THROMBOSIS RESEARCH 68; 131-136,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.


Ana C. Kempfer*, Juan Pablo Frontroth, Cristina Farias*, Emilse Bermejo and Maria A. Lazzari* Instituto de Investigaciones Hematologicas "Mariano R. Castex". Academia National de Medicina and * CONICET. Buenos Aires, Argentina. (Received

17.8.1992; accepted in original form 18.8.1992 by Editor H. Vinazzer) (Received by Executive Editorial Office 20.8.1992)

ABSTRACT In order to determine the binding of vWF, subendothelium from everted human umbilical arteries was perfused with dialysed serum containing different concentrations of purified vWF using an annular perfusion chamber at a wall shear rate of 1100 see-1 for 30 min. After perfusion, control (not perfused) and perfused vessel segments were washed and incubated with a diluted rabbit antibody against human vWF. Then the nonbound anti-vWF from both samples were used to determine indirectly vWF by EIA. Although in our experiments normal vWF serum concentrations were not enough to exert vWF binding, a substantial binding could be attained with vWF levels around 2.5 U/ml. To estimate the pre-existing subendothelial vWF amount, three different experiments were developed: a) diluted IgG from a nonimmunized rabbit, b) a diluted rabbit antibody to human vWF, c) PBS-BSA. After washing, vessel segments were incubated with rabbit antibody to human vWF. After incubation, the nonbound anti-vWF was used to determine indirectly vWF by EIA. The results obtained showed that the amount of pre-existing vWE was approximately l.lxlP U vWF/cn? subendothelium.

vWF is a plasma with multiple functions in protein haemostasis; is deficient and/or defective in vWD. Vascular vWF. The only other endothelium is the primary source of plasma type of cell that synthesizes vWF is the megakaryocyte. words : Rw immunoassay.









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The endothelium also deposits vWF into the basement membrane of blood vessels. This matrix form of vWF is important for initial attachment of platelets during vascular injury and also for adhesion of the endothelial cells to the vessel wall (1). Subendothelial vWF is responsible for approximately 40% of total platelet adhesion. Besides, plasma vWF is necessary to achieve normal adhesion (2). However the normal plasma concentration of vWF in combination with vWF in the subendothelium is not optimal. A substantial increase in platelet adhesion can be attained with vWF plasma levels up to 300% (3,4). The most realistic way in which vWF has been studied in vitro is by means of the Baumgartner perfusion chamber (5). BY using a double-perfusion setup, in with an artery was perfused first with plasma and then with platelets resuspended in an albumin solution, it has been shown that plasma vWF first binds to the vessel wall and then this bound vWF mediates platelet adhesion (8). Several groups of investigators have studied the binding of ===I vWF to the subendothelium of human umbilical arteries. This binding was rapid and could not be inhibited by a 20 fold excess of unlabeled vWF. These results indicated that the binding of vWF to the denuded arteries in this concentration range (2-20 ug/ml) was not saturable (7). In order to avoid the use of radioactive compounds, by using EIA we have developed a new method for the quantitation of vWF binding to human arterial subendothelium and for the estimation of pre-existing subendothelial vWF.

Prevarationnts. Subendothelium was obtained from human umbilical arteries which were denuded of endothelium in technique (8). Briefly arteries were situ by a balloon catheter everted on the day following endothelium denudation, immersed in at 4°C for a 0.2M Tris buffer (pH:7.4) and kept refrigerated period of up to four weeks until use. Immediately before use arteries were scrapped off with a piece of Whatman filter paper, cut in segments before using and washed with PBS (0.015M Phosphate Buffer, 0.15 M Sodium Chloride, pH:7.2 with 2.5 mM CaCl, and 1 mM MgCl, ). isolated as described vWF was Prevaration of verfusates: was purified by preparation previously (9). Type II gel filtration on Sephacryl S-1000 (Pharmacia). This preparation was solid free of fibrinogen. Purified vWF was concentrated against PVP and dialysed against PBS. Serum was obtained by collecting into glass tubes that were blood from healthy donors directly kept at 37°C for 24 h. Then serum was separated, dialysed (DS) against PBS and stored frozen before use. Preparation of perfusates was developed adding different concentrations of purified vWF to DS. Final concentrations were determined by EIA and Laurel1 techniques (10). Before using the perfusates were prewarmed for 10 minutes at 37°C. . . Perfusions were carried out in a perfusion Perfusion chamber as developed by Baumgartner (8) with an effective annular width of 1.5 mm. Constant flow was maintained at 80 ml/min, shear rate: 1100 see-z (11).

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segments were inserted into the Perfusmnts: Arteries perfusion chamber,. and PBS was perfused for 10 minutes. Perfusion out at 37°C for 30 minutes. Then the experiments were carried by perfusing buffer PBS through the arterial segment was washed chamber for 3 minutes. Finally the vessel segment was removed and for EIA 0.2% BSA in PBS (PBS-BSA) washed five times with analysis. Control vessel segments, that were not perfused, were washed (five times) with PBS-BSA for EIA analysis. . segments Studiesion of ore -. exlstlna vWF : Vessel were incubated at 37°C for 90 min in a water bath shaker with: a) l/400 diluted IgG from a nonimmunized rabbit (DAKO) (12), b) l/400 diluted rabbit antibody to human vWF (DAKO), c) PBS-BSA. Then the vessel segments were washed (ten times) with PBS-BSA for EIA analysis. ETA analysis: 0.15 ml of rabbit antibody to human vWF diluted l/4000 in PBS-BSA were added to 5 ml of PBS-BSA containing each vessel segment. The mixture was shaken at 37°C for 90 minutes. Then an aliquot (nonbound anti-vWF) of the incubation mixture was added to the vWF coated tubes as described by Ness (13) but with minor modifications: vWF was purified as described previously (9) and was diluted in 0.2M carbonate buffer pH:9.2. Goat antirabbit IgG alkaline Phosphatase Conjugate (SIGMA) was diluted l/300 and substrate solution was prepared using 1.5 mg/ml Pnitro-phenyl-phosphate (SIGMA). The reaction was stopped with 3M NaOH. The optical density was read at 405 nm wavelenght in a BEHRING EL 301 Strip Reader. In parallel experiments, serial dilutions of pooled plasma were prepared in PBS-BSA. 2.4 ml of plasma dilutions (l/100 to l/6400) were mixed with 0.094 ml of rabbit antibody to human vWF diluted l/4000 in PBS-BSA and the mixture was incubated for 90 min at 37°C. Then, similarly to the vessel segment, an aliquot of the incubation mixture was removed, added to the vWF coated tubes and analysed as it was described above.

of vWF to subendotm In order to determine the binding of vWF to subendothelium, perfusion experiments were carried out with DS containing 2.5 up to 16 U/ml of vWF. Figure 1 shows the relationship between the concentration of vWF and the binding of the protein to the subendothelium. vWF binding increased linearly with initial concentrations (2.5 U/ml) up to 4 U/ml; at vWF levels between 4 and 16 U/ml the slope of the binding curve decreased. * . les fnr the esta of ore - exystu vWF The results showed that the amount of vWF was not modified by the presence of IgG from nonimmunized rabbit compared to untreated samples. In another set of experiments we evaluated levels of vWF in vessel segments that were incubated with anti-vWF prior to the incubation with the same antibody for EIA. Unexpectly, the levels of the nonbound anti-vWF were lower than the amount of anti-vWF added, thus we detected vWF under these conditions (Table 1).




5 vWF


15 (U/ml)

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serum FIG 1: Umbilical arteries segments perfused with dialysed containing purified vWF in different concentrations (2.5, 4, 9, Perfusions were performed for 30 min at 1100 13 and 16 U/ml). set-'. The amount of bound vWF for each experiment was calculated by substracting the value obtained in the respective control sample. The bars indicate the standard error of the mean (n=4).

Comparison of IgG from nonimmunized rabbit and rabbit antibody to human vWF vessel treatments. Vessel Incubation PBS-BSA



IgG from a nonimmunized Rabbit antibody

vWF (UxlO-"/cm" subendothelium) 2.5 + 0.3


2.4 f 0.5

to human vWF

1.3 f 0.3

Difference between anti-vWF added and nonbound anti-vWF expressed as subendothelial vWF, determined in arterial segments previously incubated at 37°C for 90 min in a l/400 dilution of the various media with PBS-BSA. Data were presented as mean f SEM (n=4 - 40).




we have demonstrated the binding of vWF using an EIA method. Our results showed that the binding of vWF was dependent on the concentration of vWF present in the perfusate. The binding of vWF increased up to 16 U/ml. Other was detected by 2.5 U/ml of vWF, vWF increasing demonstrated that workers have previously improves adhesion of platelets to over 1 U/ml concentrations artery segments (41, thus our technique may be useful for studies of platelet adhesion. from the in UxlO-"/cm =, obtained The mean value of vWF, incubation of vessel segments with normal rabbit IgG were similar to the mean value obtained from untreated vessel segments. This to human vWF did not bind indicated antibody that rabbit to subendothelium (12). The results of vessel nonspecifically segments incubation with rabbit anti-vWF, prior to the incubation that the concentration with the same antibody for EIA, indicated of the antibody decreased when it was incubated 90 min at 37°C with the vessel segment, but this behaviour was independent of subendothelial vWF. The difference between both treated mean values the amount allow to estimate of pre-existing subendothelial viz and it was approximately 1.1x10-" U vWF/cm=. The different methods for the immunolocalization of pre-existing subendothelial vWF and the quantitation of Lz31 vWF binding to subendothelium has provided useful information with adequate reliability. Although this method offers no particular advantage in the quantitation of vWF binding, the ease and nonradioactive nature and possibility to estimate the pre-existing subendothelial vWF offers definite laboratory advantages.

We thank Rosa Veron for her assistance, Hemotherapy team for drawing the normal blood samples, the members of the Mater Dei Center and Ferndndez Hospital for providing the umbilical cords.


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(EIA) Thromb.

A simple enzyme-immunoassay test for von Willebrand factor binding in human arterial subendothelium.

In order to determine the binding of vWF, subendothelium from everted human umbilical arteries was perfused with dialysed serum containing different c...
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