vWD patient (106 labeled with [35S]methionine. vWF was isolated from cells cells); lane 3, platelet lysate from the baby's mother (107 platelets); and culture medium with monoclonal antibodies coupled to lane 4, plasma of the newborn type IIB vWD patient (1 ,ul); lane 5, Sepharose and was analyzed with SDS/polyacrylamide gel normal plasma (1 l1). electrophoresis (Fig. 3 Left). Both normal and IIB cells Downloaded by guest on September 29, 2021 Medical Sciences: de Groot et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3795

Table 1. vWF and fibronectin in the endothelial cell extracellular matrix Amount,* ng/cm2 Cell type vWF Fibronectin Normal 7.9 ± 2.3 350 ± 108 vWD IIB 7.2 ± 2.2 312 ± 96 *Mean ± SD (n = 4). fibronectin in the matrix of IIB cells were similar to the amounts found in the matrix of normal cells (Table 1). The extracellular matrix of 1113 or normal endothelial cells was exposed in a perfusion set-up to platelets resuspended in human albumin solution. In this way platelet adhesion de-

. it pended only on matrix vWF. Comparable numbers of platelets adhered to both matrices (Fig. 4). There was no difference in the time dependence (data not shown) or in the FIG. 2. vWF in type IIB vWD endothelial cells. (A) Indirect shear-rate dependence (Fig. 4) of platelet adhesion. The immunofluorescence pattern with a murine monoclonal antibody to question arose whether the results found with the extracel- vWF and fluorescein-labeled goat anti-mouse IgG. (x 160.) (B) Im- lular matrix of cultured cells were influenced by the culture muno electron microscopy ofendothelial cells stained for vWF. After conditions. To study this, these results were verified with incubation with a monoclonal antibody against vWF, the ultrathin perfusion studies performed with inverted arterial wall seg- cross-sections were incubated with gold-labeled protein A to indicate ments from normal and the patient's umbilical cord. There the presence of vWF. (x26,000.) was no significant difference in the number of adhered synthesized and secreted the propeptide (apparent Mr platelets between the normal vessel-wall segment and the 270,000) and the mature form (apparent Mr 220,000). To patient's vessel-wall segment when the segments were per- further analyze vWF from IIB cells, vWF secreted by the fused with platelets resuspended in human albumin solution cells was subjected to multimer analysis. To analyze vWF (Fig. 5). Addition of purified normal vWF did not introduce present inside the Weibel-Palade bodies, the cells were differences, indicating that there is no competition between stimulated for 60 min with PMA. After such treatment, vWF vWF in solution and vWF in the subendothelium. present inside the cell is secreted, without lysis of the cells An important abnormality in type IIB vWD is the increased (21). After subculturing, the cells can be used for other responsiveness ofplatelet-rich plasma to ristocetin. We won- experiments. Fig. 3 Right shows that there is no difference dered whether vWf secreted from the IIB cells would show between vWF multimeric patterns from normal and from IIB enhanced affinity for platelets in the presence of ristocetin. endothelial cells: the constitutively released vWF consists of Fig. 6 shows the binding of endothelial cell vWF to normal the low and intermediate multimeric forms, whereas vWF platelets. Separate data are presented for constitutively represent in the Weibel-Palade bodies consists predominantly leased vWF and vWF released from the Weibel-Palade of the very high multimers. bodies, because these forms ofvWF differ in their multimeric To analyze the functional properties of the vWF synthe- patterns. To study vWF present in the endothelial storage sized by the 11B cells, adhesion studies were performed with organelles, the cells were stimulated for 60 min with PMA in the extracellular matrix ofthe cells. The amounts ofvWF and RPMI-1640. Constitutively released vWF from normal and IIB cells did not show affinity for platelets in the absence of ristocetin. In the presence of ristocetin, constitutively re-

22(

20 -

a1) 10 0 C lB C lB C 11B a. CONSTITUTIVE STIMULATED RELEASE RELEASE FIG. 3. Analysis of vWF synthesized by type IIB endothelial 0 '7 cells. (Left) Immunoprecipitation of [355]methionine-labeled vWF 0 500 1000 1500 from endothelial cell lysate (106 cells). Immunoprecipitates from Shear rate, sec-1 normal (control, C) and IIB vWD cells wvere analyzed by SDS/ polyacrylamide gel electrophoresis and autoradiography. Numbers FIG. 4. Adhesion ofplatelets to extracellular matrices ofcultured at left are Mr values x 10-3. (Right) vWF multimers released from normal (o) or IIB vWD (e) endothelial cells. After the cells had normal (C) or IIB endothelial cells were analyzed by SDS/agarose reached confluence, they were removed with 0.1 M NH40H and the gel electrophoresis with detection by 1251-labeled antibodies. Con- matrix was perfused on the same day for 5 min with platelets stitutive release, vWF released during 24 hr from confluent cultures; resuspended in human albumin solution with erythrocytes added stimulated release, vWF released from cells after PMA stimulation (hematocrit, 0.4) in a rectangular perfusion chamber. Error bars for 1 hr. In each lane, 20 1.d of culture medium was loaded. represent SD for 4 determinations. Downloaded by guest on September 29, 2021 3796 Medical Sciences: de Groot et al. Proc. Natl. Acad. Sci. USA 86 (1989)

40 F

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20 0

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, 0 1 2 vWF, units/ml

FIG. 5. Platelet adhesion to artery subendothelium. After isolation, normal (o) or TIB vWD (9) umbilical artery segments were mounted on the central rod of an annular perfusion chamber. Perfusions were carried out with platelets resuspended in a human albumin solution with erythrocytes (hematocrit 0.4) and various amounts of purified normal vWF added. Shear rate, 1800 sec'; perfusion time, 5 min. leased vWF from IIB cells induced binding at lower concentrations of ristocetin than vWF from normal cells. In the 0 0 0.5 1.0 1.5 Ristocetin absence of ristocetin, addition of platelets to the high mo- - + + + + Platelets lecular weight vWF released from PMA-stimulated normal cells had no effect on the amount of vWF in the solution. FIG. 6. Binding of vWF to platelets. Normal platelets were were added to vWF from stimulated IIB cells washed free of plasma proteins and then mixed with constitutively When platelets (A) or stimulus-induced (B) released vWF, both from normal cells in the absence of ristocetin and then centrifuged, almost all and from type IIB cells. Ristocetin was then added (0-1.5 mg/ml) and of the vWF disappeared from the supernatant. the response was assessed by measuring the residual vWF after To investigate whether the binding of vWF from type IIB removal of the platelets by centrifugation. endothelial cells to platelets in the absence of ristocetin results in spontaneous platelet aggregation, the different Apparently both cell types involved in the synthesis of vWF culture supernatants were tested in an aggregometer. The express all multimeric forms, while in the plasma, the final high molecular weight forms of vWF released by stimulated destination of newly formed vWF, only the lower multimeric IIB endothelial cells induced aggregation of formalin-fixed forms are present. platelets in the absence of ristocetin (Fig. 7). Also, the There are two possibilities to explain why the high and constitutively released vWF from IIB cells gave a small intermediate forms of vWF are absent from the plasma of spontaneous agglutination in the absence of any agonist. No patients with IIB vWD. The loss could happen during the influence on platelet aggregation in the absence of ristocetin process of releasing vWF from the endothelial cells and was seen with vWF released by normal cells when tested in platelets, or the loss could start only when the newly syn- the same concentrations (Fig. 7). thesized vWF makes contact with blood components. The normal set of multimers is present in the conditioned medium of cultured endothelial cells, suggesting that the highest DISCUSSION multimers disappear after contact with the blood. Indeed, vWD subtype IIB is characterized by the absence ofthe high when washed platelets were added to culture supernatant molecular weight vWF multimers in plasma but the presence from stimulated endothelial cells, vWF in the supernatant ofall multimers in platelets. In this study, we have shown that bound to the platelets even in the absence of ristocetin. In all multimeric forms of vWF are present in the endothelial accord with this observation, the same supernatant induced cells ofa vWD IIB patient. vWF is synthesized in endothelial spontaneous platelet agglutination as measured with an ag- cells and in megakaryocytes, the precursors of platelets. gregometer (Fig. 7). Consequently, subtype IIB vWD is

FY FIG. 7. Spontaneous platelet aggregation induced by endothelial cell medium. Medium from PMA-stimulated (trace 1) or unstimulated (trace 2) IIB cells or from PMA-stimulated "A (trace 3) or unstimulated (trace 4) normal cells was added to a cuvette and, after establishment of the base- line (37TC, stirring), fixed platelets were added and aggregation was re- ; -10 1100% corded. Downloaded by guest on September 29, 2021 'l~min- Medical Sciences: de Groot et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3797

obviously caused by the synthesis of an abnormal vWF with additions or deletions in the nucleotide sequence therefore increased affinity for platelets. Two groups (30, 31) have seem unlikely. shown that vWF purified from IIB vWD plasma has a similar spontaneous affinity for platelets. Zimmerman et al. (32) We are grateful to Anne-Marie van den Hoeven, Nel Beeser- found increased vWF proteolysis in IIB vWD plasma, prob- Visser, and Harry Heynen for expert technical assistance. ably due to cleavage by platelet proteases as a consequence of the increased interaction with platelets. Although all 1. Sixma, J. J. (1987) Thrombosis and Haemostasis, eds. Verstraete, multimers are normally formed at the sites of synthesis, our M., Vermylen, J., Lijnen, R. & Arnouts, J. (Leuven Univ. Press, data suggest that the absence of the higher multimers in Leuven, Belgium), pp. 127-146. is due to a direct to 2. Cheresh, D. A. (1987) Proc. Natl. Acad. Sci. USA 84, 6471-6475. plasma binding platelets. 3. Ruggeri, Z. M. & Zimmerman, T. S. (1987) Blood 70, 895-904. Endothelial cells isolated from a patient with type IIB vWD 4. Ruggeri, Z. M. & Zimmerman, T. S. (1980)J. Clin. Invest. 65,1318- synthesized and processed vWF indistinguishably from nor- 1325. mal endothelial cells. Newly synthesized vWF is normally 5. Jaffe, E. A., Hoyer, R. L. & Nachman, R. L. (1973) J. Clin. Invest. secreted into the culture medium, multimerized, deposited in 52, 2757-2764. the extracellular matrix, and stored inside the cell in the 6. Nachman, R. L., Levine, R. & Jaffe, E. A. (1977) J. Clin. Invest. Weibel-Palade bodies After 60, 914-921. (5, 9, 10). stimulation of the IIB 7. Sadler, J. W., Shelton-Inloes, B. B., Sorace, J. M., Harlan, J. M., cells with PMA, the content of the secretory granules was Titani, K. & Davie, E. W. (1985) Proc. Natl. Acad. Sci. USA 82, immediately secreted as in normal endothelial cells. Based on 6394-6398. these studies, the IIB cells behave completely normally. 8. Wagner, D. D. & Marder, V. J. (1983) J. Biol. Chem. 258, 2065- Levene et al. (16) studied endothelial cells isolated from a 2067. patient with type IIA vWD. In those cells they found that 9. Rand, J. H., Sussman, I. I., Gordon, R. E., Chu, S. V. & Salomon, vWF was also V. (1980) Blood 55, 752-756. synthesized normally (although in higher 10. Wagner, D. D., Olmsted, J. B. & Marder, V. J. (1982) J. Cell Biol. quantities) and processed to the higher multimers. The newly 95, 355-360. synthesized molecules, however, showed an increased sen- 11. Loesberg, C., Gonsalves, M. D., Zandbergen, J., Willems, Ch., van sitivity to proteolytic enzymes. Both IIA and IIB vWD are Aken, W. G., Stel, H. V., Van Mourik, J. A. & de Groot, Ph. G. characterized by the absence of higher multimers in plasma, (1983) Biochim. Biophys. Acta 763, 160-168. but in both disorders this is not due to a in the 12. Wagner, D. D. & Marder, V. J. (1984) J. Cell Biol. 99, 2123-2130. change 13. Sporn, L. A., Marder, V. J. & Wagner, D. D. (1986) Cell 46, 185- multimerization process of the molecule. 190. In this study, we found that the vWF present in the 14. Wagner, D. D., Mayadas, T. & Marder, V. J. (1986) J. Cell Biol. extracellular matrix of the IIB vWD endothelial cells had the 102, 1320-1324. same ability to support platelet adhesion as the vWF present 15. Verwey, C. L., Hart, M. & Pannekoek, H. (1987) EMBOJ. 6, 2885- in the matrix of normal endothelial cells. However, vWF 2890. released the TIB cells into the medium showed much 16. Levene, R. B., Booyse, F. M., Chediak, J., Zimmerman, T. S., by Livingston, D. M. & Lynch, D. C. (1987) Proc. Natl. Acad. Sci. higher affinity for platelets than normal vWF. This seems a USA 84, 6550-6554. paradox, but the explanation is probably simple. Normal 17. Ruggeri, Z. M., Pareti, F. I., Mannucci, P. M., Ciaverella, N. & vWF must first adsorb to a surface or interact with ristocetin Zimmerman, T. S. (1980) N. Engl. J. Med. 302, 1047-1051. before it can bind to platelet membrane glycoprotein lb. In 18. Willems, Ch., Astaldi, G. C. B., de Groot, Ph. G., Jansen, M. C., contrast, IIB vWD secrete vWF that is already in a form that Gonsalves, M. D., Zeijlemaker, W. P., van Mourik, J. A. & van can react with Aken, W. G. (1982) Exp. Cell Res. 139, 191-197. directly platelets. However, in the extracel- 19. Maciag, T., Hoover, G. A., Stemerman, M. B. & Weinstein, R. lular matrix of both normal and IIB cells, vWF is present in (1981) J. Cell Biol. 91, 420-426. a form that can bind to platelets without ristocetin. This is 20. De Groot, Ph. G., Reinders, J. H. & Sixma, J. J. (1987) J. Cell Biol. probably the reason why no difference was found between 104, 697-704. vWF in the matrix of IIB cells and vWF in the matrix of 21. Reinders, J. H., Vervoorn, R. G., Verweij, C. L., van Mourik, normal cells. Another be J. A. & de Groot, Ph. G. (1987) J. Cell. Physiol. 133, 79-87. explanation might that two different 22. were the sensitivities of which are Reinders, J. H., de Groot, Ph. G., Gonsalves, M. D., Zandbergen, assays used, probably J., Loesberg, C. & van Mourik, J. A. (1984) Biochim. Biophys. Acta completely different. A previous study (27) showed that there 844, 361-369. is more than sufficient vWF for optimal platelet adhesion in 23. Ciaverella, G., Chiavarella, N., Antoncecchi, S., de Mattia, D., the matrix of normal endothelial cells. If there is a difference Ranieri, P., Dent, J., Zimmerman, T. S. & Ruggeri, Z. M. (1985) in platelet affinity between normal matrix vWF and IIB Blood 66, 1423-1429. matrix vWF, our perfusion system is not sensitive enough to 24. Slot, J. W. & Geuze, H. J. (1981) J. Cell Biol. 90, 533-536. detect that 25. Baumgartner, H. R. (1973) Microvasc. Res. 5, 167-179. difference. 26. Sakariassen, K. S., Bolhuis, P. A. & Sixma, J. J. (1979) Nature It has been suggested that deficiencies in the carbohydrate (London) 279, 636-638. part of the molecule are the main cause of the abnormal 27. Sakariassen, K. S., Aarts, P. A. M. M., de Groot, Ph. G., Houdijk, function of vWF in type IIB vWD (33). De Marco et al. (34) W. P. M. & Sixma, J. J. (1983) J. Lab. Clin. Med. 102, 522-535. showed that vWF isolated from IIB vWD plasma has a 28. Baumgartner, H. R., Muggli, R., Tschopp, T. B. & Turitto, V. T. normal sialic acid content and induces platelet aggregation in (1976) Thromb. Haemostas. 35, 124-138. the absence of 29. MacFarlane, D. E., Stibbe, J., Kirby, E. P., Zucker, M. B., Grant, ristocetin. Moreover, carbohydrate-modified R. A. & McPherson, J. (1975) Thromb. Diath. Haemorrh. 34, 306- vWF is very sensitive to proteases (35), whereas vWF 308. secreted into the culture medium of TIB cells is stable. A 30. De Marco, L., Girolami, A., Zimmerman, T. S. & Ruggeri, Z. M. possible defect might be a mutation affecting the attachment (1985) Proc. Natl. Acad. Sci. USA 82, 7424-7428. of a carbohydrate moiety in IIB vWF or a mutation affecting 31. Gralnick, H. R., Williams, S. B., McKeown, L. P., Rick, M. E., the Maisonneuve, P., Janneau, C. & Sultan, Y. (1985) J. Clin. Invest. sensitivity of IIB vWF for glycosidases present only in 76, 1522-1529. plasma (32). Another explanation for the defect in type IIB 32. Zimmerman, T. S., Dent, J. A., Ruggeri, Z. M. & Nannini, L. H. vWD might be a mutation causing a change in the vWF (1986) J. Clin. Invest. 77, 947-951. molecule comparable with the change induced by the inter- 33. Lawrence, J. B. & Gralnick, H. R. (1987) Blood 70, 1084-1089. action with ristocetin. If this is the case, the mutation must 34. De Marco, L., Mazzuccato, M., Del Ben, M. G., Budde, U., cause relatively little change in the primary structure, be- Federici, A. B., Girolami, A. & Ruggeri, Z. M. (1987) J. Clin. cause the molecular of Invest. 80, 475-482. apparent weights vWF immunopre- 35. Federici, A. B., Elder, J. H., De Marco, L., Ruggeri, Z. M. & cipitated from normal and from IIB cells are the same. Major Zimmerman, T. S. (1984) J. Clin. Invest. 74, 2049-2055. Downloaded by guest on September 29, 2021