Scand J Haematol(l976) 16,128-134
Acquired von Willebrand's Syndrome and Thrombopathy in a Patient with Chronic Lymphocytic Leukaemia J. L. WAUTIER, M.D.,
s. LEVY-TQLEDANO, PH.D.* & J. P. U E N , M.D.
Department of Immuno-Haematology and Laboratory of Haemostasis and Experimental Thrombosis (Chief, J. P. Caen), E.R.A. 335 C.N.R.S.,' Maitre de Recherches, Paris, France
This paper reports the biological data found during an acquired bleeding disorder occurring in a 65-year-old woman affected with chronic lymphocytic leukaemia. They consists of haemostatic abnormalities which resemble an presently undescribed association of an acquired von Willebrand's syndrome with a thrombopathy. von Willebrand abnormalities were temporarily corrected by infusion of normal cryoprecipitate and reproduced in vitro by the incubation of normal platelets with the patient's IgA. The platelet defect was partially corrected after corticotherapy. The possible relationship of the associated defects is discussed. Key words: voa Willebrand's syndrome - thrombopathy - chronic lymphocytic leukaemia - autoantibo'dy Accepted for publication December 5, 1975 Correspondence to: Dr. J. L. Wautier, Service d'lmmuno-Himatologie, HGpitaI Lariboisikre, 2, rue Ambroise Park, F-75475 Paris, Cedex 10, France
'Classical' von Willebrand's disease is a long lasting inherited familial bleeding disorder with a long bleeding time and low factor VIII procoagulant activity. Four groups of authors have reported acquired disorders which resembled von Willebrand's disease (Simone et al 1968, Veltkamp et a1 1970, Ingram et a1 1971, 1973, Mant et a1 1973). The association of constitutional von Willebrand's disease with a platelet disorder has also been described (Sultan et al 1974, Chesney et a1 1974, Weiss 1975) but is uncommon. In this paper we describe an acquired
haemorrhagic disorder in a patient with chronic lymphocytic leukaemia (CLL). This haemorrhagic disorder appeared to be an association between an acquired von Willebrand's syndrome and a platelet disorder with some similarities to storage pool disease. CASE HISTORY The 65-year-old patient was seen for the first time in 1973 and since then has been regularly examined apart from therapeutical periods. She developed spontaneous bleeding a few weeks before she came t o our department: it consisted of
IMMUNE ACQUIRED VON WILLEBRAND'S SYNDROME severe haemorrhage after tooth extraction, intraocular bleeding, frequent epistaxis, gingival bleedings and ecchymosss. She had not previously experienced abnormal bleeding and there was no family history of haemorrhagic disorders. Blood examination showed the following results: RBC 3.6 x lWp1; H b 10.6 g/dl; WBC 21.9 x 10J/,d; lymphocytes 77 %; platelets 200 x 103/ pl. The extent of bone marrow infiltration by lymphocytes was 35 %. Serum immunoelectrophoresis pattern was nolrmal as were the immunoglobulins. N o autoantibodies were detected. Lymphocyte study - lymphoblastic transformation test: lymphocyte reactivity was decreased (10 % of the control) and the maximum was delayed (120 h; control 48 h). Lymphocytes bearing immunoglobulins (B lymphocytes): 65 % (control 24-47 %). T lymphocytes 22 % (control 60-80 %). CLL was then diagnosed.
MATERIAL AND METHODS Factor V l l l procoagulant activity was measured
in a one stage assay using partial thromboplastin time and haemophilic plasma as a substrate (Sultan et a1 1974). yon Willebrand facior (ristocetin cofactor) was estimated by ristocetin induced platelet aggregation (Sultan et a1 1974). Factor V l l l related antigen was measured by an electroimmunodiffusion technique using a specific rabbit antihuman factor VIII antiserum (Sultan et a1 1974). Bleeding time and quantitative bleeding time. The
Ivy bleeding time was measured and was quantitated by estimation of the overall haemoglobin loss (Sultan et a1 1974). Platelet interaction with subendothelium. Citrated whole blood was perfused through a chamber containing everted segments of rabbit aorta previously denuded of endothelium by means of a balloon catheter. The interaction of the platelets with subendothelium was evaluated by a morphometric technique (Baumgartner et a1 1972). Normal blood
Scand J Haematol(l976) 16
129
gave values of 39 f 12 % of the surface covered by adherent platelet and 1 f 1 % of these adherent platelets fo'rmed thrombi. Platelet aggregation was studied on platelet rich plasma (PRP) or on gel filtered platelets (GFP) as previously described (Wautier et a1 1973). Gel filtered platelets were obtained by filtration on Sepharose 2B. G FP were tested in buffer or in the presence of patient or control citrated platelet poor plasma to evaluate their aggregation with the following inducers: a) ADP (Sigma); b) collagen (Stago) 5, 10, 12 ,ug/ml (final concentration); c) ristocetin (Lundbeck, Copenhagen, Denmark) 0.5, 1, 1.5, 2, 2.5 mg/ml (lot 007, 006, 004); d) thrombin 0.15 U/ml. Platelet aggregation.
Platelet aggregating activity was tested after the addition of thrommbin to PRP according to the technique described by Caen & Michel (1964).
The aggregating activity of the serum obtained after coagulation was tested on no'rmal PRP 5, 15, 45 and 60 min after the addition of thrombin. Serotonin uptake and platelet populations were studied as previously described (Rendu & Caen 1972). Immunoglobulins of the patient were isolated by
chromatography on DEAE cellulose after precipitation by ammonium sulphate (Henson 1969). The eluted fractions were concentrated using an Amicon ultrafiltration cell and tested for purity by immunoelectrophoresis and Ouchterlony analysis. After purification the IgG and IgM fractions were free of contaminant. The IgA fraction was slightly contaminated by IgG. Transfusion. The quantitative bleeding time, platelet aggregation, ristocetin cofactor, factor VIII procoagulant activity and factor VIII related antigen were tested before and 1, 3, 6 and 24 h after the end of the 45 min duration infusion of 1,200 U of cryoprecipitate. The effect of the transfusion of fresh plasma (500 ml) and platelets (6 x loll) on bleeding time and platelet behaviour was estimated 1 h and 24 h after the end of the transfusion.
130
J. L. WAUTIER, S. LEVY-TOLEDANO &, I. P. CAEN
TABLE I Haemostatic values of the patient before therapy state
I Bleeding time Haemglobin lost Platelet count Factor VIII procoagulant activity (%) Factor VIII related antigen (%) von Willebrand factor (ristocetin cofactor) (%) Anticoagulant activity
Patient 20 min 100 mg 250 x 10911 52 10-20 6-10 negative
I
Control 9 min 4 mg 200-400 10911 100 100 100 negative
RESULTS
Out of therapy Haemostatic values. The results obtained at various intervals out of therapy are summarized in Table I. Despite a normal platelet count the patient had a very long bleeding time which was associated with a significant loss of haemoglobin. Factor VIII related antigen and von Willebrand factor (ristocetin cofactor) were around 10 % of normal while the factor VIII procoagulant activity was appromximately half that of the normal range.
-.+-a
'O01
Interaction with subendothelium was reduced to approximately 50 % of the normal range, and thrombi were not found. Platelet aggregation. Platelet aggregation as induced by ADP or thrombin was only slightly reduced. Second wave of aggregation was never obtained when using these two platelet aggregation inducers. Collagen induced platelet aggregation was much diminished. Patient gel-filtered platelets resuspended in normal plasma were also unreactive to collagen, while normal platelets incubated in the patient's plasma were normally aggregated by collagen (8 pg) (Figure 1). Aggregating activity released from platelets after addition of thrombin was very reduced.
50-
The pattern of platelet populations and serotonin uptake were normal.
-
0-
-
d
1 rnin Figure 1. Platelet aggregation induced by collagen (8 p g final concentration). a) Normal GFP in patient plasma. b) Patient PRP. c) Patient GFP in normal plasma. d) Patient GFP.
Inhibitory efJect of the patient's plasma and immunoglobulins on platelet aggregation induced by ristocetin (Table 11). Normal gelfiltered platelets were aggregated by ristocetin. When normal plasma was added the velocity was not modified, however on addition of patient's plasma ristocetin induced platelet aggregation was inhibited.
131
IMMUNE ACQUIRED VON WILLEBRAND'S SYNDROME TABLE I1 Effect of the patient or control plasma or patient isolated immunoglobulins on the velocity of ristocetin (1 mg/ml)-induced aggregation of normal gel-filtered platelets (GFP) Gel-filtered platelets Product added to control gel-filtered platelets
velocity
Buffer Normal plasma Patient plasma Patient IgG (3.8 mg/ml) Patient IgM (1.7 mg/ml) Patient IgA (1.6 mg/ml)
41 43 9 30 63 0
Patient IgG (3.8 mg/ml) slightly inhibited platelet aggregation induced by ristocetin but were no longer active at a lower concentration; addition of patient IgM was without effect at 1.7 mg/ml, however, the addition of patient IgA (1.6 mg/ml) to normal gel-filtered platelets completely inhibited ristocetin induced aggregation (Table 11). Using the same procedures, the different immunoglobulins were tested for collageninduced aggregation of control human platelets and no inhibition was observed.
The factor VIII procoagulant activity had returned to a normal range 1 h after the transfusion and was stable for 24 h. Factor VIII related antigen reached 40 % of the normal range 1 h after the transfusion, remained at this level for 3 h, and then decreased; the v m Willebrand factor (ristocetin cofactor) increased during the 3 h after transfusion and had maintained its level 24 h later. In parallel, platelet aggregation induced by ristocetin reached its maximum 6 h after the beginning of the transfusion.
Efect of corticotherupy (Table 111) After treatment with prednisone (15 mg per day for a month), the bleeding time remained longer than 15 min; however, pla-----
____V l l l procoagulant activity V l l l related antigen - van Willebrand's factor
Eflect of transfusion
On the platelet disorder. Platelet behaviour was not affected by transfusion of fresh plasma or cryoprecipitate. The transfusion of normal platelets did not modified the platelet aggregation induced by collagen while the platelet count was increased by 20 %. On von Willebrand's disease. The results are summarized in Figure 2. 1 h after the end of the transfusion, the bleeding time was shortened but returned to the starting level 3 h later. The loss of haemoglobin was parallel to the reduction of bleeding time.
bleeding time
- Hb lust in mg
I/ hours
/
1
3
-. \
6
~
24
Figure 2. Effect of the infusion of plasma on the bleeding time, the loss of haernoglobin, the factor VIII procoagulant activity (------) factor VIII related antigen (-.-) and von Willebrand's factor (-) (ristocetin cofactor).
132
J. L. WAUTIER, S . LEVY-TOLEDANO & J. P. CAEN
TABLE I11 Effect of corticotherapy (15 mg daily for 1 month) on factor V l l l procoagulant activity, factor VIlI related antigen, von Willebrand's factor (ristocetin cofactor) and collagen-induced aggregation in platetet-rick plasma Results are expressed in a percentage of the control values
I
I
Factor VIII procoagulant activity Factor VIII related antigen von Willebrand factor (ristocetin cofactor) Platelet aggregation induced by collagen (20 PI) Bleeding time (Ivy) (min)
telet aggregation induced by collagen was improved. The velocity rose from 10 to 50 % of the normal value. The following modifications were observed in von Willebrand factor abnormalities: factor VIII procoagulant activity was normal after corticotherapy (92 %); factor VIII related antigen rose from 12 to 20 %. Ristocetin cofactor was increased, rising from 10 % befosre corticotherapy to 24 % after.
Corticotherapy Before 52 12 10 10
>15
I
After 92 20 24 50
>15
VIII related antigen and von Willebrand factor (ristocetin cofactor) were estimated to be only 10 %. In addition, in our patient there was a virtual absence of collagen-induced platelet aggregation and an absence of release of aggregating activity after thrombin treatment. This is consistent with storage pool disease, however, in our paient the serotonin uptake was normal while it has previously been considered as decreased in albinism and storage pool disease (Weiss DISCUSSION et a1 1974). In a recent paper, Weiss (1975) This acquired haemorrhagic syndrome found described 5 patients with von Willebrand's in a 65-year-old patient affected with CLL disease who had a decreased collagen plateseems to be associated with a plasma defect let aggregation. However, only one of these and a platelet disorder. patients had associated low levels of the The simultaneous decrease of factor VIII f x t o r VIII procoagulant activity, factor procoagulant activity, factor VIII-related VIII related antigen and von Willebrand antigen and ristocetin cofactor and of the factor (ristocetin cofactor). platelet adhesion to rabbit subendothelium An association of platelet and plasmatic are consistent with the diagnosis of an ac- disorders is infrequent but known. Platelet quired von Willebrand's syndrome. abnormalities have been described in mild However, the case of acquired von Wille- haemophilia (Chesney et a1 1974) and in brand's syndrome described in this paper is dominant inherited factor VIII deficiency different from the cases previously descTibed associated with thrombocytopathic throm(Shone et a1 1968, Veltkamp et a1 1970, bolcytopenia (Sultan et a1 1974). In this Ingram et a1 1971, 1973, Mant et a1 1973) latter disease, platelet and factor VIII abin that the factor VIII procoagulant activity normalities are linked and transmitted in was only 50 % decreased while he factor an autosomal dominant fashion.
IMMUNE ACQUIRED VON WILLEBRAND’S SYNDROME
The mechanism of the acquired von Willebrand’s disease may be due to the appearance of von Willebrand factomr-inhibitor in the plasma of our patient with CLL. The transfusion of cryoprecipitate resulted in a rise in factor VIII procoagulant activity; but the correction of bleeding time, factor VIII related antigen and von Willebrand factor (ristocetin cofactor) lasted only a short time. The fact that patient plasma and patient IgA inhibited platelet aggregation induced by ristocetin, and that corticotherapy improved the symptoms of the von Willebrand syndrome are in good agreement with the presence of an inhibitor against the von Willebrand factor. Two hypotheses can be proposed concerning the inhibitory activity of patient IgA: (a) There is a non-specific reaction between patient IgA and the von Willebrand factor. The patient IgA may have possessed an abnormal structure which was not detected by immunoelectrophoresis and which may have reacted preferentially with the von Willebrand factor. (b) A more attractive hypothesis is that the patient IgA may be an antibody directed specifically against the von Willebrand factor. It has previously been shown that antibodies against the von Willebrand factor were not found (Rossman & Brody 1974) or were infrequent (Blomback & Egberg 1975) in patients with the von Willebrand’s disease, in contrast to the antibodies found in patients with haemophilia. This may be due to the low antigenicity of the human von Willebrand factor compared with the protein responsible for factor VIII procoagulant activity.
133
However, autoantibodies against the von Willebrand factor protein have been observed in patients with lymphocyte dysfunctions: systemic lupus erythematosus (Simone et al 1968, Ingram et a1 1971, 1973), dysproteinaemia (Mant et a1 1973) or with a hypersensitivity reaction (Veltkamp et a1 1970). Our patient with CLL, like other patients with CLL, had an abnormal repartition of T and B lymphocyte populations, and some lymphocyte functions were abnormal. These abnormalities may therefore involve the production of an autoantibody against the von Willebrand factor or the loss of the control of immunoglobulin production. An immunological explanation of an acquired von Willebrand’s syndrome associated with monoclonal gammapathy has also been proposed by Mant et a1 (1973); but they could not demonstrate that the immunoglobulin possessed an antibody activity against the von Willebrand factor, and they suggested the possibility of an humoral or a cellular mechanism. A direct cellular interaction seems improbable in our patient because the lymphocytes of the patient had no inhibitory effect on normal platelet aggregation induced by ristocetin (unpublished data). The platelet disorder would seem not to be explained by an immunological mechanism, since the incubation of patient immunoglobulins with normal gel-filtered platelets did not reproduce the platelet abnormalities. However, the platelet defects were improved by corticotherapy, which is known to depress immunological reaction involving a decrease in immunoglobulin levels and in the cellular immunity response. It has been shown in animal experiments (Clancy & Firkin 1973) that antiplatelet antibodies are capable of producing platelet abnormalities
134
J. L. WAUTIER, S. LEVY-TOLEDANO & J. P. CAEN
simil3r to those observed in storage pool disease. An attractive hypothesis for explaining acquired plasma and platelet disorder in this patient affected with CLL is that an immune complex consisting of V O n Willebrand factor and anti-von Willebrand factor react platelets and their reactivity to collagen. ACKNOWLEDGEMENTS We are grateful to Mrs. Y. Sultan and J. Simeon (Laboratory HCmostase, Hapita1 Saint-Louis, Paris) for performing the measurement of factor VIII related antigen by the Laurel1 technique, and to E. Bodevin, R. Bredoux and H. Souchon for their technical assistance. We also wish t o thank Dr. A. Nurden for his help in the preparation of the manuscript. REFERENCES Baumgartner H R & Haudenschild C M (1975) Adhesion of platelets t o subendothelium. Ann N Y Acad Sci 201, 22-36. Blornback M & Egberg N (1975) Two cases of von Willebrand’s disease with specific inhibitors of ristocetin induced aggregation of normal platelets. Vth Congr Int SOCThromb Haemost, Paris, July 1975, p 278. Caen J & Michel H (1964) L‘activitB agrCgante du s6rum. Rev Fr Et Clin Biol 9, 1001-02. Chesney C, Colman R W & Pechet L (1974) A syndrome of platelet release abnormality and mild hemophilia. Blood 43, 821-30. Clancy R & Firkin B (1973) Antibody induced thrombocytopathy. Thromb Res 3, 375-87. Henson P M (1969) The adherence of leucocytes and platelets induced by fixed IgG antibody or complement. Immunology 16, 107-21. Ingram G I, Kinston P J, Leslie J & Bowie J W
(1971) Four cases of acquired von Willebrand’s syndrome. Br Haematol 21, 189-99. Ingram G I, Prentice C R, Forbes C D & Leslie J (1973) Low factor VIII like antigen in acquired Willebran& syndrome and response to treatment. B r J Haematol 25, 13540. K a e b C A (1974) Kernoff P B A, R i m C R Transfusion and gel filtration studies in von Willebrand’s disease. Br J Haematol 28, 357-70. Mant M J, Hirsh J, Gauldie J, Bienenstock J, Pineo G F & Luke K M (1973) Von Willebrand’s syndrome presenting as an acquired bleeding disorder in association with a monoclonal gammapathy. Blood 42, 429-36. Rendu F & Caen J P (197273) Effect of reserpine on I4C serotonin uptake by human platelet populations. Haemostasis 1, 161-68. Rossman R E & Brody J I (1974) Reactivity to factor VIII concentrates of lymphocytes from patients with haemophilia and van Willebrand’s disease. Br J Haematol 28, 131-36. Simone J V, Cornet J A & Abildgaard C F (1968) Acquired von Willebrand’s syndrome in systemic lupus erythematosus. Btood 31, 806-12. Sultan Y, Bernal-Hoyos E, Levy-Toledano S, Jeanneau C & Caen J P (1974) Dominant inherited familial factor VIII deficiency associated with thrombocytopathic thrombocytopenia. Pathol Biol (Paris) 22, 27-36. Veltkamp J J, Stevens P, Plas M & Loeliger E A (1970) Production site of bleeding factor. Thromb Diath Haemorrh 23, 412. Wautier J L, Tobelem G M, Peltier A P & Caen J P (1973) Evidence for C1 on human platelets. Haemostasis 2, 281-86. Weiss H J (1975) Abnormalities of factor VIII and platelet aggregation use of ristocetin in diagnosing the von Willebrand’s syndrome. Blood 45, 403-12. Weiss H J, Tschopp T B, Rogers J & Brand H (1974) Studies of platelet 5 hydroxytryptamine in storage pool disease and albinism. J Clin Invest 54, 421-32.
Acquired von Willebrand's Syndrome and Thrombopathy in a Patient with Chronic Lymphocytic Leukaemia J. L. WAUTIER, M.D.,
s. LEVY-TQLEDANO, PH.D.* & J. P. U E N , M.D.
Department of Immuno-Haematology and Laboratory of Haemostasis and Experimental Thrombosis (Chief, J. P. Caen), E.R.A. 335 C.N.R.S.,' Maitre de Recherches, Paris, France
This paper reports the biological data found during an acquired bleeding disorder occurring in a 65-year-old woman affected with chronic lymphocytic leukaemia. They consists of haemostatic abnormalities which resemble an presently undescribed association of an acquired von Willebrand's syndrome with a thrombopathy. von Willebrand abnormalities were temporarily corrected by infusion of normal cryoprecipitate and reproduced in vitro by the incubation of normal platelets with the patient's IgA. The platelet defect was partially corrected after corticotherapy. The possible relationship of the associated defects is discussed. Key words: voa Willebrand's syndrome - thrombopathy - chronic lymphocytic leukaemia - autoantibo'dy Accepted for publication December 5, 1975 Correspondence to: Dr. J. L. Wautier, Service d'lmmuno-Himatologie, HGpitaI Lariboisikre, 2, rue Ambroise Park, F-75475 Paris, Cedex 10, France
'Classical' von Willebrand's disease is a long lasting inherited familial bleeding disorder with a long bleeding time and low factor VIII procoagulant activity. Four groups of authors have reported acquired disorders which resembled von Willebrand's disease (Simone et al 1968, Veltkamp et a1 1970, Ingram et a1 1971, 1973, Mant et a1 1973). The association of constitutional von Willebrand's disease with a platelet disorder has also been described (Sultan et al 1974, Chesney et a1 1974, Weiss 1975) but is uncommon. In this paper we describe an acquired
haemorrhagic disorder in a patient with chronic lymphocytic leukaemia (CLL). This haemorrhagic disorder appeared to be an association between an acquired von Willebrand's syndrome and a platelet disorder with some similarities to storage pool disease. CASE HISTORY The 65-year-old patient was seen for the first time in 1973 and since then has been regularly examined apart from therapeutical periods. She developed spontaneous bleeding a few weeks before she came t o our department: it consisted of
IMMUNE ACQUIRED VON WILLEBRAND'S SYNDROME severe haemorrhage after tooth extraction, intraocular bleeding, frequent epistaxis, gingival bleedings and ecchymosss. She had not previously experienced abnormal bleeding and there was no family history of haemorrhagic disorders. Blood examination showed the following results: RBC 3.6 x lWp1; H b 10.6 g/dl; WBC 21.9 x 10J/,d; lymphocytes 77 %; platelets 200 x 103/ pl. The extent of bone marrow infiltration by lymphocytes was 35 %. Serum immunoelectrophoresis pattern was nolrmal as were the immunoglobulins. N o autoantibodies were detected. Lymphocyte study - lymphoblastic transformation test: lymphocyte reactivity was decreased (10 % of the control) and the maximum was delayed (120 h; control 48 h). Lymphocytes bearing immunoglobulins (B lymphocytes): 65 % (control 24-47 %). T lymphocytes 22 % (control 60-80 %). CLL was then diagnosed.
MATERIAL AND METHODS Factor V l l l procoagulant activity was measured
in a one stage assay using partial thromboplastin time and haemophilic plasma as a substrate (Sultan et a1 1974). yon Willebrand facior (ristocetin cofactor) was estimated by ristocetin induced platelet aggregation (Sultan et a1 1974). Factor V l l l related antigen was measured by an electroimmunodiffusion technique using a specific rabbit antihuman factor VIII antiserum (Sultan et a1 1974). Bleeding time and quantitative bleeding time. The
Ivy bleeding time was measured and was quantitated by estimation of the overall haemoglobin loss (Sultan et a1 1974). Platelet interaction with subendothelium. Citrated whole blood was perfused through a chamber containing everted segments of rabbit aorta previously denuded of endothelium by means of a balloon catheter. The interaction of the platelets with subendothelium was evaluated by a morphometric technique (Baumgartner et a1 1972). Normal blood
Scand J Haematol(l976) 16
129
gave values of 39 f 12 % of the surface covered by adherent platelet and 1 f 1 % of these adherent platelets fo'rmed thrombi. Platelet aggregation was studied on platelet rich plasma (PRP) or on gel filtered platelets (GFP) as previously described (Wautier et a1 1973). Gel filtered platelets were obtained by filtration on Sepharose 2B. G FP were tested in buffer or in the presence of patient or control citrated platelet poor plasma to evaluate their aggregation with the following inducers: a) ADP (Sigma); b) collagen (Stago) 5, 10, 12 ,ug/ml (final concentration); c) ristocetin (Lundbeck, Copenhagen, Denmark) 0.5, 1, 1.5, 2, 2.5 mg/ml (lot 007, 006, 004); d) thrombin 0.15 U/ml. Platelet aggregation.
Platelet aggregating activity was tested after the addition of thrommbin to PRP according to the technique described by Caen & Michel (1964).
The aggregating activity of the serum obtained after coagulation was tested on no'rmal PRP 5, 15, 45 and 60 min after the addition of thrombin. Serotonin uptake and platelet populations were studied as previously described (Rendu & Caen 1972). Immunoglobulins of the patient were isolated by
chromatography on DEAE cellulose after precipitation by ammonium sulphate (Henson 1969). The eluted fractions were concentrated using an Amicon ultrafiltration cell and tested for purity by immunoelectrophoresis and Ouchterlony analysis. After purification the IgG and IgM fractions were free of contaminant. The IgA fraction was slightly contaminated by IgG. Transfusion. The quantitative bleeding time, platelet aggregation, ristocetin cofactor, factor VIII procoagulant activity and factor VIII related antigen were tested before and 1, 3, 6 and 24 h after the end of the 45 min duration infusion of 1,200 U of cryoprecipitate. The effect of the transfusion of fresh plasma (500 ml) and platelets (6 x loll) on bleeding time and platelet behaviour was estimated 1 h and 24 h after the end of the transfusion.
130
J. L. WAUTIER, S. LEVY-TOLEDANO &, I. P. CAEN
TABLE I Haemostatic values of the patient before therapy state
I Bleeding time Haemglobin lost Platelet count Factor VIII procoagulant activity (%) Factor VIII related antigen (%) von Willebrand factor (ristocetin cofactor) (%) Anticoagulant activity
Patient 20 min 100 mg 250 x 10911 52 10-20 6-10 negative
I
Control 9 min 4 mg 200-400 10911 100 100 100 negative
RESULTS
Out of therapy Haemostatic values. The results obtained at various intervals out of therapy are summarized in Table I. Despite a normal platelet count the patient had a very long bleeding time which was associated with a significant loss of haemoglobin. Factor VIII related antigen and von Willebrand factor (ristocetin cofactor) were around 10 % of normal while the factor VIII procoagulant activity was appromximately half that of the normal range.
-.+-a
'O01
Interaction with subendothelium was reduced to approximately 50 % of the normal range, and thrombi were not found. Platelet aggregation. Platelet aggregation as induced by ADP or thrombin was only slightly reduced. Second wave of aggregation was never obtained when using these two platelet aggregation inducers. Collagen induced platelet aggregation was much diminished. Patient gel-filtered platelets resuspended in normal plasma were also unreactive to collagen, while normal platelets incubated in the patient's plasma were normally aggregated by collagen (8 pg) (Figure 1). Aggregating activity released from platelets after addition of thrombin was very reduced.
50-
The pattern of platelet populations and serotonin uptake were normal.
-
0-
-
d
1 rnin Figure 1. Platelet aggregation induced by collagen (8 p g final concentration). a) Normal GFP in patient plasma. b) Patient PRP. c) Patient GFP in normal plasma. d) Patient GFP.
Inhibitory efJect of the patient's plasma and immunoglobulins on platelet aggregation induced by ristocetin (Table 11). Normal gelfiltered platelets were aggregated by ristocetin. When normal plasma was added the velocity was not modified, however on addition of patient's plasma ristocetin induced platelet aggregation was inhibited.
131
IMMUNE ACQUIRED VON WILLEBRAND'S SYNDROME TABLE I1 Effect of the patient or control plasma or patient isolated immunoglobulins on the velocity of ristocetin (1 mg/ml)-induced aggregation of normal gel-filtered platelets (GFP) Gel-filtered platelets Product added to control gel-filtered platelets
velocity
Buffer Normal plasma Patient plasma Patient IgG (3.8 mg/ml) Patient IgM (1.7 mg/ml) Patient IgA (1.6 mg/ml)
41 43 9 30 63 0
Patient IgG (3.8 mg/ml) slightly inhibited platelet aggregation induced by ristocetin but were no longer active at a lower concentration; addition of patient IgM was without effect at 1.7 mg/ml, however, the addition of patient IgA (1.6 mg/ml) to normal gel-filtered platelets completely inhibited ristocetin induced aggregation (Table 11). Using the same procedures, the different immunoglobulins were tested for collageninduced aggregation of control human platelets and no inhibition was observed.
The factor VIII procoagulant activity had returned to a normal range 1 h after the transfusion and was stable for 24 h. Factor VIII related antigen reached 40 % of the normal range 1 h after the transfusion, remained at this level for 3 h, and then decreased; the v m Willebrand factor (ristocetin cofactor) increased during the 3 h after transfusion and had maintained its level 24 h later. In parallel, platelet aggregation induced by ristocetin reached its maximum 6 h after the beginning of the transfusion.
Efect of corticotherupy (Table 111) After treatment with prednisone (15 mg per day for a month), the bleeding time remained longer than 15 min; however, pla-----
____V l l l procoagulant activity V l l l related antigen - van Willebrand's factor
Eflect of transfusion
On the platelet disorder. Platelet behaviour was not affected by transfusion of fresh plasma or cryoprecipitate. The transfusion of normal platelets did not modified the platelet aggregation induced by collagen while the platelet count was increased by 20 %. On von Willebrand's disease. The results are summarized in Figure 2. 1 h after the end of the transfusion, the bleeding time was shortened but returned to the starting level 3 h later. The loss of haemoglobin was parallel to the reduction of bleeding time.
bleeding time
- Hb lust in mg
I/ hours
/
1
3
-. \
6
~
24
Figure 2. Effect of the infusion of plasma on the bleeding time, the loss of haernoglobin, the factor VIII procoagulant activity (------) factor VIII related antigen (-.-) and von Willebrand's factor (-) (ristocetin cofactor).
132
J. L. WAUTIER, S . LEVY-TOLEDANO & J. P. CAEN
TABLE I11 Effect of corticotherapy (15 mg daily for 1 month) on factor V l l l procoagulant activity, factor VIlI related antigen, von Willebrand's factor (ristocetin cofactor) and collagen-induced aggregation in platetet-rick plasma Results are expressed in a percentage of the control values
I
I
Factor VIII procoagulant activity Factor VIII related antigen von Willebrand factor (ristocetin cofactor) Platelet aggregation induced by collagen (20 PI) Bleeding time (Ivy) (min)
telet aggregation induced by collagen was improved. The velocity rose from 10 to 50 % of the normal value. The following modifications were observed in von Willebrand factor abnormalities: factor VIII procoagulant activity was normal after corticotherapy (92 %); factor VIII related antigen rose from 12 to 20 %. Ristocetin cofactor was increased, rising from 10 % befosre corticotherapy to 24 % after.
Corticotherapy Before 52 12 10 10
>15
I
After 92 20 24 50
>15
VIII related antigen and von Willebrand factor (ristocetin cofactor) were estimated to be only 10 %. In addition, in our patient there was a virtual absence of collagen-induced platelet aggregation and an absence of release of aggregating activity after thrombin treatment. This is consistent with storage pool disease, however, in our paient the serotonin uptake was normal while it has previously been considered as decreased in albinism and storage pool disease (Weiss DISCUSSION et a1 1974). In a recent paper, Weiss (1975) This acquired haemorrhagic syndrome found described 5 patients with von Willebrand's in a 65-year-old patient affected with CLL disease who had a decreased collagen plateseems to be associated with a plasma defect let aggregation. However, only one of these and a platelet disorder. patients had associated low levels of the The simultaneous decrease of factor VIII f x t o r VIII procoagulant activity, factor procoagulant activity, factor VIII-related VIII related antigen and von Willebrand antigen and ristocetin cofactor and of the factor (ristocetin cofactor). platelet adhesion to rabbit subendothelium An association of platelet and plasmatic are consistent with the diagnosis of an ac- disorders is infrequent but known. Platelet quired von Willebrand's syndrome. abnormalities have been described in mild However, the case of acquired von Wille- haemophilia (Chesney et a1 1974) and in brand's syndrome described in this paper is dominant inherited factor VIII deficiency different from the cases previously descTibed associated with thrombocytopathic throm(Shone et a1 1968, Veltkamp et a1 1970, bolcytopenia (Sultan et a1 1974). In this Ingram et a1 1971, 1973, Mant et a1 1973) latter disease, platelet and factor VIII abin that the factor VIII procoagulant activity normalities are linked and transmitted in was only 50 % decreased while he factor an autosomal dominant fashion.
IMMUNE ACQUIRED VON WILLEBRAND’S SYNDROME
The mechanism of the acquired von Willebrand’s disease may be due to the appearance of von Willebrand factomr-inhibitor in the plasma of our patient with CLL. The transfusion of cryoprecipitate resulted in a rise in factor VIII procoagulant activity; but the correction of bleeding time, factor VIII related antigen and von Willebrand factor (ristocetin cofactor) lasted only a short time. The fact that patient plasma and patient IgA inhibited platelet aggregation induced by ristocetin, and that corticotherapy improved the symptoms of the von Willebrand syndrome are in good agreement with the presence of an inhibitor against the von Willebrand factor. Two hypotheses can be proposed concerning the inhibitory activity of patient IgA: (a) There is a non-specific reaction between patient IgA and the von Willebrand factor. The patient IgA may have possessed an abnormal structure which was not detected by immunoelectrophoresis and which may have reacted preferentially with the von Willebrand factor. (b) A more attractive hypothesis is that the patient IgA may be an antibody directed specifically against the von Willebrand factor. It has previously been shown that antibodies against the von Willebrand factor were not found (Rossman & Brody 1974) or were infrequent (Blomback & Egberg 1975) in patients with the von Willebrand’s disease, in contrast to the antibodies found in patients with haemophilia. This may be due to the low antigenicity of the human von Willebrand factor compared with the protein responsible for factor VIII procoagulant activity.
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However, autoantibodies against the von Willebrand factor protein have been observed in patients with lymphocyte dysfunctions: systemic lupus erythematosus (Simone et al 1968, Ingram et a1 1971, 1973), dysproteinaemia (Mant et a1 1973) or with a hypersensitivity reaction (Veltkamp et a1 1970). Our patient with CLL, like other patients with CLL, had an abnormal repartition of T and B lymphocyte populations, and some lymphocyte functions were abnormal. These abnormalities may therefore involve the production of an autoantibody against the von Willebrand factor or the loss of the control of immunoglobulin production. An immunological explanation of an acquired von Willebrand’s syndrome associated with monoclonal gammapathy has also been proposed by Mant et a1 (1973); but they could not demonstrate that the immunoglobulin possessed an antibody activity against the von Willebrand factor, and they suggested the possibility of an humoral or a cellular mechanism. A direct cellular interaction seems improbable in our patient because the lymphocytes of the patient had no inhibitory effect on normal platelet aggregation induced by ristocetin (unpublished data). The platelet disorder would seem not to be explained by an immunological mechanism, since the incubation of patient immunoglobulins with normal gel-filtered platelets did not reproduce the platelet abnormalities. However, the platelet defects were improved by corticotherapy, which is known to depress immunological reaction involving a decrease in immunoglobulin levels and in the cellular immunity response. It has been shown in animal experiments (Clancy & Firkin 1973) that antiplatelet antibodies are capable of producing platelet abnormalities
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J. L. WAUTIER, S. LEVY-TOLEDANO & J. P. CAEN
simil3r to those observed in storage pool disease. An attractive hypothesis for explaining acquired plasma and platelet disorder in this patient affected with CLL is that an immune complex consisting of V O n Willebrand factor and anti-von Willebrand factor react platelets and their reactivity to collagen. ACKNOWLEDGEMENTS We are grateful to Mrs. Y. Sultan and J. Simeon (Laboratory HCmostase, Hapita1 Saint-Louis, Paris) for performing the measurement of factor VIII related antigen by the Laurel1 technique, and to E. Bodevin, R. Bredoux and H. Souchon for their technical assistance. We also wish t o thank Dr. A. Nurden for his help in the preparation of the manuscript. REFERENCES Baumgartner H R & Haudenschild C M (1975) Adhesion of platelets t o subendothelium. Ann N Y Acad Sci 201, 22-36. Blornback M & Egberg N (1975) Two cases of von Willebrand’s disease with specific inhibitors of ristocetin induced aggregation of normal platelets. Vth Congr Int SOCThromb Haemost, Paris, July 1975, p 278. Caen J & Michel H (1964) L‘activitB agrCgante du s6rum. Rev Fr Et Clin Biol 9, 1001-02. Chesney C, Colman R W & Pechet L (1974) A syndrome of platelet release abnormality and mild hemophilia. Blood 43, 821-30. Clancy R & Firkin B (1973) Antibody induced thrombocytopathy. Thromb Res 3, 375-87. Henson P M (1969) The adherence of leucocytes and platelets induced by fixed IgG antibody or complement. Immunology 16, 107-21. Ingram G I, Kinston P J, Leslie J & Bowie J W
(1971) Four cases of acquired von Willebrand’s syndrome. Br Haematol 21, 189-99. Ingram G I, Prentice C R, Forbes C D & Leslie J (1973) Low factor VIII like antigen in acquired Willebran& syndrome and response to treatment. B r J Haematol 25, 13540. K a e b C A (1974) Kernoff P B A, R i m C R Transfusion and gel filtration studies in von Willebrand’s disease. Br J Haematol 28, 357-70. Mant M J, Hirsh J, Gauldie J, Bienenstock J, Pineo G F & Luke K M (1973) Von Willebrand’s syndrome presenting as an acquired bleeding disorder in association with a monoclonal gammapathy. Blood 42, 429-36. Rendu F & Caen J P (197273) Effect of reserpine on I4C serotonin uptake by human platelet populations. Haemostasis 1, 161-68. Rossman R E & Brody J I (1974) Reactivity to factor VIII concentrates of lymphocytes from patients with haemophilia and van Willebrand’s disease. Br J Haematol 28, 131-36. Simone J V, Cornet J A & Abildgaard C F (1968) Acquired von Willebrand’s syndrome in systemic lupus erythematosus. Btood 31, 806-12. Sultan Y, Bernal-Hoyos E, Levy-Toledano S, Jeanneau C & Caen J P (1974) Dominant inherited familial factor VIII deficiency associated with thrombocytopathic thrombocytopenia. Pathol Biol (Paris) 22, 27-36. Veltkamp J J, Stevens P, Plas M & Loeliger E A (1970) Production site of bleeding factor. Thromb Diath Haemorrh 23, 412. Wautier J L, Tobelem G M, Peltier A P & Caen J P (1973) Evidence for C1 on human platelets. Haemostasis 2, 281-86. Weiss H J (1975) Abnormalities of factor VIII and platelet aggregation use of ristocetin in diagnosing the von Willebrand’s syndrome. Blood 45, 403-12. Weiss H J, Tschopp T B, Rogers J & Brand H (1974) Studies of platelet 5 hydroxytryptamine in storage pool disease and albinism. J Clin Invest 54, 421-32.
