Britishloumal of Haematology, 1975, 29, 605.
The von Willebrand Syndrome PENELOPE STABLEFORTH, JANE HUGHES, ELAINEWILSON AND KATHARINE M. DORMANDY
Department of Haematology, Royal Free Hospital, London (Received 3 June 1974; acccptedf o r publication 2 3 September 1974) SUMMARY. Five patients with an original diagnosis of voii Willebrand’s disease are dcscribed because their levels of factor VIII related protein, Ristocetin-induced platelet aggregation andlor family studies differed from the main group of patients with classical von Willebrand’s disease. Two had normal levels of factor VIII related protein with reduced Ristocetin aggregation when this was tested in platelet rich plasma. In one, however, this was due to a plasnia dcfect and in the otlier to a plate let abnormality. After cryoprecipitate infusion all abnormal tests were corrected in both these patients. The first patient,however,failed to show a secondary rise of factor VIII whereas the second showed a secondary rise or’both factor VIII and of factor VIII related protein. The other three cases, who wcrc all very severely affected, have been separated from the main group as none of their families was segregating for classical voii Willebrand’s disease. It is suggested that the term von Willebrand’s discase sliould be confined to those patients who have reduced factor VIII related protein and Ristocetin aggrcgation, and that voii Willebrand’s syndrome should be used for the various sub-groups that are emerging. The reports of Howard & Firkin (1971) and Howard et ul(1973b) that in a platelet rich plasnia (T’RP) system the antibiotic Ristocetiii aggregated normal platelets but not those of patients with von Willebrand’s disease (vwd), led us to reinvestigate our patients with this coiidition. Investigations included nieasurcinent of Ristocctin aggregation and factor VIII rclatcd protein (VIII RP), as well as standard platelet and coagulation studies. SUBJECTS Control Subjects Forty-two volunteers from the hospital staff without a history of hacmostatic disorder were used as controls. One volunteer was used as a control on each occasion a patient was tested.
Patients and Relatives Fifteen propositi and 15 of their affected relatives form the basis of this investigation. 27 of these, from 12 kindreds, were of intermediate sevcrity while three, from three other separate families, were extremely severe cases (Graham cf a / , 1973). In 26 the original diagnosis was based on finding a proloiigcd bleeding tiinc (BT), a low level of factor VIII and reduced Correspondence: Dr K. M. Dormandy, Haeinophilia Centre,* Department of Haematology, The Royal Free Hospital, Pond Street, London NW3 2QG. * Iritcrnational Haemophilia Training Centre, World Fcdcration of Haemophilia.
605
Penelope Stableforth et a1
606
platelet retention. Four patients with haemorrhagic diathescs who had !ow levels of factor VIII and reduced platelet retention (cases 6, 11, 20 and 26) were included, although the BT was consistently normal ; all these four had affected relatives with prolonged bleeding times (Table I). Eleven clinically unaffected relatives of the five cases of special interest were also investigated. The histories of cases 17 and 19, whose VIII RP and/or Ristocetin aggregation was later shown to give different results from the main group, are as follows: Case 17 (family tree, Fig I), a 63-year-old woman with a history of epistaxes and bleeding I
m
ITL: P
h
b
FIG I. Pedigree of case 17 (111 IS). In Figs 1-5, square symbols represent malcs and round symbols females; hatching indicntes history of bleeding and stippling indicates evidence of trait; a triangle within a symbol indicates tested and normal, and a number within a symbol represents that number of clinically unaffected persons; the propositus (affected) is indicated by an arrow.
after dental extraction. An artificial menopause had been induced at 44 years of age because of uncontrollable menorrhagia requiring blood transfusions. The diagnosis of vWd was based on the following findings: bleeding time grcater than 20 min; platelet count 250 ooo/pl, platelet retention 9%, factor VIII 32%. Platelet aggregation was normal with ADP, adrenaline, collagen and bovine fibrinogen. Case 19 (family tree, Fig z),a 24-year-old girl with a history of epistaxes, menorrhagia and bleeding after dental extraction. At 17 years of age, 12 units of blood had been required during an operation for the removal of a cyst from her hip. The diagnosis of vWd was originally made on the basis of the following findings: bleeding time 18 min; platelet count 180 ooo/pl, platelet retention IO%, factor VIII tested on five separate occasions ranged between 3 2 and 42%. The blood film showed normal platelet morphology; platelet aggregation was normal with ADP, adrenaline, collagen and bovine fibrinogen. The family trees of cases 28, 29 and 30, the extreme cases, are shown in Figs 3, 4 and 5. Cryoprecipitate infusions were given to three patients (cases 14, 17 and 19). The bleeding time, platelet retention, factor VIII, VIII RP and Ristocetin aggregation were monitored before and after the infusions. MATERIALS AND METHODS
Blood was taken through an Abbott No.
I
butterfly needle into a plastic syringe, and
The von Willebrand Syndrome
607
I
II
m
/ FIG2. Pedigree of case 19 (IV I).
I
v
II
m FIG 3. Pedigree of case 28 (111 4).
I
I
I
m
m FIG4. Pedigree of case 29 (IV
10).
immediately transferred into a polystyrcnc pot containing 3 . 8 j (w/v) trisodiuin citrate at a ratio of 9 parts blood to I part trisodium citrate. Platelet rich plasma (PRP)was prepared by centrifuging the citrated blood at room temperature at 600 g for 5 min at a time and removing the PRP aftcr each centrifugation. Platclct poor plasma (PPP)was prepared by centrifuging the citratcd blood at 4°C at 10000 g for 5 min.
Penelope Stableforth et a1
608 I
IT
m
Ill
x FIG 5. Pedigree of case 30 (IV 2).
Washed platelets were prepared from citrated plasma by dispensing I ml aliquots of PRP into polystyrene tubes and adding 0.2 ml of 0.077Methylene diamine tetra acetic acid (EDTA). This was centrifuged at 3500 g for 20 min and the supernatant discarded. I ml of EDTA was added and this was then centrifuged again for 20 min. This procedure was repeated twice. Cryoprecipitate was prepared from normal and haeniophilic plasma by the method of Pool & Shannon (1965).Purifedfactor VIIIwas prepared by the method of Van Mourik & Mochtar (1970).Ristocetin powder (H. Lundbeck & Co., Copenhagen) stored at 4°C in the dark was made up in isotonic saline and used at final concentrations of 1.0mg/ml and 1.5 mg/ml. Adenosine diphosphate (ADP) (Sigma) was dissolved in isotonic saline to give concentrations from I x I O - ~M to I x I O - ~M. Adrenaline (Evans) was dissolved in isotonic saline to give concentrations from I x I O - ~to I x I O - ~M. Collagen (Sigma)was made into a suspension using isotonic saline. Factor-VIII assays were performed by the two-stage assay of Denson (1966)(Diagen kit) using a porcine standard. Factor VIII related protein (VIII R P ) was measured by the Laurel1 technique (1966)on whole plasma. The antibody used was raised in rabbits against human cryoprecipitate and purified by absorption with v W plasma from a very severely affected patient with a factor VIII of less than I%. A frozen plasma standard was used from a pool of 12 normal donors of both sexes between the ages of 20 and 50. Bleeding time ( B T )was by the Ivy method (Ivy et al, 1935).(The longest of the three times was taken as the bleeding time, normal 2-8 min.) Platelet retention was determined by a modified Helleni technique. 2 ml citrated whole blood was passed through a column containing I g of glass beads type 07c-5005, Minnesota Mining and Manufacturing Company, at a speed of 1.5 ml/min and collected in a tube containing 2.5 mg dried sequestrene (normal range 20-40%). Platelet aggregation was measured by the turbidometric method of Born (1962,1970).The platelet counts of control and test PRP were adjusted with PPP to within 50 ooo/pl. I ml PRP was placed in a cuvette and aggregation was recorded for 5 min after the addition of
The von Willebrand Syndrome 0.1ml ofRistocetin. The results werc recorded as a
609
perccntagc of thc total light transmitted by
the test in comparison with that of the control. RESULTS Table I summarizes the results and shows the related patients. The control rangc for Ristocetin aggregation at 1.0mg/ml was 20-70% ; whilc that of the vW patients was from o to 10%. In the mixing tests 29 of the 30 patients showed a reduced Ristocetin aggregation when control platelets were suspended in VWPPP, but normal aggregation with VWplatelets in control PPP. In case 19, the only exception among the patients, thc findings wcre rcversed, TABLE 1. Results of investigations on v W patients and their affected relatives
B T (min)
Case no.
Platelet retention
Factor V I I I
VIII RP
(%)
(%I
(Yo)
Ristocetin PRP
(I
wlg/nil) a'qgregation
c Platelets/ t Plasma
(yo)
t Platelets/ c Plasma
__-
Normal range
2-8
[::
____
4 M SF 6 M 7F 8F PF
LIO M 11 M
[::: [:; L 16 M I7 F 18 F I9 F
[::r 22
:[
F
;
18 20
+ + + +
20-40
~
~
Z
I1
IS IS
4
O
O
60-160
20-70
c platelets+c plasma
>20
18
2
2
30
21
4
S
2
S
28 28
2
3 3
I
I2
IS
4
I4 I7 4 7
I0 I0
I4 18 18 42 16
22
21
18g IS 8
IS
I3
I0
I1
24 24
I7
The von Willebrand Syndrome PENELOPE STABLEFORTH, JANE HUGHES, ELAINEWILSON AND KATHARINE M. DORMANDY
Department of Haematology, Royal Free Hospital, London (Received 3 June 1974; acccptedf o r publication 2 3 September 1974) SUMMARY. Five patients with an original diagnosis of voii Willebrand’s disease are dcscribed because their levels of factor VIII related protein, Ristocetin-induced platelet aggregation andlor family studies differed from the main group of patients with classical von Willebrand’s disease. Two had normal levels of factor VIII related protein with reduced Ristocetin aggregation when this was tested in platelet rich plasma. In one, however, this was due to a plasnia dcfect and in the otlier to a plate let abnormality. After cryoprecipitate infusion all abnormal tests were corrected in both these patients. The first patient,however,failed to show a secondary rise of factor VIII whereas the second showed a secondary rise or’both factor VIII and of factor VIII related protein. The other three cases, who wcrc all very severely affected, have been separated from the main group as none of their families was segregating for classical voii Willebrand’s disease. It is suggested that the term von Willebrand’s discase sliould be confined to those patients who have reduced factor VIII related protein and Ristocetin aggrcgation, and that voii Willebrand’s syndrome should be used for the various sub-groups that are emerging. The reports of Howard & Firkin (1971) and Howard et ul(1973b) that in a platelet rich plasnia (T’RP) system the antibiotic Ristocetiii aggregated normal platelets but not those of patients with von Willebrand’s disease (vwd), led us to reinvestigate our patients with this coiidition. Investigations included nieasurcinent of Ristocctin aggregation and factor VIII rclatcd protein (VIII RP), as well as standard platelet and coagulation studies. SUBJECTS Control Subjects Forty-two volunteers from the hospital staff without a history of hacmostatic disorder were used as controls. One volunteer was used as a control on each occasion a patient was tested.
Patients and Relatives Fifteen propositi and 15 of their affected relatives form the basis of this investigation. 27 of these, from 12 kindreds, were of intermediate sevcrity while three, from three other separate families, were extremely severe cases (Graham cf a / , 1973). In 26 the original diagnosis was based on finding a proloiigcd bleeding tiinc (BT), a low level of factor VIII and reduced Correspondence: Dr K. M. Dormandy, Haeinophilia Centre,* Department of Haematology, The Royal Free Hospital, Pond Street, London NW3 2QG. * Iritcrnational Haemophilia Training Centre, World Fcdcration of Haemophilia.
605
Penelope Stableforth et a1
606
platelet retention. Four patients with haemorrhagic diathescs who had !ow levels of factor VIII and reduced platelet retention (cases 6, 11, 20 and 26) were included, although the BT was consistently normal ; all these four had affected relatives with prolonged bleeding times (Table I). Eleven clinically unaffected relatives of the five cases of special interest were also investigated. The histories of cases 17 and 19, whose VIII RP and/or Ristocetin aggregation was later shown to give different results from the main group, are as follows: Case 17 (family tree, Fig I), a 63-year-old woman with a history of epistaxes and bleeding I
m
ITL: P
h
b
FIG I. Pedigree of case 17 (111 IS). In Figs 1-5, square symbols represent malcs and round symbols females; hatching indicntes history of bleeding and stippling indicates evidence of trait; a triangle within a symbol indicates tested and normal, and a number within a symbol represents that number of clinically unaffected persons; the propositus (affected) is indicated by an arrow.
after dental extraction. An artificial menopause had been induced at 44 years of age because of uncontrollable menorrhagia requiring blood transfusions. The diagnosis of vWd was based on the following findings: bleeding time grcater than 20 min; platelet count 250 ooo/pl, platelet retention 9%, factor VIII 32%. Platelet aggregation was normal with ADP, adrenaline, collagen and bovine fibrinogen. Case 19 (family tree, Fig z),a 24-year-old girl with a history of epistaxes, menorrhagia and bleeding after dental extraction. At 17 years of age, 12 units of blood had been required during an operation for the removal of a cyst from her hip. The diagnosis of vWd was originally made on the basis of the following findings: bleeding time 18 min; platelet count 180 ooo/pl, platelet retention IO%, factor VIII tested on five separate occasions ranged between 3 2 and 42%. The blood film showed normal platelet morphology; platelet aggregation was normal with ADP, adrenaline, collagen and bovine fibrinogen. The family trees of cases 28, 29 and 30, the extreme cases, are shown in Figs 3, 4 and 5. Cryoprecipitate infusions were given to three patients (cases 14, 17 and 19). The bleeding time, platelet retention, factor VIII, VIII RP and Ristocetin aggregation were monitored before and after the infusions. MATERIALS AND METHODS
Blood was taken through an Abbott No.
I
butterfly needle into a plastic syringe, and
The von Willebrand Syndrome
607
I
II
m
/ FIG2. Pedigree of case 19 (IV I).
I
v
II
m FIG 3. Pedigree of case 28 (111 4).
I
I
I
m
m FIG4. Pedigree of case 29 (IV
10).
immediately transferred into a polystyrcnc pot containing 3 . 8 j (w/v) trisodiuin citrate at a ratio of 9 parts blood to I part trisodium citrate. Platelet rich plasma (PRP)was prepared by centrifuging the citrated blood at room temperature at 600 g for 5 min at a time and removing the PRP aftcr each centrifugation. Platclct poor plasma (PPP)was prepared by centrifuging the citratcd blood at 4°C at 10000 g for 5 min.
Penelope Stableforth et a1
608 I
IT
m
Ill
x FIG 5. Pedigree of case 30 (IV 2).
Washed platelets were prepared from citrated plasma by dispensing I ml aliquots of PRP into polystyrene tubes and adding 0.2 ml of 0.077Methylene diamine tetra acetic acid (EDTA). This was centrifuged at 3500 g for 20 min and the supernatant discarded. I ml of EDTA was added and this was then centrifuged again for 20 min. This procedure was repeated twice. Cryoprecipitate was prepared from normal and haeniophilic plasma by the method of Pool & Shannon (1965).Purifedfactor VIIIwas prepared by the method of Van Mourik & Mochtar (1970).Ristocetin powder (H. Lundbeck & Co., Copenhagen) stored at 4°C in the dark was made up in isotonic saline and used at final concentrations of 1.0mg/ml and 1.5 mg/ml. Adenosine diphosphate (ADP) (Sigma) was dissolved in isotonic saline to give concentrations from I x I O - ~M to I x I O - ~M. Adrenaline (Evans) was dissolved in isotonic saline to give concentrations from I x I O - ~to I x I O - ~M. Collagen (Sigma)was made into a suspension using isotonic saline. Factor-VIII assays were performed by the two-stage assay of Denson (1966)(Diagen kit) using a porcine standard. Factor VIII related protein (VIII R P ) was measured by the Laurel1 technique (1966)on whole plasma. The antibody used was raised in rabbits against human cryoprecipitate and purified by absorption with v W plasma from a very severely affected patient with a factor VIII of less than I%. A frozen plasma standard was used from a pool of 12 normal donors of both sexes between the ages of 20 and 50. Bleeding time ( B T )was by the Ivy method (Ivy et al, 1935).(The longest of the three times was taken as the bleeding time, normal 2-8 min.) Platelet retention was determined by a modified Helleni technique. 2 ml citrated whole blood was passed through a column containing I g of glass beads type 07c-5005, Minnesota Mining and Manufacturing Company, at a speed of 1.5 ml/min and collected in a tube containing 2.5 mg dried sequestrene (normal range 20-40%). Platelet aggregation was measured by the turbidometric method of Born (1962,1970).The platelet counts of control and test PRP were adjusted with PPP to within 50 ooo/pl. I ml PRP was placed in a cuvette and aggregation was recorded for 5 min after the addition of
The von Willebrand Syndrome 0.1ml ofRistocetin. The results werc recorded as a
609
perccntagc of thc total light transmitted by
the test in comparison with that of the control. RESULTS Table I summarizes the results and shows the related patients. The control rangc for Ristocetin aggregation at 1.0mg/ml was 20-70% ; whilc that of the vW patients was from o to 10%. In the mixing tests 29 of the 30 patients showed a reduced Ristocetin aggregation when control platelets were suspended in VWPPP, but normal aggregation with VWplatelets in control PPP. In case 19, the only exception among the patients, thc findings wcre rcversed, TABLE 1. Results of investigations on v W patients and their affected relatives
B T (min)
Case no.
Platelet retention
Factor V I I I
VIII RP
(%)
(%I
(Yo)
Ristocetin PRP
(I
wlg/nil) a'qgregation
c Platelets/ t Plasma
(yo)
t Platelets/ c Plasma
__-
Normal range
2-8
[::
____
4 M SF 6 M 7F 8F PF
LIO M 11 M
[::: [:; L 16 M I7 F 18 F I9 F
[::r 22
:[
F
;
18 20
+ + + +
20-40
~
~
Z
I1
IS IS
4
O
O
60-160
20-70
c platelets+c plasma
>20
18
2
2
30
21
4
S
2
S
28 28
2
3 3
I
I2
IS
4
I4 I7 4 7
I0 I0
I4 18 18 42 16
22
21
18g IS 8
IS
I3
I0
I1
24 24
I7
