THROMBOSIS RESEARCH @ Pergamon
14;
495-500
Press Ltd.1979.
Printed
in Great Britain 0049-3848/79/
BRIEF
0301-0495
$02.00/o
COMMUNICATION
INHIBITION OF WILLEBRAND FACTOR IN VON WILLEBRAND DISEASE
S. Maragall, R. Castillo, A. Ordinas F. Liendo and M. Rodrfgues Facultad de Medicina. Hospital ClXnico y Provincial Barcelona. Spain (Received 8.5.1978; in revised form 18.11.1978. Accepted by Editor Y.V. Sultan
INTRODUCTION
Six cases of congenital von Willebrand disease with homologous
IgG antibodies to von Willebrand factor (VIIIR:WF) have been re cently described (1, 2, 3, 4). In three of those cases, the ant& bodies precipitated factor VIII related antigen (VIIIR:AG) from normal plasma (2). We now report a new case of an inhibitor directed towards VIIIR:WF and VIIIR:AG in a polytransfused patient with severe von Willebrand disease. The inhibitory capacity was found in the Ig fraction of the patient's plasma. The antibodies inhibited the Ristocetin-induced aggregation of normal platelet rich plasma (PRP) and also precipitated factor VIII related antigen from normal and hemophilic plasmas. PATIENT AND METHODS A.R., born in 1958, had the diagnosis of von Willebrand disease made at the age of two after an episode of epistaxis. A bleeding time according to Ivy's method was greater than 30 minutes and factor VIII procoagulant activity (VIII:C) was 0.07 u/ml . The parents were asymptomatic and had normal bleeding times and VIII:C . The patient subsequently developed frequent bleeding episodes including haematuria, bruises and haemarthrosis which on several occasions required treatment with human plasma and cryoprecipitate. In 1975, after noting poor clinical response to cryoprecipitate, an inhibitor of ristocetin-induced aggregation was demonstrated. 495
496
INHIBITION
OF vWF IN VW DISEASE
Vo1.14,No.2/3
For the next two years the patient did not require treatment and the inhibitor disappeared. In 1977, however, she required therapy for menometrorrhagla. Within 48 hours after transfusion the inhibitor reappeared. The bleeding time was determined according to Ivy's technique as modified by Borchgrevink (51. The VIII:C was assayed by the onestage kaolin activated partial thromboplastin time method. The VIIIR:AG.was measured by quantitative immunoelectrophoresis according to Laurell's (6) using rabbit anti-VIII antibody at 0.3 % (Behring). Crossed antigen-antibody electrophoresis was performed on the plasma samples according to the technique of Sultan et al. (7). The VIIIR:WF was measured using platelets which were gel-filtered and then washed once (8). The results of the three activities of factor VIII were expressed in units per ml using a pool of normal human plasma kept at -80X, as a control. One unit corresponds to the concentration of VIII:C, VIIIR:AG and VIIIR:WF in one ml of the control pool. Normal values were : VIIIR:AG 1.08 u/ml (SD + 0.4); VIIIR:WF 1.11 u/ml (SD + 0.32); VIII:C 0.98 u/ml (SD +-0.30). Platelet VIIIR:AG was determined by the method of Howard et al. (9). Immunoglobulins (Ig) from the patient's plasma was obtained from the heated serum by precipitation in 50 % saturated ammonium sulphate. Inhibitory activities The inhibition of the ristocetin and the bovine factor VIII-induced aggregation of normal PRP was performed with 100 pl of the various dilutions of plasma or Ig from the patient + 200 ~1 of normal PRP + 10 ~1 of the.aggregating agent solution. The final concentration of ristocetin (H.Lundbeck & Co., Den-. mark) was 1.2 mg/ml and that of bovine factor VIII (S.Maw & Son, Barnet,England) was 0.3 mg/ml. Precipitation of the VIIIR:AG was assessed by centrifugatlng a mixture of normal and patient plasma previously incubated at 379C for one hour and at ~PC for 16 hours, and then assaying the supernatant with rabbit antiserum according to the Laurel1 technique. The level of inhibitor to VIIIR:AG was quantitated by incubating a pool of normal plasmas with different dilutions of the patient's plasma. Precipitation of VIIIR:AG by the patient's antiserum was assayed according to the Laurel1 technique (6) and double immunodiffusion method. Inhibition of VIII:C was measured by the method of Biggs and Bidwell (10). RESULTS When the inhibitor was found for the first time, the patient's plasma had unmeasurable levels of VIIIR:AG (co.05 u/ml) and VIIIR:WF (CO.05 u/ml). The VIII:C was 0.02 u/ml and VIIIR:AG was not found in the platelets. _ At that time, a dilution 1:16 of the patient's plasma still in hlbited the ristocetin-induced aggregation of normal PRP (Fig.11
Vol.lk,No.2/3
FIG.
INRIRITION OF vWF IN VW DISEASE
497
1
Inhibition of ristocetin-induc ed platelet aggregation of normal PRP by the patient's plasma. 200 pl of normal PRP were mixed with 100 pl of various dilutions of the patient's plasma and then 10 pl of ristocetin (1.2 mg/ml final concentration) were added. Curve E corresponds to the control system. Curves A,B, C and D were performed with the patient's plasma dilutions of 1:2, 1:4, 1:B and 1:16 respectively.
The bovine factor VIII-induced aggregation of normal PRP was also inhibited by the patient's plasma but with less intensity. The Ig fraction of the patient's plasma inhibited the ristocetin and the bovine factor VIII-induced aggregation of normal PRP to the same extent. Inhibition of the VIIIR:WF activity oc, cured immediately;no incubation was needed. when the patient's plasma and normal plasma were incubated as described in the methods and the supernatant tested by the Laurel1 technique, no peak was observed. This suggests that precipitationof normal VIIIR:AG occured during incubation (Fig 2). Guchterlony immunodiffusionalso failed to show a precipiting line between normal cryoprecipitateand patient's plasma.
FIG. 2 Inhibition of VIIIR:AG precipltation with normal plasma : 1. Normal plasma diluted in buffer 1:21 2. Equal mixture of patient's plasma and normal plasma; 3. Plas ma from severe von Willebrand disease.
INHIBITION
498
FIG.
Vol.lh,No.2/3
OF vWF IN VW DISEASE
3
*
Factor VIII and inhibitor levels of activity after the transfusion of cryoprecipitates (3.000 u). A - VIII:C levels E - VIIIR:WF levels I and inhibitor to VIIIR:WF h------d and C - VIIIR:AG )----------I inhibitor to VIIIR:AG T)---------_E The inhibitor was not detectad before the transfusion.
Z' VU P 4LA_ 1
a
4 &I i 4__&&LJ U I
I
I II ,1 .. C
•l
Two years later, when the patient required treatment with cryoprecipitate (60 u/Kg weight), the initial increment in VIIIR:AG, VIIIR:WF-and VIII:C appeared normal after the transfusion, although the increase of the VIII:C was not as progressive as us, al (Fig. 3A). The inhibitor, which had not been detected before the transfusion, reappeared after 48 hours. A fortnight later, the Inhibitor against VIIIR:WF activity was active at a plasma dilution of 1:50 (Fig. 3B). In the Laurel1 electroimmunoassay, after incubation of the patient's plasma and normal plasma no peak was detected until a dilution of 1:64 or greater of the patient's plasma (Fig. 3C). At this time, the inhibitor precipitated VIIIR:AG in immunodiffusion (Fig. 4) and immunoelectropho resls (Fig. 5)
FIG.
4
Double immunodiffusion agarose.
in
A - Precipitation line between normal cryoprecipitate and patient's plasma B - L& ne of identity between homo logous antibodies (patientis plasma) and heterologous antibodies (rabbit).
Vo1.14,No.2/3
INHIBITION
499
OF vWF IN VW DISEASE
FIG. 5 Precipitation of VIIIR:AG with the patient's serum (titre anti-VIIIR:AG 1:64; final dilution 1:250).
.
1. 10 ~1 of cryopreclpitate (10 u/ml). from seve 2. 10 ~1 of cryopreclpltate re von Willebrand disease.
DISCUSSION was probably affected by a double heterozygous severe type of von Willebrand disease since VIIIR:AG and VIIIR:WF we re apparently completely absent , and VIIIR:AG was also missing in the platelets. Moreover, the father showed an abnormal augmeE ted mobility of the VIII antigen in crossed immunoelectrophoresis although VIIIR:AG was 0.85 u/ml; VIIIR:WF was 1.10 u/ml and VIII:C was 1.00 u/ml . On the other hand the mother had VIIIR:AG 0.50 u/ml (below normal); VIIIR:WP 1.00 u/ml and VIII:C 1.00 u/ml
The patient
Among the six previously reported cases of acquired inhibitors in patients with von Willebrand disease , three showed inhibitory activity against VIIIR:WP, but no precipitating properties against VIIIR:AG (1, 3, 4). The inhibitor presently described is identical to those reported by Mannucci et al. (2) as they show precipitating properties against normal VIIIR:AG, in addition to inhibition of VIIIR:WP . The inhibitor that we have studied prevents a normal PRP from aggregating in presence of ristocetin and bovine factor VIII and precipitates VIIIR:AG in three systems : 1. double immunodiffusion in agarose. 2. immunoelectrophoresis. and 3. by inhibiting the precipitation of factor VIIIR:AG with heterologous antibody using the Laurel1 technique after incubation of normal plasma with the patient's plasma. This suggests that our patient developed an homologous inhibitor to factor VIIIR:AG and VIIIR:WP . The fact that this inhibitor had weak VIII:C neutralizing activity (which was not detected the first time) does not prove that it is a specific anti-VIII:C inhibitor. It is more likely that the potent precipitating inhibitor also inhibits the site of VIII:C . This is supported by the finding that the neutralizing activity against VIII:C is not time dependent as occurs with virtually all the inhibitors found in hemophilic subjects. Very little is known about precipitating antibodies in human pathology. Interestingly the inhibitor disappeared during two years without treatment. We do not know the life span of this i,n hibitor as the patient was not tested during the.two years.
INHIBITION OF vWF IN VW DISEASE
vo1.14,No.2/3
However,
it is obvious that this kind of inhibitor can disappear and follow the inmune secondary response when a transfusion is given (after 48 hours with a maximum of 15 days). It is to be noted that when large doses of factor VIII concentrateswere infused to this patient, no fever or pain or shivers were observed, as happens In inmune complexe diseases. REFERENCES 1.
EGBERG, N. and BLOMBACK, M. On the characterizationof acquired inhibitors to ristocetin-inducedplatelet aggregation found in patients with von WLllebrand's disease. Thromb. Res. 9, 527, 1976.
2. MANNUCCI, P.M., MEYER, D., RUGGERI, Z.M., KOUTTS, J., CIAV& RELLA, U. and LAVERGNE, J.M. Precipitating antibodies in von Willebrand's disease. Nature, 262, 141, 1976. 3. SARJI, R.E., STRATTON, R.D., WAGNER, R.H. and BRINKHOUS, K.M. Nature of von Wlllebrand factor : A new assay and a specific inhibitor. Proc. Nat. Acad. Sci. USA, 71, 8, 2937, 1974. 4. STRATTON, R.D., WAGNER, R.H., WEBSTER, W.P. and BRINKHOUS, K.M. Antibody nature of circulating inhibitor of plasma von Willebrand factor. Proc. Nat. Acad. Sci. USA, 72, 10, 4167, 1975. 5. BORCHGREVINK, C.F. A method for measuring platelet adhesiveness In vivo. Acta Med. Stand., 168, 157, 1960. 6. LAURELL, C.B. Electroimmunoassay.Stand, J. Clin. Lab. Invest 29 (suppl.) 124, 21, 1972. 7. SULTAN, Y., SIMBON, J. and CAEN, J. Electrophoreticheterogeneity of normal factor VIII/van Willebrand protein, and abnormal electrophoreticmobility in patients with von Wills brand disease. J. Lab. Clin. Med., 87, 2, 185, 1976. 8. TANGEN, O., BERMAN, H.J. and MARFEY, P. Gel filtration : a new technique for separation of blood platelets from plasma. Thromb. Diath. Haemorrh., 25, 268, 1971. 9. HOWARD, M.A., MONTGOMERY, DC. and HARDISTY, R.M. Factor VIII-related antigen in platelets. Thromb. Res., 4:5, 617, 1974. 10. BIGGS, R. and BIDWELL, E.B. A method for the study of antihaemophilic globulin inhibitors with reference to six cases. Brit. J. Haemat., 5, 379, 1959.
14;
495-500
Press Ltd.1979.
Printed
in Great Britain 0049-3848/79/
BRIEF
0301-0495
$02.00/o
COMMUNICATION
INHIBITION OF WILLEBRAND FACTOR IN VON WILLEBRAND DISEASE
S. Maragall, R. Castillo, A. Ordinas F. Liendo and M. Rodrfgues Facultad de Medicina. Hospital ClXnico y Provincial Barcelona. Spain (Received 8.5.1978; in revised form 18.11.1978. Accepted by Editor Y.V. Sultan
INTRODUCTION
Six cases of congenital von Willebrand disease with homologous
IgG antibodies to von Willebrand factor (VIIIR:WF) have been re cently described (1, 2, 3, 4). In three of those cases, the ant& bodies precipitated factor VIII related antigen (VIIIR:AG) from normal plasma (2). We now report a new case of an inhibitor directed towards VIIIR:WF and VIIIR:AG in a polytransfused patient with severe von Willebrand disease. The inhibitory capacity was found in the Ig fraction of the patient's plasma. The antibodies inhibited the Ristocetin-induced aggregation of normal platelet rich plasma (PRP) and also precipitated factor VIII related antigen from normal and hemophilic plasmas. PATIENT AND METHODS A.R., born in 1958, had the diagnosis of von Willebrand disease made at the age of two after an episode of epistaxis. A bleeding time according to Ivy's method was greater than 30 minutes and factor VIII procoagulant activity (VIII:C) was 0.07 u/ml . The parents were asymptomatic and had normal bleeding times and VIII:C . The patient subsequently developed frequent bleeding episodes including haematuria, bruises and haemarthrosis which on several occasions required treatment with human plasma and cryoprecipitate. In 1975, after noting poor clinical response to cryoprecipitate, an inhibitor of ristocetin-induced aggregation was demonstrated. 495
496
INHIBITION
OF vWF IN VW DISEASE
Vo1.14,No.2/3
For the next two years the patient did not require treatment and the inhibitor disappeared. In 1977, however, she required therapy for menometrorrhagla. Within 48 hours after transfusion the inhibitor reappeared. The bleeding time was determined according to Ivy's technique as modified by Borchgrevink (51. The VIII:C was assayed by the onestage kaolin activated partial thromboplastin time method. The VIIIR:AG.was measured by quantitative immunoelectrophoresis according to Laurell's (6) using rabbit anti-VIII antibody at 0.3 % (Behring). Crossed antigen-antibody electrophoresis was performed on the plasma samples according to the technique of Sultan et al. (7). The VIIIR:WF was measured using platelets which were gel-filtered and then washed once (8). The results of the three activities of factor VIII were expressed in units per ml using a pool of normal human plasma kept at -80X, as a control. One unit corresponds to the concentration of VIII:C, VIIIR:AG and VIIIR:WF in one ml of the control pool. Normal values were : VIIIR:AG 1.08 u/ml (SD + 0.4); VIIIR:WF 1.11 u/ml (SD + 0.32); VIII:C 0.98 u/ml (SD +-0.30). Platelet VIIIR:AG was determined by the method of Howard et al. (9). Immunoglobulins (Ig) from the patient's plasma was obtained from the heated serum by precipitation in 50 % saturated ammonium sulphate. Inhibitory activities The inhibition of the ristocetin and the bovine factor VIII-induced aggregation of normal PRP was performed with 100 pl of the various dilutions of plasma or Ig from the patient + 200 ~1 of normal PRP + 10 ~1 of the.aggregating agent solution. The final concentration of ristocetin (H.Lundbeck & Co., Den-. mark) was 1.2 mg/ml and that of bovine factor VIII (S.Maw & Son, Barnet,England) was 0.3 mg/ml. Precipitation of the VIIIR:AG was assessed by centrifugatlng a mixture of normal and patient plasma previously incubated at 379C for one hour and at ~PC for 16 hours, and then assaying the supernatant with rabbit antiserum according to the Laurel1 technique. The level of inhibitor to VIIIR:AG was quantitated by incubating a pool of normal plasmas with different dilutions of the patient's plasma. Precipitation of VIIIR:AG by the patient's antiserum was assayed according to the Laurel1 technique (6) and double immunodiffusion method. Inhibition of VIII:C was measured by the method of Biggs and Bidwell (10). RESULTS When the inhibitor was found for the first time, the patient's plasma had unmeasurable levels of VIIIR:AG (co.05 u/ml) and VIIIR:WF (CO.05 u/ml). The VIII:C was 0.02 u/ml and VIIIR:AG was not found in the platelets. _ At that time, a dilution 1:16 of the patient's plasma still in hlbited the ristocetin-induced aggregation of normal PRP (Fig.11
Vol.lk,No.2/3
FIG.
INRIRITION OF vWF IN VW DISEASE
497
1
Inhibition of ristocetin-induc ed platelet aggregation of normal PRP by the patient's plasma. 200 pl of normal PRP were mixed with 100 pl of various dilutions of the patient's plasma and then 10 pl of ristocetin (1.2 mg/ml final concentration) were added. Curve E corresponds to the control system. Curves A,B, C and D were performed with the patient's plasma dilutions of 1:2, 1:4, 1:B and 1:16 respectively.
The bovine factor VIII-induced aggregation of normal PRP was also inhibited by the patient's plasma but with less intensity. The Ig fraction of the patient's plasma inhibited the ristocetin and the bovine factor VIII-induced aggregation of normal PRP to the same extent. Inhibition of the VIIIR:WF activity oc, cured immediately;no incubation was needed. when the patient's plasma and normal plasma were incubated as described in the methods and the supernatant tested by the Laurel1 technique, no peak was observed. This suggests that precipitationof normal VIIIR:AG occured during incubation (Fig 2). Guchterlony immunodiffusionalso failed to show a precipiting line between normal cryoprecipitateand patient's plasma.
FIG. 2 Inhibition of VIIIR:AG precipltation with normal plasma : 1. Normal plasma diluted in buffer 1:21 2. Equal mixture of patient's plasma and normal plasma; 3. Plas ma from severe von Willebrand disease.
INHIBITION
498
FIG.
Vol.lh,No.2/3
OF vWF IN VW DISEASE
3
*
Factor VIII and inhibitor levels of activity after the transfusion of cryoprecipitates (3.000 u). A - VIII:C levels E - VIIIR:WF levels I and inhibitor to VIIIR:WF h------d and C - VIIIR:AG )----------I inhibitor to VIIIR:AG T)---------_E The inhibitor was not detectad before the transfusion.
Z' VU P 4LA_ 1
a
4 &I i 4__&&LJ U I
I
I II ,1 .. C
•l
Two years later, when the patient required treatment with cryoprecipitate (60 u/Kg weight), the initial increment in VIIIR:AG, VIIIR:WF-and VIII:C appeared normal after the transfusion, although the increase of the VIII:C was not as progressive as us, al (Fig. 3A). The inhibitor, which had not been detected before the transfusion, reappeared after 48 hours. A fortnight later, the Inhibitor against VIIIR:WF activity was active at a plasma dilution of 1:50 (Fig. 3B). In the Laurel1 electroimmunoassay, after incubation of the patient's plasma and normal plasma no peak was detected until a dilution of 1:64 or greater of the patient's plasma (Fig. 3C). At this time, the inhibitor precipitated VIIIR:AG in immunodiffusion (Fig. 4) and immunoelectropho resls (Fig. 5)
FIG.
4
Double immunodiffusion agarose.
in
A - Precipitation line between normal cryoprecipitate and patient's plasma B - L& ne of identity between homo logous antibodies (patientis plasma) and heterologous antibodies (rabbit).
Vo1.14,No.2/3
INHIBITION
499
OF vWF IN VW DISEASE
FIG. 5 Precipitation of VIIIR:AG with the patient's serum (titre anti-VIIIR:AG 1:64; final dilution 1:250).
.
1. 10 ~1 of cryopreclpitate (10 u/ml). from seve 2. 10 ~1 of cryopreclpltate re von Willebrand disease.
DISCUSSION was probably affected by a double heterozygous severe type of von Willebrand disease since VIIIR:AG and VIIIR:WF we re apparently completely absent , and VIIIR:AG was also missing in the platelets. Moreover, the father showed an abnormal augmeE ted mobility of the VIII antigen in crossed immunoelectrophoresis although VIIIR:AG was 0.85 u/ml; VIIIR:WF was 1.10 u/ml and VIII:C was 1.00 u/ml . On the other hand the mother had VIIIR:AG 0.50 u/ml (below normal); VIIIR:WP 1.00 u/ml and VIII:C 1.00 u/ml
The patient
Among the six previously reported cases of acquired inhibitors in patients with von Willebrand disease , three showed inhibitory activity against VIIIR:WP, but no precipitating properties against VIIIR:AG (1, 3, 4). The inhibitor presently described is identical to those reported by Mannucci et al. (2) as they show precipitating properties against normal VIIIR:AG, in addition to inhibition of VIIIR:WP . The inhibitor that we have studied prevents a normal PRP from aggregating in presence of ristocetin and bovine factor VIII and precipitates VIIIR:AG in three systems : 1. double immunodiffusion in agarose. 2. immunoelectrophoresis. and 3. by inhibiting the precipitation of factor VIIIR:AG with heterologous antibody using the Laurel1 technique after incubation of normal plasma with the patient's plasma. This suggests that our patient developed an homologous inhibitor to factor VIIIR:AG and VIIIR:WP . The fact that this inhibitor had weak VIII:C neutralizing activity (which was not detected the first time) does not prove that it is a specific anti-VIII:C inhibitor. It is more likely that the potent precipitating inhibitor also inhibits the site of VIII:C . This is supported by the finding that the neutralizing activity against VIII:C is not time dependent as occurs with virtually all the inhibitors found in hemophilic subjects. Very little is known about precipitating antibodies in human pathology. Interestingly the inhibitor disappeared during two years without treatment. We do not know the life span of this i,n hibitor as the patient was not tested during the.two years.
INHIBITION OF vWF IN VW DISEASE
vo1.14,No.2/3
However,
it is obvious that this kind of inhibitor can disappear and follow the inmune secondary response when a transfusion is given (after 48 hours with a maximum of 15 days). It is to be noted that when large doses of factor VIII concentrateswere infused to this patient, no fever or pain or shivers were observed, as happens In inmune complexe diseases. REFERENCES 1.
EGBERG, N. and BLOMBACK, M. On the characterizationof acquired inhibitors to ristocetin-inducedplatelet aggregation found in patients with von WLllebrand's disease. Thromb. Res. 9, 527, 1976.
2. MANNUCCI, P.M., MEYER, D., RUGGERI, Z.M., KOUTTS, J., CIAV& RELLA, U. and LAVERGNE, J.M. Precipitating antibodies in von Willebrand's disease. Nature, 262, 141, 1976. 3. SARJI, R.E., STRATTON, R.D., WAGNER, R.H. and BRINKHOUS, K.M. Nature of von Wlllebrand factor : A new assay and a specific inhibitor. Proc. Nat. Acad. Sci. USA, 71, 8, 2937, 1974. 4. STRATTON, R.D., WAGNER, R.H., WEBSTER, W.P. and BRINKHOUS, K.M. Antibody nature of circulating inhibitor of plasma von Willebrand factor. Proc. Nat. Acad. Sci. USA, 72, 10, 4167, 1975. 5. BORCHGREVINK, C.F. A method for measuring platelet adhesiveness In vivo. Acta Med. Stand., 168, 157, 1960. 6. LAURELL, C.B. Electroimmunoassay.Stand, J. Clin. Lab. Invest 29 (suppl.) 124, 21, 1972. 7. SULTAN, Y., SIMBON, J. and CAEN, J. Electrophoreticheterogeneity of normal factor VIII/van Willebrand protein, and abnormal electrophoreticmobility in patients with von Wills brand disease. J. Lab. Clin. Med., 87, 2, 185, 1976. 8. TANGEN, O., BERMAN, H.J. and MARFEY, P. Gel filtration : a new technique for separation of blood platelets from plasma. Thromb. Diath. Haemorrh., 25, 268, 1971. 9. HOWARD, M.A., MONTGOMERY, DC. and HARDISTY, R.M. Factor VIII-related antigen in platelets. Thromb. Res., 4:5, 617, 1974. 10. BIGGS, R. and BIDWELL, E.B. A method for the study of antihaemophilic globulin inhibitors with reference to six cases. Brit. J. Haemat., 5, 379, 1959.
