Significance and quantitative analysis of von Willebrand factor in human platelets

THROMBOSIS RESEARCH 65; 631-640,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

SIGNIFICANCE

AND QUANTITATIVE

ANALYSIS

HUMAN

OF VON WILLEBRAND

FACTOR

IN

PLATELETS

Th. Eller, P. Brauer, J. Albert, J. Mayer, F. Keller Central Laboratory of the Medical University Hospital Wtirzburg, FRG

(Received 18.7.1991; accepted in revised form 7.1.1992 by Editor H.A. Vinazzer)

ABSTRACT

The von Willebrand factor (vWF) is found in plasma and in platelets. The concentration and multimeric composition of the vWF in platelets of 160 patients with bleeding tendency were examined sines very little is known about the platelet vWF. For quantitative analysis of the platelet vWF, a modified ELISA was established. A reference range from 70% - 130% of platelet vWF concentration consisidered normal was established by examining 80 healthy blood donors. 16.9% of the 160 patients showed a decreased vWF concentration in platelets only, while all the other coagulation parameters were normal. 3 of our patients belong to the same family and sug esting an autosomal dominant genetic transmission for the von Willebran 8 disease type l-3. The data also suggeste, that a quantitative and qualitative analysis of the vWF in plasma and platelets is required for an exact diagnosis of the von Willebrand disease.

INTRODUCTION

Due to the complexity of the von Willebrand factor (vWF) the manifestations of the von Willebrand disease (vWD) varies extensivly. The vWD can only be classified by quantitative and qualitative analysis of the vWF in plasma and platelets (1 . The multimers of the glycoprotein vWF occuring in lasma and platelets vary in their mo 1ecular weights from 0.5 to 20 million Daltons (2). In Raemostasis, the vWF has two functions. First the vWF is a carrier of the coagulation FVIII:C in the intrinsic system. Second, the large vWF multimers support the adhesion of the platelets to the subendothelium as well as aggregation of platelets (2,3). During protein biosynthesis of the vWF present in subendothelium and megacaryocytes, a dimer of vWF is formed first. These dimers are glycosylated and then polymerise to

Keywords: von Willebrand factor, platelets, von Willebrand disease, ELISA

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multimers including the formation of disulfide bonds. The vWF synthesized in megacaryoctes is stored in the a-granules of the platelets during thrombopoesis (4). Since the biological significance of the vWF in the platelets is uncertain, we analysed the multimeric structure and the vWF concentration in the platelets of all patients who were referred to our out-patient department for haemostaseology in order to examine a bleeding tendency. This study should explain the significance of the vWF in platelets and emphazise the importance of a complete analysis. MATERIALS AND METHODS Patients For the present study 160 patients refered to our out-patient department for the examination of possible haemostaseological disorders, were recruited. In order to establish our reference range for a normal vWF concentration in platelets, 60 healthy blood donors from the department of transfusion medicine of the surgical university hospital were tested. Platelet preparation Citrate blood was centrifuged at 107 x g for 20 minutes to obtain platelet rich plasma, which was removed with a Pasteur pipette. The platelets were washed twice with TrisEDTA-buffer (0.05M Tris, 0.15M NaCI, 0.1% EDTA-Na, pH 7.3) and then twice with Trisbuffer (0.05M Tris, 0.15M NaCl pH 7.3). Prior to the last precipitation, platelets were counted with an automatic blood cell counter. The precipitate was resus ended in a calculated amount of buffer to result in a platelet concentration of 1*lo6 Pul using 0.05M Tris 0.15M NaCI, 3% Albumin (pH 7.3) as resuspension buffer. Lysis of the platelets was carried out with 125~14% Triton-X-l OO-solution per 1ml platelet suspension. This solution was incubated for 30min at 37OC. The lysate was stored at -7OOCuntil analysis. After thawing, remaining membrane fragments were precipitated by a short centrifugation (5). Electra lmmuno Diffusion EID The quantitative analysis of the vWF in platelets by electroimmuno diffusion has been performed according to published procedures (6,7). However, only 0.1% antibody (AntiFVlllass.AG from Behring Werke AG Marburg) was added to the agarose gel solution. All other conditions were as described. Enzyme Immunoassay ELISA A modified enzyme immunoassay for quantitative determination of vWF in platelets was performed using the autoanalyser ES 22 and a manual of Boehringer Mannheim Corp. In our analysis, plasma (25~1)was replaced by 5Oul of the platelet lysate. All other steps of the analysis were performed according to the Boehringer Mannheim manual (6). In order to obtain a calibration curve, a pool of platelet lysate from 20 healthy donors was used in the EID as well as in the ELISA. The ELISA calibration curve is shown in Fig.1. To avoid interferences by Triton-X-100 (used for lysing the platelets), the final Triton concentration was always 0.5% (v/v) (8,9).

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Extlnctlon ‘.*a

100

60

160

% of normal +

mean values

Fig. 1: Calibration curve for the ELISA determination of platelet vWF

Separation of the vWF-Multimers The vWF-multimers were separated with a discontinuous SDS agarose gel and subsequent immunostaining (collecting gel: 0.8% agarose in 0.125 M Tris-HCI, 0.1% SDS pH 6.8; separation gel: 1.5% agarose in 0.375 M Tris-HCI, 0.1% SDS pH 8.8) according to a modified method (10). The electrophoresis was performed with a buffer of 0.05M Tris, 0.384M Glycin,O.l% SDS and 5mM NaCI. For sample preparation, 4Oul plasma or platelet lysate were mixed with 5Oul of buffer (0.01 M Tris-HCI, 1mM EDTA, 2% SDS pH 8.0) 1Oul Bromphenolblue (0.2% solution in buffer) and 100~12% seaplaque agarose solution and incubated for 1Omin at 60°C. 4Oul sample was applied to the gel and the electrophoresis was carried out overnight at 12.5mA and 14OC. After electrophoresis the gel was washed for 1 h in dest. H,O, pressed and dried. Then the gel was incubated in 1OOmlPBS-Tween-buffer with 1% humanglobulin, 500~1anti-vWF (Anti-Rllllas. AG of Behring Werke AG Marburg) was added and the incubation proceeded overnight. Prior to the next overnight incubation with a l/500 dilution of a biotin marked IgG antibody (Anti-rabbi IgG, Sigma Chemie Munich) the gel was washed with PBS-Tween for 24h. After washing three times for 40min with PBSTween the gel was incubated 4h in a l/500 dilution of Avidin-Peroxidase (Sigma Chemie Munich). Finallv it was washed for 30min and stained under visual control with 4-chlornaphtoi/H 0, solution (80mg 4-chlor-naphtol dissolved in 20ml methanol, added to lOOmI PBS, mixed with 6Oul H,O,) (10,ll).

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RESULTS Correlation between EID and ELBA The correlation of the immunologic determinations of vWF in platelets between the EID and ELISA was carried out on 22 pairs of results indicating a correlation coefficient of 0.999 (Fig.2). All other statistical parameters are listed in tab. 1.The evaluation of the correlation according to Passing Bablock (12) shows no significant deviation from ideal results. Therefore, the results o 6tained wtih either EID or ELISA can be considered as comparable.

0

20

40

00

80

100

120

140

180

180

EID % d.N.

Fig.2: Correlation between EID and ELISA TABLE 1: Statistical data of the correlation EID - ELISA

pair of results

22

mean values of diff.

2.5%

slope

1.008

intercept

1.814

correlation

0.999

PO0

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Concentrationof the von-Willebrand-Factorin platelets In order to establish a reference range, blood samples obtained from 60 healthy blood donors from the department of transfusion medicine of the university hospit@ were tested for the concentration of vWF in the platelets. Values of 42 to 200% per 1*IO platelets (a pool of 20 healthy donors is equivalent to 100% per 1*lo6 platelets) were found. According to the statistical evaluation 95% of the results ranged between 61 and 200% representing the 2.5% and the 97.5Ohpercentile, resectivly. The median was found to be 112% (tab.2). The vWF concentration in plasma was also analysed in a part of the colletive (n =45). For the 2.5% and 97.5% percentile values, 61 and 200% were found again and the median was 106%. Due to a positive skewness and a positive curtosis, both distributions were assest as squeezed and left shifted (13). According to these results the reference range for the concentration of vWF in platelets was considered to be 70 to 130%. Leaving a borderline zone of 50 and 70%, for which controls are necessary.

TABLE 2: Statistical data of the healthy donors

number of donors

80

median of platelet vWF cont.

112%

standard deviation

41.2%

2.5% perzentile

61%

97.5% perzentile

200%

35 (21.6%) of the 160 patients being examined for a bleeding tendency in our out patient department for haemostaseology revealed a decreased vWF concentration in platelets (Fig.3). Except a bleeding tendency in the patient’s history as well as a bleeding time varying from upper limit to prolonged there were no hints of a coagulation disorder in 27 of these patients (16.9%). A decreased concentration of vWF in platelets was found and classified as type l-3 vWD in these patients. The average value of vWF concentration in the platelets of our patient collective was found to be at 41.5% (tab.3).

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number

<10

20

40

aa

60

30 blood

80 100 120 140 % d. N. per 1 MO Thr/ml donors

m

160

180

200

vWD type l-3, l-l

Fig.3: Comparison of healthy blood donors with vWD patients (types I-1 and l-3) with respect to their platelet vWF concentration TABLE 3: Patients with decreased vWF concentration in platelets

number of examined patients

160

patients with decreased platelet vWF cont.

35

(21.8%)

isolated decrease of,vWF in platelets

27

(16.9%)

14

(8.8%)

._

patients

in the borderline

zone

mean value of patients with vWD type I-l, I-3

41.5%

In 14 of the 35 patients with a decreased vWF concentration in platelets the results were found in the borderline zone. 5 of those patients were repeatedly tested. In three of them

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the results could be confirmed. In the other 2 normal results were found in the plateJets, but patho ical values in plasma. This rompted us to assume a modification ofvWD from type 9I- to i-2. In another, case a cEange from i-2 to l-3 was found. Muitimeric analysis of von-Wiliebrand-Factorin platelets Fig. 4 shows the multimeric analysis of vWF present in plasma (lane 1) or in platelets (lane 2) of healthy volunteers. The platelet vWF has 34 additional large multimeric bands. For near1 all of the tested patients with a bleeding tendency, this kind of multimeric pattern, coulJ be demonstrated although a different staining intensity was Visible.in one patient with type I vWD a missing tri lett structure of the multimeric pattern in the plasma as well as in the platelets was foun cp.

Origin

Fig.4: Multimeric anal sis of vWF in plasma (lanel) and platelets rlane 2) of a healthy volunteer

Front Lane 7

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Genetic transmissionof the vWD type l-3 In Fig.5 a family tree is presented. In three members of this family a vWD type l-3 (G 1.l, G 2.1, G 3.1) could be detected. In an additional female (G 2.2) of this family, another known bleeding tendency could not be examined until1 now. Since the vWD type l-3 is present at least once in each generation of this family and is apparently transmitted in a straight line without a generation gap, an autosomal dominant genetic transmission of the vWD type l-3 is assumed (14).

0

healthy

worn.”

healthy

I.”

cl

Fig.5: Family tree of patients with vWD l-3

The clinical significance of the platelet vWF is still unclear (3). According to our results and other studies (15,16) the reason could be a release of vWF from the platelets during the first phase of haemostasis in order to am lify the coagulation processes. During a vascular injury the large vWF multimeres &ind to the collagen fibers of the subendothel cells first. Then the platelets are activated by binding to the vWF and release additional vWF into the plasma. This amplifies the primary haemostasis in the area surrounding the point of injury. In patients with vWD Typ l-3 this amplification is taking place in a reduced manner and therefore results in a bleeding tendency. This theory is su ported by the correlation between bleeding time and platelet vWF concentration, which Ras been found by Gralnick and co-workers. However, this correlation is not found between the vWF concentration in plasma and the bleeding time. The authors conclude, that a sufficent vWF concentration in the platelets could at least partially compensate a lack of vWF in the plasma (15). Consequently the vWF concentration in the platelets would be of great importance. However, this significant correlation between platelet vWF concentration and the bleeding time could not be reproduced in the patients we tested. Out of 160 patients 27 (16.9Oh)showed no pathological change in the coagulation factors in plasma, but a decreased concentration of vWF in the platelets. Without testing the

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platelet vWF concentration, the bleeding tendency of these patients could not have been clarified. The discovery of an autosomal dominant genotype of the type l-3 vWD emphasizes the importance of the vWF concentration in platelets. These findings are in good agreement with the results of other authors who also describe an autosomal dominant genotype to the vWD type I (17). The change of vWD type l-3 to vWD l-2 and l-2 to l-3, respectively, observed in three cases prompts us to sug est that the vWF concentration in plasma and platelets may not be considered as isolate ! parameter. For the regulation of primary haemostasis, the total pool of vWF available appears to be important. These observations are supported by the results of Gralnick et al and d’Alessio et al (15,16). Although the present results cannot completely explain the significance of the vWF in platelets, they emphasize that the analysis of the vWF multimers as well as the quantitative analysis of the vWF concentration in platelets is an important laboratory parameter for the diagnosis of the vWD.

Acknowledaement

We thank Mrs. Gertrud Mora-Maza for translating of this manuscript.

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