Type IB von willebrand's disease and pregnancy: Comparison of analytical methods of von willebrand factor for classification of von willebrand's disease subtypes

THROMBOSIS RESEARCH 50; 409-418, 1988 0049-384B/BB $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

TYPE IB VON WILLEBRAND'S DISEASE AND PREGNANCY: COMPARISON OF ANALYTICAL METHODS OF VON WILLEBRAND FACTOR FOR CLASSIFICATION OF VON WILLEBRAND'S DISEASE SUBTYPES

Hoyu

Takahashi,

Nobuyuki

Hayashi*

and

Akira

First Department of Internal Medicine and "Department Gynecology, Niigata University School of Medicine, Niigata

951,

Shibata of Obstetrics and l-754 Asahimachi,

Japan

(Received 18.1.1988; Accepted in revised form 23.2.1988 by Editor H. Yamazaki)

ABSTRACT The clinical factor VIII patient with vWf multimers

course and and bleeding

changes in von Willebrand time during pregnancy are

type IB von Willebrand's disease were present in plasma with

factor described

(vWD) in relatively

(vWf), in a

whom all reduced

content of the high-molecular-weight ristocetin cofactor was elevated to some

multimers. At term, extent (from the baseline and below the never normalized Bleeding time remained prolonged.

value of 5 U/d1 to 23 U/dl), but level of vWf antigen (49 U/dl). In addition, abnormal distribution in the multimer size of plasma vWf was never corrected. These findings contrast with responses during pregnancy reported in type IA The patient was infused during labor with 400 ml of fresh frozen plasma, and blood loss was approximately 800 ml. Analytical techniques of vWf by crossed immunoelectrophoresis, gel filtration on Sepharose 2B and SDS-agarose gel electrophoresis for the classification of vWD subtypes were compared. Multimer favorable individuals.

analysis by differentiation

SDS-agarose of type

gel

IB from

electrophoresis type IIA vWD.

was

needed

for

INTRODUCTION von Willebrand's disease (vWD) is an autosomally inherited disorder affecting both males and females. It is associated with tendencies for abnormal bleeding: in females, bleeding symptoms hypermenorrhea, abnormal postpartum bleeding and bleeding

Key words:

von

Willebrand's

von

Willebrand

disease, factor

type

multimers, 409

IB von Willebrand's pregnancy,

delivery

bleeding variable include during

disease,

410

TYPE

IB vWB AND PREGNANCY

Vol. 50, No. 3

spontaneous abortions. This disorder is characterized by a deficiency of or structural defect in von Willebrand factor (vWf), a long flexible filamentous glycoprotein which mediates platelet adhesion to exposed subendothelium and subsequent platelet plug formation at the sites of vascular injury (1.2). vWf circulates in normal plasma as a series of multimers in which the repeating protomer is a dimer of the 270 kd subunit of 2,050 amino acid residues. composed The high-molecular-weight multimers of vWf preferentially bind to platelets in the presence of ristocetin and to collagen, indicating the greater efficacy of these multimers in hemostasis (1). vWD is a heterogeneous disorder. In type I vWD all multimeric forms of vWf are present in plasma, while patients with type II vWD lack the high-molecular-weight multimers in plasma. Several subtypes have been identified within both type I and type II vWD (1.2). In type IA vWD all vWf multimers are present in plasma in normal relative whereas the high-molecular-weight multimers are relatively proportion, decreased

in

type

IB vWD

(3).

Several cases of vWD in pregnancy have been reported. However, only a few cases have been thoroughly documented on the basis of molecular abnormalities in subtypes of vWD (4-8). various Therefore, little information has been obtained concerning the changes in vWf, correction of bleeding time and hemorrhagic complications during delivery in each vWD This report describes the clinical course and coagulation subtype. profiles of a pregnant patient with type IB vWD. In addition, analytical techniques for vWf multimer sizing were compared.

MATERIALS AND METHODS Venous blood was collected and mixed with l/IO volume of 0.129 M Platelet-rich plasma (PRP) was prepared from the trisodium citrate. titrated whole blood by centrifugation at 175 g for 10 minutes at room temperature, and platelet-poor plasma by centrifugation at 2,000 g for 20 minutes

at 4°C.

The bleeding time was measured by the method of Duke (9) with a Platelet counts were performed normal upper limit of less than 5 minutes. normal an electronic counter (Coulter Electronics, Hialeah, FL, USA): Platelet retention to glass beads column Fa"nge is 157,000-388,000/ 1. is 66.5 t was determined by the me t hod of Hellem II (10); normal value Platelet aggregation studies were performed in PRP 9.5% (mean + SD). (250,000 platelets/pi) Ristocetin was obtained

by the turbidimetric method using from H. Lundbeck Co. (Copenhagen,

an aggregometer. Denmark).

VIII activity (VIII:C) was assayed by a one-stage method (11). Factor was quantitated by rocket immunoelectrophoresis ;L;Ii)antigen (vWf:Ag) macroscopic Ristocetin cofactor (RCof) was measured with a . For tilt-tube method (13) using formalin-fixed normal platelets (14). these concentrations, 1 unit (U) was defined as the amount present in 1 ml Normal ranges of VIII:C, vWf:Ag and RCof were of pooled normal plasma. 55-185

U/dl, Crossed

49-181

U/d1

and

52-153

immunoelectrophoresis.

barbital buffer pH 8.6 tanks, 0.05 M barbital

U/dl,

respectively.

Agarose

at a concentration buffer pH 8.6 was

was

of 1%. used.

dissolved

in

0.025

M

In the electrophoresis Electrophoresis in the

Vol.

50,

No.

3

first

dimension

blue)

migrated

dimension was washed, dried

IB

TYPE

was to

performed

a length

carried out and stained

vWB

at of

AND

5 V/cm 40

until the marker Electrophoresis

mm.

at 1 mA/cm for with Coomassie

The multimeric composition of vWf sulfate (SDS)-agarose gel electrophoresis of Ruggeri & Zimmerman (15) system 1.5-mm-thick and ID-cm-long running gel

411

PREGNANCY

18 hours. brilliant

dye in

The plates blue.

(bromphenol the second were

then

was

analyzed by sodium dodecyl using a discontinuous buffer with slight modifications. A consisted of either 1.4% SeaKem

HGT(P)

agarose (FMC, Rockland. ME, USA) or LGT agarose (Sigma Chemical MO, USA) and 2.5-cm-long stacking gel consisted of 0.8% co., St. Louis, Electrophoresis was performed horizontally at 0.5 mA/cm HGT(P) agarose. and at 16°C in LKB Multiphor apparatus (LKB-Produkter AB, Bromma, Sweden) Following the until the tracking dye reached near the end of the gel. electrophoresis, vWf multimers an immunoperoxidase technique Gel filtration. Two (Pharmacia Fine Chemicals, (Pharmacia).

The

elution

>lI

>30 13.4

were visualized directly on the as previously described (16).

gel

using

ml

of plasma was applied to a 1.6 x 30 cm column Uppsala, Sweden) packed with Sepharose 2B buffer was a mixture of 9 parts of 0.15 M sodium

16

>20

18.5

8.9

Platelet retention (XI

DOYS after delivery

Birth

Duration of gestation in weeks

(min)

>15 Z-13 Bleeding tlm

7

1.5 25.8

FIG. 1 Clinical

course

and

vWD during pregnancy, frozen plasma.

coagulation labor

and

profiles after

in a patient

delivery.

FFP

with denotes

type

IB

fresh

412

TYPE

IB vWB AND

PREGNANCY

chloride and one part of 0.05 M imidazole run at 29 ml/hour and fractions of 2.6 ml VIII:C and vWf:Ag. Protein was monitored

Vol.

50,

buffer

pH 7.35. The column immediately assayed were by absorbance at 280 nm.

No.

was for

PATIENT The

patient

is a 27-year-old

nasal bleeding, bleeding tonsillectomy at age 3. because

of

abdominal

woman

from trivial At age 26, pain

and

with

vWD.

wounds,

she nausea.

and was She

She

had

prolonged admitted underwent

histories bleeding to

a an

of after

hospital emergency

operation. Intraperitoneal bleeding (approximately 500 ml) was noticed, but the bleeding site was obscure. At that time she underwent an appendectomy, and severe bleeding at the surgical wound site developed She was admitted to Niigata requiring blood transfusion (1,600 ml). University Hospital, Niigata at age 27 for detailed examination of the bleeding tendency. The hemostatic evaluation on the patient revealed that she had vWD (see below). VIII:C Her father had similar abnormalities:

FIG. 2 Crossed immunoelectrophoresis of vWf in plasma from a normal subject (A), from the pregnant gestation

type IB vWD patient in nonstate (B), at 35 weeks of (C) and 1.5 hours after

delivery (D) and in the cord blood from the infant (E). The anode is to the right in the first dimension, second and at the top in the dimension.

3

Vol.

was

50,

44

No.

TYPE

3

U/dl,

vWf:Ag

23

U/d1

IB vWB AND

and

RCof

413

PREGNANCY

7 U/dl.

During the At the age of 27 years, the patient became pregnant. hemostatic evaluation was performed at periodic intervals. The pregnancy, At 41 weeks of gestation, delivery normal. course of pregnancy was She 100 ml. started and blood loss during the first stage of labor was A normal was then transfused with 400 ml of fresh frozen plasma (Fig. 1). The baby girl weighing 3,130 g was delivered by vacuum extraction. varices. resulting in bleeding from vulvar episiotomy was also performed, No excess Total blood loss during delivery was approximately 800 ml. bleeding

occurred

postpartum,

and

the

patient

was

discharged

with

her

baby. RESULTS

Hemostatic Prior bleeding decreased

Evaluation to

in Non-Pregnant

pregnancy,

time (>30 platelet

coagulation

minutes), retention

State data

on

normal platelet rate (13.4-26.3%).

FIG.

the

patient

count low

showed

prolonged

(173,000-252,000/~1), VIII:C (24-36 U/dl),

3

Multimeric composition of plasma vWf analyzed by SDS-1.4% HGT agarose gel electrophoresis and visualized by an immunoperoxidase staining. Plasma samples from a normal subject (A), from the type IB vWD patient in non-pregnant state (B), at 30 (C), 38 (D), 40 (E), 41 (F) weeks of gestation, after infusion of fresh frozen plasma (G) and 11 days after delivery (H) and from the cord blood (I) were analyzed. The arrow indicates the origin of the running is at the bottom. gel, and the anode

414

TYPE IB vWB AND PREGNANCY

low vWf:Ag aggregation

(7-21 U/dl), in PRP with

Vol. 50, No. 3

markedly low RCof (4-5 U/dl) and 1.25 mg/ml ristocetin on several

absent

platelet

determinations. Platelet aggregation induced by ADP, collagen, epinephrine and bovine vWf was normal. Crossed immunoelectrophoresis against antiserum to vWf demonstrated that the patient's plasma was lacking in the slow-moving, high-molecular-weight forms of vWf (Fig. 2). Multimer analysis of plasma vWf by SDS-agarose gel electrophoresis revealed only a faint staining of all multimers; visualization

Changes

the (Fig.

concentration 3).

of

vWf

was

insufficient

for

clear

in vWf and Bleeding Time During Pregnancy

Changes in factor VIII/vWf-related activities, bleeding time and platelet retention rate in the patient during prepartum period and in postpartum period are depicted in Fig. 1. During gestation VIII:C increased gradually and reached to normal level, but vWf:Ag and RCof were Just prior to infusion of fresh frozen plasma below normal even at term. at 41 weeks' gestation, plasma levels of VIII:C, vWf:Ag and RCof were 70 Duke bleeding time remained U/dl, 49 U/d1 and 23 U/dl, respectively. and platelet retention to glass beads was not prolonged (>20 minutes), Following the plasma infusion, bleeding time was shortened to normalized. 7 minutes,

and vWf:Ag and RCof were elevated to 98 U/d1 and Plasma levels of VIII:C, vWf:Ag and RCof declined respectively. during postpartum period (Fig. 1).

FIG.

50 U/dl, gradually

4

Gel filtration on Sepharose 28 of normal plasma (A) and plasma samples from the type IB vWD patient at 40 weeks of gestation IF,' and 1.5 hours after delivery

.

Fraction

number

Vol.

50,

No.

Qualitative studied

IB vWB AND

TYPE

3

with

of

characteristics

crossed

415

PREGNANCY

plasma

vWf

during

pregnancy

were

immunoelectrophoresis,

gel filtration on Sepharose 2B immunoelectrophoresis Crossed electrophoresis. and SDS-agarose gel lower-molecular-weight revealed the predominance of the fast-migrating, forms of vWf in the patient's plasma studied at 35 and 41 weeks of The plasma sample obtained 1.5 hours after delivery (3 hours gestation. after the infusion of fresh frozen plasma) showed a modest increase in the less anodic forms as compared with preinfusion plasma (Fig. 2). Gel filtration on Sepharose 26 showed a delayed elution of vWf in plasma at 40 Some vWf weeks' gestation when compared with that in normal plasma. eluted near the void volume when post-transfusion plasma was applied (Fig. electrophoresis Multimeric analysis of vWf by SDS-agarose gel 4). revealed patient's

the presence of the high-molecular-weight multimers in the plasma obtained during pregnancy (30-41 weeks' gestation). A the concentration of the high-molecular-weight reduction in The electrophoretic mobility of the was also noted (Fig. 3).

relative multimers satellite subbands of individual repeating from that in normal plasma, demonstrating not aberrant. Plasma

prepared

from

the

cord

multimers was indistinguishable that the triplet structure was

blood

of

the

infant

contained

98

U/d1

VIII:C, 130 U/d1 vWf:Ag and 115 U/d1 RCof. Crossed immunoelectrophoresis arc (Fig. 2). shift of the precipitin showed a pronounced cathodic SDS-agarose gel electrophoresis demonstrated the presence of abnormally high-molecular-weight vWf multimers which was larger in size than those present in normal plasma (Fig. 3).

DISCUSSION vWD is quantitative Recently have

a bleeding disorder and/or qualitative

developed

laboratory

with a complex abnormalities of techniques

hemostatic vWf are the

including

defect. cause of

multimer

permitted

classification of this disorder into several (IA-C, IIA-H, III and others) (1.2). The patient presented laboratory findings suggestive of type IIA vWD; markedly

bleeding markedly

time. absent ristocetin-induced decreased RCof, vWf:Ag higher

mobility on was elevated predominance

crossed during of the

platelet aggregation RCof, and vWf with When plasma (Fig. 2).

than

The vWD.

analysis subtypes here had prolonged PRP, in a faster vWf level

immunoelectrophoresis pregnancy, gel filtration studies also supported lower-molecular-weight forms of vWf in plasma

the (Fig.

However, analysis by SDS-agarose gel electrophoresis multimer 4). revealed the presence of the high-molecular-weight multimers (Fig. 3), demonstrating that the patient had type I vWD. The relative diminution of the high-molecular-weight multimers, normal triplet structure of each VWf multirner, and RCof lower than the level of vWf:Ag indicate that she belongs to type IB category electrophoresis should be diagnosing the subtypes of filtration profiles did not in this patient. In pregnancy, individuals. may

obviate

The the

(3).

vWf and factor spontaneous rise

need

for

These

results

imply

that

SDS-agarose

performed in each patient before vWD. Crossed immunoelectrophoresis permit classification of type IB vWD,

replacement

VIII in vWf

rise and

therapy

in both healthy factor VIII during at

delivery.

gel

finally or gel at least

and vWD pregnancy

Deliveries

are

416

IB vWB AND

TYPE

uneventful

without

supportive

therapy

Vol.

PREGNANCY

in

some

suffer from bleeding complications (4-8,17-21). the fact that the increases in vWf:Ag, RCof and

replacement section. experiences subtypes

No.

patients, but others may This would be related to VIII:C are variable among

subtypes and from patient to patient. Lipton et al. al. (7) have suggested that RCof and/or bleeding predictors for risk of bleeding in pregnant patients, (6) have stressed that during delivery is low

50,

(21) and Chediak time are the while Conti et

the most important determinant factor VIII. Furthermore, the

of

abnormal

et best al.

bleeding

prophylactic therapy is controversial during vaginal or Cesarean These opinions would be resulted from different clinical in the management of pregnant vWD patients with different

and

with

different

use of delivery

obstetric

problems.

Here we described the course and outcome of pregnancy in a woman with The changes in hemostatic parameters were different from type IB vWD. type IA patients. In type IA patients, there is a progressive improvement as well as a correction of the plasma levels of vWf:Ag, RCof and VIII:C

of the bleeding time during pregnancy (6.7). In our type IB patient, however, the elevation of vWf:Ag was modest, the level of RCof was not proportional to the vWf:Ag level, and bleeding time remained prolonged at term (Fig. 1). The hemostatically most active, high-molecular-weight vWf multimers did not become predominant and remained relatively reduced during higher

the gestation (Fig. 3). risk of abnormal bleeding

Thus, type IB vWD patients during delivery than type

may

IA

have a patients.

She

was infused during labor with fresh frozen plasma, and blood loss was Additionally, vWf multimers larger than normal were limited to 800 ml. found in the cord blood from the infant (Figs. 2 and 3). They would be derived from endothelial cells under stimuli (22,23) and collected before processing to the circulating plasma. gel electrophoresis

somewhat smaller multimers normally found in the Both crossed immunoelectrophoresis and SDS-agarose were sensitive to detect the unusually large vWf

multimers. In female vWD patients, their subtypes should be evaluated by before analysis of vWf as early as possible, preferably The classification of vWD subtypes may permit, at least to pregnancy.

multimer

some extent, the prediction of changes in vWf hemostatic outcome during delivery. However,

and only

factor VIII and a limited number

the of

patients in pregnancy have been characterized by their underlying subtypes In each of vWD (4-8), and more clinical experience must be accumulated. should be monitored during case, the clinical and laboratory findings pregnancy, labor and after delivery, because different subtypes of the disease and diffrent obstetric problems may result in variable outcome.

ACKNOWLEDGMENTS This work was supported in part bv Grants-in Research from the Min\stry of Education,"Science and

Aid for Culture of

Scientific Japan.

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3