THROMBOSIS Printed
RESE_XRCH in G-ear Britain
EFFECT Magnus
Vol.
12,
pp.
629-61>,
19,8
Per.gamon Press, Ltd.
OF DEXTRMJ 70 ON FACTOR VIII AP;D PLATELET FUP!CTION IN von WILLEBRAND'S DISEASE aberq,
Ulla iiedner and Sven-Erik
Berqentz
Department of Plastic Surgery and Coagulation Laboratory, Allmanna Sjukhuset, 214 01 ?!almB, Sweden
(Received 29.11.1977; in revised form 26.1.19~8. Accepted by Editor H.C. Godal)
ABSTRACT Dextran given intravenously to two patients with mild von Willebrand's disease and two healthy controls impaired the factor VIII properties measured, i.e. it decreased the factor VIII-related antigen and the ristocetin co-factor activity. In the patients the variables fell from borderline to abnormally low levels accompanied by a prolongation of the bleedinq time. Infusion of factor VIII concentrate reversed the dextraninduced impairment of factor VIII and the prolonged bleeding time. The findings lend support to the view that dextran induces a defective platelet function in vivo by interfering with the factor VIII molecular complex. They also show that patients with von Willebrand's disease should not be qiven dextran as it might potentiate their bleeding diathesis.
INTRODUCTION The importance of factor VIII for normal platelet function has recently been emphasized (1). Aberg et al (2) found that 500 ml 6 % dextran 70 given intravenously impaired some of the properties-.of factor VIII, especially the one necessary for platelet aggregation by the antibiotic ristocetin, i.e. it created a situation similar to that in one variant of von Willebrand's disease (type III) (3). It was therefore considered worthwhile to study the effect of infusion of dextran in von Willebrand's disease in which the factor VIII molecule is defective, or present in ahnormal1.y low concentrations, in an
629
effort to clarify the association between on factor VIII and platelet function.
LABORATORY
the effect
of dextran
XETHODS
Blood was collected and titrated plasma prepared as described by Nilsson (4). The factor VIII coagulant activity (VIII:C) was assessed from the normalizing effect on the recalcification time of platelet-rich hemophilia A plasma containing less than 1 % of the normal factor VIII activity. The activity was expressed as a percentage of that of pooled plasma from 20 healthy individuals. The normal range was 60-160 i. The error of the method at different levels has been calculated by Nilsson (4). The factor VIII-related antigen (VIIIR:Ag) was determined with a specific rabbit antiserum against the factor VIII-related antigen. The normal range was 50-170 % and the error of the method 8.2 % (5). The ristocetin co-factor activity (VIIIR:vW/RCF/) was determined quantitatively according to Zuzel et al (6). This method uses formalin-fixed platelets and dilution of test samples in von Willebrand plasma lacking ristocetin co-factor activity. The activity of each test sample was always measured in two dilutions and expressed as a percentage of that of a normal plasma pool, which was defined as 100 S (in 86 normal controls the range was 43-204 % and the error of the method 9.6 %I. Bleeding time (BT) was determined with Ivy's method. The normal value was 6-12 min (7). Fibrinogen was determined according to Nilsson and Olow (8) and fibrin/fibrinogen degradation products (FDP) according to Nilghn (9).
MATERIAL Two patients with von Willebrand's disease and two healthy volunteers were studied. Relevant data concerning the patients are given in Table I. The patients had the classical form of von Willebrand's disease, called type I. This is characterized by decreased VIII:C and VIIIR:Aq as well as VIIIR:vW(RCF). The disease was mild in both patients, as shown by the borderline BT. The patients belong to families 20 and 42 in the Swedish register of patients with von Willebrand's disease (10). Infusion of 500 ml of 6 % dextran 70 (MacrodexR, Pharmacia, Uppsala, Sweden) was carried out within one hour. Four hours later factor VIII concentrate (AHF, Kabi, Sweden,fraction I-O, prepared according to the glycine method of Blomback and Blomback (ll,), was given intravenously in a dose of 8 U/kg body weight to the controls and 12 U/kg body weight to the patients. Blood samples were obtained before and at various intervals after the infusion of dextran and factor VIII concentrate, respectively. Informed consent was obtained.
631
TABLE
I
Some basic data about the two patients with von P7illebrand's disease. The family numbers refer to the family to which the patients belong in the Swedish register of von willebrand's disease. Family number
Sex
Age years
VIII:C 93
VIIIR:Ag %
VIIIR:vW (RCF) 9
BT min
20
oh
36
40
51
36
12
42
oh
28
58
48
32
11
RESULTS Dextran invariably lowered VIIIR:Ag and VIIIR:vW(RCF). In the patients also VIII:C was decreased (Fig. 1). The lowest values were found 2-4 hours after the dextran infusion. In the one control in whom this test was performed the BT remained unchanged and in both patients it was markedly prolonged at four hours after the infusion. Infusion of factor VIII concentrate raised VIIIR:Ag, VIIIR:vW(RCF) as well as VIII:C at the same time as it shortened the prolonged BT. The changes in the hematocrit and fibrinogen are shown in Fig. 2. No FDD were found.
DISCUSSION In a previous investigation it was shown that 500 ml 6 % dextran 70 given intravenously to healthy volunteers significantly decreased VIIIR:Ag and VIIIR:vW(RCF)(2). The mean VIII:C was unchanged although individual variations existed. These findings were confirmed in the present study, which showed the effect of dextran in two normal subjects and two patients with von Willebrand's disease. The two patients had a mild variant of von Willebrand's disease type I (3), as seen from the moderate Dextran decrease in the VIII:C, VIIIR:Ag and VIIIR:vW(RCF). caused a further decrease which was proportionate to that found in the normal subjects. But since the patients had lower initial concentrations they also had lower values than the controls 4 hours after the infusion of dextran. This explains the prolonged BT in the patients. In the control in whom this test was performed the changes were not severe enough to prolong the BT, a finding confirming previous reports on the effect of dextran in subjects with normal hemostasis (12, 13). As expected (2), dextran did not reach its maximal effect until some hours after the infusion.
i-01.12,?r’o.i,
FIG. 1 VIII:C, VIIIR:Ag, VIIIR:vW(RCF) and BT before and after infusion of dextran and AHF.
-tic
t % 50 40
-
Controls Pat. with v.Willebrand’s
30 Fib. g/l
disease
1 ----.__ y--r------c= ---_e
FIG. 2 Fibrinogen and hematocrit before and after infusion of dextran and AHF.
-
1 1
v01.12,s0.s
DESTR_:xd
F. i-111 IS +A--DISE.-;S-E
433
The mechanism underlying the effect of dextran on factor (Fig. 2) caused by VIII is obscure (2). The slight hemodilution dextran cannot explain this effect since isotonic saline given in amounts which caused a similar hemodilution did not influence factor VIII (2). The effect of dextran therefore seems to be due to a specific property of this substance. The subjects studied were fasting and consequently the depressing effect of food on VIIIR:vW(RCF) (14) can be excluded. There was no proteolysis as seen from the fact that TrasylolR, given before and during infusion of dextran did not influence the impairment of factor VIII (unpublished observation). The findings that dextran did not affect factor VIII in vitro and that the maximal effect was obtained at four hours after infusion suggest that dextran interferes with the metabolism of factor VIII (2). Thus, dextran influenced the properties of factor VIII in patients with von Willebrand's disease as it does in healthy subjects. In the patients the variables fell from borderline values to abnormally low levels resulting in a prolongation of the BT and a deterioration of the disease from a mild to a more (fraction I-O, severe form. Infusion of factor VIII concentrate AHF, Kabi) reversed not only the decrease in VIIIR:Ag and VIIIR:vW(RCF) of the controls and the von Willebrand patients, but also the prolonged BT of the latter. The findings lend support to the view that dextran temporarily impairs platelet function in vivo by interfering with the factor VIII molecular complex. The results also show that dextran infusion should not be given to patients with von Willebrand's disease, as they might develop a severe bleeding diathesis.
ACKNOWLEDGEMENT This investigation was supported by grants from the Swedish Medical Research Council (B78-19X-00087-14A, B78-17X-00759-13B).
REFERENCES 1.
WEISS,H.J. Platelet function. New Engl.
physiology and abnormalities J. Med. 293, 580, 1975.
of platelet
Effect of dextran on 2. &3ERG,M., HEDNER,U., and BERGENTZ,S.-E. factor VIII (antihemophilic factor) and platelet function. Ann. Surg. 1978 (In press). 3. NILSSON,I.M. and HOLMBERG,L. Biochemical and clinical aspects on factor VIII. First Florence Conf. on Haemostasis and Thrombosis, May 1977. Thrombos. Haemostas. 1978 (In press). 4. NILSSON,I.M. Haemorrhagic & Sons, London, 1974.
and Thrombotic
Diseases.
5. HOLMBERG,L. and NILSSON,I.M. Genetic variants brand's disease. Brit. med. J. 3, 317, 1972.
John Wiley
of von Wille-
DESTR.&Sd
F.VIII
IS \-V-DISUSE
Yol.
12,x0.4
r ZC'ZEL,M., XILSSO?:,I.M.., and ABERG,M. Plasma ristocetin co6. factor: Assay and kinetic analysis of platelet agglutination. Thrombos. Res., 1978 (In press). 7. NILSSON,I.M., MP.GNUSSON,S., and BORCHGREVINK,C. The Duke and Ivy methods for determination of the bleeding time. Thrombos. Diathes. haemorrh. 10, 223, 1963. 8. NILSSON,I.M. and OLOW,B. Determination of fibrinogen and fibrinogenolytic activity. Thrombos. Diathes. haemorrh. 8, 297, 1962. 9. NILEHN,J.-E. Separation and estimation of "split products" of fibrinogen and fibrin in human serum. Thrombos. Diathes. haemorrh. 18, 487, 1967. 10. SILWER,J. von Willebrand's Stand. Suppl. 238, 1973. 11. BLOMBACK,B. fibrinogen.
disease
in Sweden.
and BLOMBXCK,M. Purification Arkiv Kemi. 10, 415, 1956.
Acta Paediat.
of human
and bovine
12. CRONBERG,S., ROBERTSON,B., NILSSON,I.M., and NILEHN,J.-E. Suppressive effect of dextran on platelet adhesiveness. Thrombod. Diathes. haemorrh. 16, 384, 1966. 13. WEISS,H.J. The effect of clinical dextran on platelet aggreand ADP release in man: In vivo and in gation,adhesion, vitro studies. J. Lab. clin. Med. 69, 37, 1967. 14. SARJI,K.E., GRAVES,J.M., and COLWELL,J.A. von Willebrand factor activity in normal subjects: sex,difference and variability. Thrombos. Res. 7, 885, 1975.
RESE_XRCH in G-ear Britain
EFFECT Magnus
Vol.
12,
pp.
629-61>,
19,8
Per.gamon Press, Ltd.
OF DEXTRMJ 70 ON FACTOR VIII AP;D PLATELET FUP!CTION IN von WILLEBRAND'S DISEASE aberq,
Ulla iiedner and Sven-Erik
Berqentz
Department of Plastic Surgery and Coagulation Laboratory, Allmanna Sjukhuset, 214 01 ?!almB, Sweden
(Received 29.11.1977; in revised form 26.1.19~8. Accepted by Editor H.C. Godal)
ABSTRACT Dextran given intravenously to two patients with mild von Willebrand's disease and two healthy controls impaired the factor VIII properties measured, i.e. it decreased the factor VIII-related antigen and the ristocetin co-factor activity. In the patients the variables fell from borderline to abnormally low levels accompanied by a prolongation of the bleedinq time. Infusion of factor VIII concentrate reversed the dextraninduced impairment of factor VIII and the prolonged bleeding time. The findings lend support to the view that dextran induces a defective platelet function in vivo by interfering with the factor VIII molecular complex. They also show that patients with von Willebrand's disease should not be qiven dextran as it might potentiate their bleeding diathesis.
INTRODUCTION The importance of factor VIII for normal platelet function has recently been emphasized (1). Aberg et al (2) found that 500 ml 6 % dextran 70 given intravenously impaired some of the properties-.of factor VIII, especially the one necessary for platelet aggregation by the antibiotic ristocetin, i.e. it created a situation similar to that in one variant of von Willebrand's disease (type III) (3). It was therefore considered worthwhile to study the effect of infusion of dextran in von Willebrand's disease in which the factor VIII molecule is defective, or present in ahnormal1.y low concentrations, in an
629
effort to clarify the association between on factor VIII and platelet function.
LABORATORY
the effect
of dextran
XETHODS
Blood was collected and titrated plasma prepared as described by Nilsson (4). The factor VIII coagulant activity (VIII:C) was assessed from the normalizing effect on the recalcification time of platelet-rich hemophilia A plasma containing less than 1 % of the normal factor VIII activity. The activity was expressed as a percentage of that of pooled plasma from 20 healthy individuals. The normal range was 60-160 i. The error of the method at different levels has been calculated by Nilsson (4). The factor VIII-related antigen (VIIIR:Ag) was determined with a specific rabbit antiserum against the factor VIII-related antigen. The normal range was 50-170 % and the error of the method 8.2 % (5). The ristocetin co-factor activity (VIIIR:vW/RCF/) was determined quantitatively according to Zuzel et al (6). This method uses formalin-fixed platelets and dilution of test samples in von Willebrand plasma lacking ristocetin co-factor activity. The activity of each test sample was always measured in two dilutions and expressed as a percentage of that of a normal plasma pool, which was defined as 100 S (in 86 normal controls the range was 43-204 % and the error of the method 9.6 %I. Bleeding time (BT) was determined with Ivy's method. The normal value was 6-12 min (7). Fibrinogen was determined according to Nilsson and Olow (8) and fibrin/fibrinogen degradation products (FDP) according to Nilghn (9).
MATERIAL Two patients with von Willebrand's disease and two healthy volunteers were studied. Relevant data concerning the patients are given in Table I. The patients had the classical form of von Willebrand's disease, called type I. This is characterized by decreased VIII:C and VIIIR:Aq as well as VIIIR:vW(RCF). The disease was mild in both patients, as shown by the borderline BT. The patients belong to families 20 and 42 in the Swedish register of patients with von Willebrand's disease (10). Infusion of 500 ml of 6 % dextran 70 (MacrodexR, Pharmacia, Uppsala, Sweden) was carried out within one hour. Four hours later factor VIII concentrate (AHF, Kabi, Sweden,fraction I-O, prepared according to the glycine method of Blomback and Blomback (ll,), was given intravenously in a dose of 8 U/kg body weight to the controls and 12 U/kg body weight to the patients. Blood samples were obtained before and at various intervals after the infusion of dextran and factor VIII concentrate, respectively. Informed consent was obtained.
631
TABLE
I
Some basic data about the two patients with von P7illebrand's disease. The family numbers refer to the family to which the patients belong in the Swedish register of von willebrand's disease. Family number
Sex
Age years
VIII:C 93
VIIIR:Ag %
VIIIR:vW (RCF) 9
BT min
20
oh
36
40
51
36
12
42
oh
28
58
48
32
11
RESULTS Dextran invariably lowered VIIIR:Ag and VIIIR:vW(RCF). In the patients also VIII:C was decreased (Fig. 1). The lowest values were found 2-4 hours after the dextran infusion. In the one control in whom this test was performed the BT remained unchanged and in both patients it was markedly prolonged at four hours after the infusion. Infusion of factor VIII concentrate raised VIIIR:Ag, VIIIR:vW(RCF) as well as VIII:C at the same time as it shortened the prolonged BT. The changes in the hematocrit and fibrinogen are shown in Fig. 2. No FDD were found.
DISCUSSION In a previous investigation it was shown that 500 ml 6 % dextran 70 given intravenously to healthy volunteers significantly decreased VIIIR:Ag and VIIIR:vW(RCF)(2). The mean VIII:C was unchanged although individual variations existed. These findings were confirmed in the present study, which showed the effect of dextran in two normal subjects and two patients with von Willebrand's disease. The two patients had a mild variant of von Willebrand's disease type I (3), as seen from the moderate Dextran decrease in the VIII:C, VIIIR:Ag and VIIIR:vW(RCF). caused a further decrease which was proportionate to that found in the normal subjects. But since the patients had lower initial concentrations they also had lower values than the controls 4 hours after the infusion of dextran. This explains the prolonged BT in the patients. In the control in whom this test was performed the changes were not severe enough to prolong the BT, a finding confirming previous reports on the effect of dextran in subjects with normal hemostasis (12, 13). As expected (2), dextran did not reach its maximal effect until some hours after the infusion.
i-01.12,?r’o.i,
FIG. 1 VIII:C, VIIIR:Ag, VIIIR:vW(RCF) and BT before and after infusion of dextran and AHF.
-tic
t % 50 40
-
Controls Pat. with v.Willebrand’s
30 Fib. g/l
disease
1 ----.__ y--r------c= ---_e
FIG. 2 Fibrinogen and hematocrit before and after infusion of dextran and AHF.
-
1 1
v01.12,s0.s
DESTR_:xd
F. i-111 IS +A--DISE.-;S-E
433
The mechanism underlying the effect of dextran on factor (Fig. 2) caused by VIII is obscure (2). The slight hemodilution dextran cannot explain this effect since isotonic saline given in amounts which caused a similar hemodilution did not influence factor VIII (2). The effect of dextran therefore seems to be due to a specific property of this substance. The subjects studied were fasting and consequently the depressing effect of food on VIIIR:vW(RCF) (14) can be excluded. There was no proteolysis as seen from the fact that TrasylolR, given before and during infusion of dextran did not influence the impairment of factor VIII (unpublished observation). The findings that dextran did not affect factor VIII in vitro and that the maximal effect was obtained at four hours after infusion suggest that dextran interferes with the metabolism of factor VIII (2). Thus, dextran influenced the properties of factor VIII in patients with von Willebrand's disease as it does in healthy subjects. In the patients the variables fell from borderline values to abnormally low levels resulting in a prolongation of the BT and a deterioration of the disease from a mild to a more (fraction I-O, severe form. Infusion of factor VIII concentrate AHF, Kabi) reversed not only the decrease in VIIIR:Ag and VIIIR:vW(RCF) of the controls and the von Willebrand patients, but also the prolonged BT of the latter. The findings lend support to the view that dextran temporarily impairs platelet function in vivo by interfering with the factor VIII molecular complex. The results also show that dextran infusion should not be given to patients with von Willebrand's disease, as they might develop a severe bleeding diathesis.
ACKNOWLEDGEMENT This investigation was supported by grants from the Swedish Medical Research Council (B78-19X-00087-14A, B78-17X-00759-13B).
REFERENCES 1.
WEISS,H.J. Platelet function. New Engl.
physiology and abnormalities J. Med. 293, 580, 1975.
of platelet
Effect of dextran on 2. &3ERG,M., HEDNER,U., and BERGENTZ,S.-E. factor VIII (antihemophilic factor) and platelet function. Ann. Surg. 1978 (In press). 3. NILSSON,I.M. and HOLMBERG,L. Biochemical and clinical aspects on factor VIII. First Florence Conf. on Haemostasis and Thrombosis, May 1977. Thrombos. Haemostas. 1978 (In press). 4. NILSSON,I.M. Haemorrhagic & Sons, London, 1974.
and Thrombotic
Diseases.
5. HOLMBERG,L. and NILSSON,I.M. Genetic variants brand's disease. Brit. med. J. 3, 317, 1972.
John Wiley
of von Wille-
DESTR.&Sd
F.VIII
IS \-V-DISUSE
Yol.
12,x0.4
r ZC'ZEL,M., XILSSO?:,I.M.., and ABERG,M. Plasma ristocetin co6. factor: Assay and kinetic analysis of platelet agglutination. Thrombos. Res., 1978 (In press). 7. NILSSON,I.M., MP.GNUSSON,S., and BORCHGREVINK,C. The Duke and Ivy methods for determination of the bleeding time. Thrombos. Diathes. haemorrh. 10, 223, 1963. 8. NILSSON,I.M. and OLOW,B. Determination of fibrinogen and fibrinogenolytic activity. Thrombos. Diathes. haemorrh. 8, 297, 1962. 9. NILEHN,J.-E. Separation and estimation of "split products" of fibrinogen and fibrin in human serum. Thrombos. Diathes. haemorrh. 18, 487, 1967. 10. SILWER,J. von Willebrand's Stand. Suppl. 238, 1973. 11. BLOMBACK,B. fibrinogen.
disease
in Sweden.
and BLOMBXCK,M. Purification Arkiv Kemi. 10, 415, 1956.
Acta Paediat.
of human
and bovine
12. CRONBERG,S., ROBERTSON,B., NILSSON,I.M., and NILEHN,J.-E. Suppressive effect of dextran on platelet adhesiveness. Thrombod. Diathes. haemorrh. 16, 384, 1966. 13. WEISS,H.J. The effect of clinical dextran on platelet aggreand ADP release in man: In vivo and in gation,adhesion, vitro studies. J. Lab. clin. Med. 69, 37, 1967. 14. SARJI,K.E., GRAVES,J.M., and COLWELL,J.A. von Willebrand factor activity in normal subjects: sex,difference and variability. Thrombos. Res. 7, 885, 1975.
