Ann Hematol (1992) 65:253-259
Annals of
Hematology 9 Springer-Verlag 1992
Original article Pharmacokinetics and hemostatic effect of different factor V l l l / v o n Willebrand factor concentrates in von Willebrand's disease type III S. L e t h a g e n , E. B e r n t o r p , a n d I . M . Nilsson
Department for CoagulationDisorders, Universityof Lund, Malm0 General Hospital, S-21401 Malm6, Sweden Received July 3, 1992/Accepted September 18, 1992
Summary. Four different plasma-derived concentrates composed of coagulation factor VIII (FVIII) and von Willebrand factor (vWF) of varying quality (Hemate-P, Behring; Profilate, Alpha; and FVIII-VHP-vWF, C.R.T.S Lille), or almost purified vWF (Facteur Willebrand, C.R.T.S Lille) and one recombinant FVIII concentrate (Recombinate, Baxter) were given, in doses of 30-60 IU VIII: C/kg or 70-110 IU RCof/kg, to five patients with von Willebrand's disease type III, in order to evaluate the role of the vWF in factor FVIII concentrates. All plasma concentrates except Profilate had a multimeric vWF pattern almost similar to that of normal plasma. Bleeding time (b.t.), VIII 9C, vWF: Ag, ristocetin cofactor activity, and multimeric pattern of the plasma-vWF were followed for 72 h. Both Duke b.t. and the multimeric pattern in plasma normalized after infusion of Hemate-P, FVIIIVHP-vWF, and Facteur Willebrand and, to a lesser extent, after Profilate. As expected, in response to Recombinate there was no effect on primary hemostasis, and the half-life of FVIII procoagulant activity (VIII:C) was very short. Normalization of the vWF is important not only for improving the primary hemostasis, but also for maintaining the plasma FVIII concentration on a high level, both by reducing the elimination rate of infused FVIII and via a secondary release of endogenous FVIII. If a prompt hemostatic effect is required, we recommend a concentrate containing both FVIII and all vWF multimers, but for prophylactic treatment, pure vWF may be used. Key words: von Willebrand's disease type III -
von
Willebrand factor - Factor VIII concentrates
Correspondence to: S. Lethagen,Department for CoagulationDisorders, University of Lund, Malmt3 General Hospital, S-21401 MalmO, Sweden
Introduction
Von Willebrand's disease (vWD) was first described by Erik von Willebrand [40] as an inherited bleeding disorder characterized by nose bleeding, menorrhagia, bleeding after tooth extractions and from wounds, and by a prolonged bleeding time despite a normal platelet count. In the 1950s, Nilsson and co-workers [25] showed that infusion of fraction I-O to patients with vWD both corrected the decreased FVIII concentration, the prolonged bleeding time, and the capillary bleeding, and induced a secondary increase in FVIII activity [26]. vWD was found to be caused by deficiency of the von Willebrand factor (vWF), which was present in plasma from both healthy individuals and patients with hemophilia A. In mild forms of vWD type I, where the vWF is qualitatively normal but decreased in concentration, treatment with 1-deamino-8-D-arginine vasopressin (DDAVP) is usually effective [18, 24]. As patients with vWD type III have no endogenous synthesis of vWF and manifest no response to DDAVP [29], they are dependent on replacement therapy in the event of severe bleeding. The highly purified commercial concentrates used in the treatment of hemophilia A have been found unsuitable for the treatment of vWD, as they have been shown to be ineffective in controlling the bleeding time in patients with vWD [7, 23], probably because they lack the high-molecularweight multimers of vWF [30], and they fail to support platelet adhesion to subendothelium in a perfusion chamber [38]. During recent years, several factor concentrates for use in vWD have been described. Hemate-P (Behring) has been reported to contain all multimers of the vWF [4] and to be efficient and safe for the treatment of vWD [1, 5, 37]. A French concentrate containing all vWF multimers and only a low concentration of FVIII [11, 21] and a British FVIII concentrate (8Y) [9, 31] have both been reported to shorten the prolonged bleeding time and to be hemostatically effective in vWD. The aim of the present study was to investigate the effect of factor concentrates of different composition and
254
Concentrates tested
q u a l i t y o f v W F a n d F V I I I o n h e m o s t a t i c variables in patients with v W D type III, a n d to evaluate the i m p o r t a n c e o f the presence o f the largest v W F m u l t i m e r s . A n o t h e r a i m was to evaluate the half-life o f V I I I : C after administ r a t i o n o f r e c o m b i n a n t F V I I I to patients with v W D type III, as c o m p a r e d with t h a t in severe h e m o p h i l i a A patients.
The following concentrates were studied: Hemate-P (Behring), prepared from cryoprecipitate by AI(OH)3 adsorption and glycin e precipitation, and virus inactivated with wet heat (60~ for 10 h), Profilate (Alpha) prepared from cryoprecipitate by AI(OH)3 adsorption, polyethylene glycol and glycine precipitation, and virus inactivated with solvent detergent inactivation (SDI); F VIII-VHPvWF and Facteur Willebrand (C.R.T.S. Lille), both prepared from cryoprecipitate by ion-exchange chromatography and affinity chromatography and virus inactivated with SDI [8] Recombinate (Baxter), a recombinant factor VIII concentrate manufactured with DNA technology using Chinese hamster ovary cells (CHO) and purified by ion-exchange and affinity chromatography.
Materials and methods Study population Five patients with vWD type III were included. Their clinical data are given in Table 1. In addition, three patients with severe hemophilia A, 25-39 years of age, were given recombinant FVIII.
Dosages FVIII concentrates were administered at dosages of 30-63 IU VIII : C/kg body wt. and Facteur Willebrand at dosages of 71ll0 IU Ristocetin cofactor activity/kg body wt. Each concentrate was given to three patients, except for Hemate-P, which was given to all five patients and served as a reference. Each patient received three concentrates, except for patient number 1, who received all five concentrates. Each patient was given about the same dosage of the different FVIII concentrates (Table 2). The patients were not bleeding and were not given any factor concentrates during a period of at least 1 week before receiving each study drug.
Table 1. Patient data Patient number
Sex
Age Body weight (years) (kg)
Baseline concentration of VIII:C vWF:Ag (IU/ml) (IU/ml)
1 2 3 4 5
Male Male Male Male Female
24 20 17 23 41
0.03 0.02 0.09 0.02 0.03
74 82 67 65 63
< < < <
1200 410
420 n.d. 480
0.40
0.63
0.63
2.23
1.53
0.87
2.02
1.78
0.98
150
> 1200
n.d.
0.37 0.70 0.75
0.07 0.73 0.62
0.03 0.72 0.56
0 1.23 2.36
0 0.54 0.65
0 0.37 0.29
0 0.59 0.37
0 0.27 0.25
> 1200 540 300
n.d. 600 1080
n.d. 600 > 1200
0.39
0.62
0.22
0.14
0 1.00 1.05 (10') 0.41
FVIII-VHP-vWF 2400 [30] Hemate-P 4000 [60] Profilate 4000 [60]
0.49
0.43
0.12
0.11
300
600
720
1.35 1.39
1.07 1:04
1.01 0.91
3.32 3.50
1.14 1.74
0.83 0.90
0.98 0.87
0.56 0.69
360 270
540 210
540 180
0.63
0.99
1.22
1.71
1.04
1.01
2.08 2.18 (10') 2.88
Facteur Willebrand 7360 [110] Hemate-P 2600 [40] Facteur Willebrand 4600 [71] Recombinate 2770 [43] Hemate-P 2500 [40] FVIII-VHP-vWF 3300 [52] Recombinate 4000 [63]
1.39
1.16
360
240
840
0.86 0.37
0.90 0.59
0.89 0.73
1.75 1.52
1.20 0.69
0.72 0.69
2.25 1.45
0.96 0.63
0.58 0.66
540 300
960 495
960 > 1200
0.29 1.30 1.12
0.04 1.11 1.16
0.03 0.79 0.85
0 2.55 1.57
0 1.05 0.53
0 0.56 0.31
0 1.92 1.59
0 0.78 0.63
0 0.50 0.33
> 1200 240 550
n.d. 480 1080
n.d. 480 > 1200
0.51
0.08
0.04
0
0
0
0
0
0
> 1200
n.d.
Concentrate Dosage IU[IU/kg] (VIII:C or RCof for Facteur Willebrand)
VIII: C IU/ml after 1h 12 h
Hemate-P 2960 [40] Profilate 3000 [40] FVIII-VHP-vWF 3300 [43] Facteur WiUebrand 5520 [74] Recombinate 2959 [40] Hemate-P 2500 [30] Profilate 2500 [30]
0.59 0.84 0.84
Duke bleeding time s after 10rain 12h 24h
n.d.
255
Assays
In vivo studies
Venou~ blood for coagulation analysis was collected into siliconeco~ted vacutainer tubes (Becton Dickinson) with a volume of 5 ml, containing 0.5 ml 0.129 M sodium citrate. Platelet-poor plasma (PPP) was obtained after centrifugation at 2000x g for 10 min. Plasma not immediately analyzed was frozen and kept at - 7 0 ~ until assessed. Platelet-rich plasma (PRP) was obtained after centrifugation at 275 x g for 10 min. Bleeding time was measured both with a Simplate-II device (normal range: 360-720 s) and according to Duke's method (norreal range: 60-300 s). Factor VIII clotting activity (VIII: C) was assayed with a chromogenic substrate method (Coatest Factor VIII, KabiVitrum) (normal range: 0.5-2.1 1U/ml) [17] and a one-stage clotting assay (normal range: 0.5-1.7 IU/ml) [28]. Von Willebrandfactor antigen (vWF:Ag) was measured with an IRMA using a monoclonal antibody against vWF (normal range: 0.5-2.0 IU/ml) [15, 35]. Standard plasma from 20 healthy subjects was calibrated against the 13th British standard (85/573) for analyses of VIII : C and the 1st British standard (85/644) for analyses of vWF:Ag. Results were expressed in IU/ml. The multimeric structure of vWF (multimeric sizing) in PPP was analyzed with sodium dodecyl sulfate agarose gel electrophoresis (1.9070 agarose concentration) as described elsewhere [34]. The multimeric bands were identified with a 125I-labeled mouse monoclonal antibody followed by autoradiography [15]. Ristocetin cofactor activity (RCof) was determined with a method described by Zuzel and co-workers [41] (normal range: 0.50-1.60 IU/ml) using formalin-fixed normal platelets and the patient's PPP. Platelets were fixed by incubating 1 part of PRP with 1 part of formalin in Tris-saline buffer (8 mg/ml formalin) for 18 h at 4~ The platelets were washed and resuspended in Trissaline buffer containing 0.15 M NaC1 and 0.1 M Tris, pH 7.4. Pharmacokinetics: The half-life of VIII : C was calculated as described by BjOrkman and co-workers [6]. Adverse events were checked for. All patients enrolled into the study gave their informed consent, and the study was approved by the Ethics Committee of the Medical Faculty, University of Lund.
P l a s m a c o n c e n t r a t i o n s o f V I I I : C, v W F : A g , R C o f a n d the p o s t - i n f u s i o n D u k e b l e e d i n g times are shown in Table 2. F i g u r e 2 shows the r e s p o n s e in v W F : A g , R C o f , V I I I - C a n d D u k e b l e e d i n g t i m e in p a t i e n t 1, w h o received all five concentrates. B o t h D u k e a n d S i m p l a t e II bleeding times exceeded 1200 s in all p a t i e n t s before t h e i n f u s i o n o f f a c t o r concentrates. D u k e b l e e d i n g t i m e s h o r t e n e d in response to all concentrates except R e c o m b i n a t e , w h i c h d i d n o t affect t h e b l e e d i n g time. W i t h t h e exception o f p a t i e n t 4, w h o m a n i f e s t e d a r e m a r k a b l y p o o r response to H e m a t e - P , t h e s h o r t e n i n g o f bleeding t i m e lasted l o n g e r after H e m a t e - P ( 2 4 - 3 6 h ) t h a n after F V I I I - V H P - v W F ( 4 - 2 4 h ) o r Facteur W i l l e b r a n d ( 8 - 2 4 h) (Table 2). A s can be seen f r o m Fig. 2, the s h o r t e n i n g o f b l e e d i n g t i m e after P r o f i l a t e was
Results In vitro studies T h e in vitro p r o p e r t i e s o f the different concentrates are given in Table 3. T h e m u l t i m e r i c v W F p a t t e r n was a l m o s t n o r m a l in vitro in H e m a t e - P , F V I I I - V H P - v W F , a n d Facteur W i l l e b r a n d , whereas P r o f i l a t e lacked the largest m u l timers (Fig. 1). N o v W F was detected in R e c o m b i n a t e .
Table 3. In vitro properties of the FVIII/vWF concentrates studied Concentrate
VIII : C (IU/ml)
Hemate-P Profilate FVIII-VHP-vWF Facteur Willebrand Recombinate
28 33 19 10 68
vWF : Ag IRMA (IU/ml)
RCof IU/ml
Multimeric pattern
69 108 40 68
56 105 43 43
Large Large Large Large
< 0.001
0
present absent present present
No vWF
Fig. 1. Multimeric structure of the vWF in normal plasma (NP) and in plasma from patient 1 with vWD type III before (B) and: 1, 4, 12, and 24 h after administration of HematexP, Facteur Willebrand, and Profilate and in vitro in the same three concentrates
256
VIII=C (IU/ml)
Vlll:C
(lU/rnl
1.0
1.0-
0.5
0.80.6-
vWF=Ag IRMA (IU/ml)
0.40.21.5
O" ....
Before 1
0.5
a,
13
i
12
//
,
24
//
3'6
,;,
48h
b
Fig. 3. Effect of administration on VIII 9C in plasma of three patients with vWD type III: patient 1 (40 IU/kg) (9 9 patient 4 ([] []), and patient 5 (A ~); and in three patients with severe hemophilia A: patients H1 (47 IU/kg) (o e), H2 (61 IU/kg) (m l), and H3 (35 IU/kg) (zx. zx)
Rcof (IU/ml)
1.5
0.5 Duke BT (see)
50OI Before
2 4
8
12
24
36
48
72 h
Fig. 2. Effect of administration of Hemate-P (9 9 F VIIIVHP-vWF ([] ~), Facteur Willebrand (& ... A), Profilate (e e), and Recombinate ( " B) on VIII:C, vWF:Ag (IRMA), and RCof in plasma and Duke bleeding time in patient 1, with vWD type III
of shorter duration, except when the dosage was large (60 IU VIII:C/kg; Table 2). The Simplate II bleeding time did not shorten after any of the concentrates (not shown). Except for Recombinate, which contains no vWF, all concentrates produced large increases in plasma vWF : Ag concentrations, peak levels usually occurring within the first hour (Fig. 2). Figure 1 shows the multimeric pattern of the vWF in plasma in patient 1: Almost all multimers appeared after Hemate-P, Facteur Willebrand, and FVIII-VHP-vWF, and were still detectable after 4 h, but after 12 h the largest multimers had disappeared. After Profilate the largest multimers were lacking. As expected, no vWF multimers were seen in patients receiving Recombinate (not shown), vWF was undetectable in platelets both before and after infusion of all concentrates (not shown), as demonstrated earlier [16]. RCof was increased by all concentrates except Recombinate, the highest levels being detected after Facteur Willebrand. Profilate produced increases in RCof similar to or slightly higher than those produced by Hemate-P, but in patient 2 the duration of the increase was very short
after Profilate. There was no correlation between the increase in RCof and the multimeric pattern of vWF in plasma. The plasma concentration of VIII:C was increased after all five concentrates and remained high for long periods after Hemate-P, FVIII-VHP-vWF, Facteur Willebrand, and Profilate (Table 2 and Fig. 2). Facteur Willebrand, which contains only small amounts of FVIII, induced the lowest initial increases in VIII:C (0.320.56 IU/ml after 10 min and 0.37-0.63 IU/ml after 1 h) but the VIII'C concentrations continued to increase for another 24-48 h (Fig. 2). In the vWD patients the duration of the V I I I ' C response after Recombinate was very short, the half-life being only 3 h 55 min, 5 h 4 min, or 5 h 55 min, as compared with 8 h 9 min, 10 h 1 min, or 11 h 7 min in the patients with severe hemophilia A (Fig. 3). No adverse events were seen after any of the concentrates.
Discussion
In vWD, the aim of treatment is to correct the defect in primary hemostasis, the prolonged bleeding time, and the FVIII deficiency. In mild vWD type I, this can be achieved by administration of DDAVP, but in vWD type III, where the vWF is completely absent and the FVIII concentration very low, plasma concentrates must be given. The first concentrates to become available were human fraction I - O (AHF-Kabi) [25] and cryoprecipitate [3, 32], both of which contain FVIII and the full complement of vWF multimers [4, 30] and have been found to promote platelet adhesion to the subendothelium [38]. Because of insufficient virus inactivation, AHF-Kabi is no longer produced, and the use of cryoprecipitate is questionable. Therefore, it is important to evaluate the efficacy of FVIII concentrates of higher purity and with a higher degree of virus safety for the treatment of patients with vWD type III. The aim of the present study was to investigate the effect of factor concentrates of different composition and
257 quality of vWF and FVIII on hemostatic variables in patients with vWD type III. Comparisons between concentrates should therefore be done primarily in individual patients. As Hemate-P was given to all five patients it may serve as a reference. The recovery of VIII 9C, vWF : Ag, and RCof differed markedly between the concentrates. The complex nature of the vWF protein complicates pharmacokinetic evaluation of FVIII/vWF concentrates in vWD, as the concentration of vWF.'Ag or the RCof in plasma does not necessarily reflect the biological effectiveness of the vWF, which is dependent on the multimeric structure of the vWF. The improvement of multimeric vWF pattern in plasma and shortening of bleeding time are important determinants of hemostatic capacity of concentrates and cannot be described with ordinary pharmacokinetic variables. It has been suggested that normal multimeric structure of the concentrates is a prerequisite for normalization of the bleeding time [4, 201. Studies with the annular perfusion chamber technique [2, 36] have shown one of the primary functions of vWF to be mediation of platelet interactions with the subendothelium, the largest vWF multimers being the most active [38]. Moreover, Federici and co-workers [10] found the largest vWF multimers to have stronger affinity than the smaller multimers for the platelet receptors glycoprotein Ib and I I b / I I I a . We found Hemate-P, FVIII-VHP-vWF, and Facteur Willebrand to have a multimeric vWF pattern in vitro almost similar to that of normal plasma and to temporarily normalize the multimeric vWF-pattern in plasma after intravenous administration. These three concentrates manifested lasting effects on Duke bleeding times and on the vWF and RCof concentrations in plasma, indicating that they may be clinically effective in connection with bleeding caused by impaired primary hemostasis in vWD, mainly bleeding from mucous membranes. Our findings are in agreement with those of earlier studies [5, 11]. Berntorp and Nilsson [5] found Hemate-P to temporarily correct the hemostatic defect in 11 patients with vWD (7/11 with type III) and to give satisfactory hemostasis in connection with childbirth and major surgery. Goudemand and co-workers [11] found Facteur Willebrand to temporarily correct the bleeding time, greatly improve the plasma vWF multimeric pattern, increase RCof, and give satisfactory hemostasis in connection with different bleeding episodes in 9 vWD patients (1/9 with type III). Profilate, on the other hand, shortened the Duke bleeding time despite the lack of the largest multimers and despite the fact that the multimeric structure in plasma did not normalize, though the effect on the bleeding time was of shorter duration than that of the other concentrates. The relatively short duration of Profilate's effect on bleeding times is probably due to its lack of the large multimers. The presence of the intermediate and small multimers of the vWF, which are also capable of mediating platelet adhesion, explains the fact that Profilate nevertheless shortened the bleeding time. Rodhegiero and co-workers [33] recently argued that normalization of the bleeding time is required only in cases of mucosal, especially gastrointestinal bleeding,
and that good postsurgical hemostasis had been obtained in several cases, even in connection with tooth extraction, despite the fact that the Ivy bleeding time was not corrected, provided that a sufficiently high FVIII concentration was obtained. We found, however, that none of the concentrates was able to shorten the Simplate II bleeding time, even if the multimeric pattern was normalized in plasma. The Duke bleeding time is a less sensitive test and is probably clinically more useful for evaluating the risk of excessive operative blood loss in patients with type III vWD [27]. Both Berntorp and Nilsson [5] and Goudemand and co-workers [11] used the Duke bleeding time method in their type-Ill patients. The amount and quality of platelet vWF are important determinants of primary hemostasis, bleeding time and severity of bleeding symptoms in vWD [12, 16, 19]. As known, platelets in vWD type III lack vWF. There was no uptake of vWF in platelets after any of the concentrates given in this study, which may explain why the Simplate II bleeding time does not normalize even if the vWF in plasma is normal. We found that Hemate-P, FVIII-VHP-vWF, Profilate, and Recombinate, which all contain FVIII, induced large initial FVIII increases due to their FVIII content. Facteur WiUebrand, which contains only small amounts of FVIII, induced a lower initial increase. It has long been known that administration of vWF to patients with vWD type III induces a secondary increase of FVIII in plasma [25], probably by enhancing secretion [13]. The secondary increase in FVIII was most evident after administration of Facteur Willebrand, as it contains only small amounts of FVIII, a rapid release of FVIII from endogenous reservoirs being seen already after 10 min, and the plasma concentrations of V I I I ' C peaked after 24-48 h, suggesting that the FVIII release continued, possibly until an optimal ratio between the amount of FVIII and vWF subunits in plasma had been established. Similar secondary FVIII increases were observed after administration of all vWF-containing concentrates (Fig. 2). A tendency was observed toward a faster drop in VIII:C concentrations after Profilate, which may have been due to a faster elimination rate of vWF. It is obvious that the half-life of FVIII is very short when the patient is devoid of vWF, as shown by the difference in response to the administration of Recombinate between the yon Willebrand patients and the hemophilia patients (Fig. 3). The normalization of plasma vWF is of importance not only for primary hemostasis, but also for maintaining the increased FVIII concentration at a high level for a long period of time. This is achieved both by a slower elimination rate of the infused FVIII due to binding to vWF [14, 39] and as a result of the vWF-induced release of endogenous FVIII [13, 25]. To sum up, several factor concentrates are now available which are suitable for treatment of patients with vWD. The present findings show that even if a FVIII concentrate lacking the largest multimers may have some hemostatic effect, it is advantageous to administer a concentrate consisting of all vWF multimers in order to achieve a more durable hemostatic effect. A pure vWF concentrate induces a long-lasting FVIII increase and
258 m a y therefore be used prophylactically, b u t in the event
o f bleeding o r as cover for surgery, w h e n a m o r e p r o m p t effect is necessary, a c o n c e n t r a t e also c o n t a i n i n g F V I I I s h o u l d be a d m i n i s t e r e d .
Acknowledgements. The investigation was supported by the Swedish Medical Research Council (grant no. 00087), the Medical Faculty, University of Lund, Malm6 General Hospital Research Foundations, and the Agnes and Nils Nilsson Foundation.
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17. Lethagen S, Ostergaard H, Nilsson IM (1986) Clinical application of the chromogenic assay of factor VIII in haemophilia A, and different variants of yon Willebrand's disease. Scand J Haematol 37:448-453 18. Mannucci PM, Ruggeri ZM, Pareti FI, Capitanio A (1977) 1-Deamino-8-D-arginine vasopressin: a new pharmacological approach to the management of haemophilia and von Willebrand's disease. Lancet 1:869-872 19. Mannucci PM, Lombardi R, Bader R, et al. (1985) Heterogeneity of type-I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor. Blood 66: 796802 20. Mannucci PM, Moia M, Rebulla O, Altieri D, Monteagudo J, Castillo (1987) Correction of the bleeding time in treated patients with severe von Willebrand disease is not solely dependent on the normal multimeric structure of plasma yon Willebrand factor. Am J Hematol 25:55-65 21. Mazurier C, Jorieux S, de Romeuf C, Samor B, Goudemand M (1991) In vitro evaluation of a very-high-purity, solvent/ detergent-treated, von Willebrand factor concentrate. Vox Sang 61:1-7 22. Nilsson IM (1984) Von Willebrand's disease from 1926-1983. Scand J Haematol 33:21-43 23. Nilsson IM, Hedner U (1977) Characteristics of various factor VIII concentrates used in treatment of hemophilia A. Br J Haematol 37:543-557 24. Nilsson IM, Lethagen S (1991) Current status of DDAVP formulations and their use. In: Lusher JM, Kessler CM (eds) Hemophilia and von Willebrand's disease in the 1990s. Elsevier Science Publishers, Amsterdam, pp 443-453 25. Nilsson IM, Blomb~tck B, Jorpes E, Blomb~ck M, Johansson SA (1957) Von Willebrand's disease and its correction with human plasma fraction I-O. Acta Med Scand 159: 179188 26. Nilsson IM, Blomb~tck M, Blomb~ick B (1959) Von Willebrand's disease in Sweden. Its pathogenesis and treatment. Acta Med Scand 164:263-278 27. Nilsson IM, Magnusson S, Borchgrevink C (1963) The Duke and Ivy methods for determination of the bleeding time. Thromb Diathes Haemorrh 10:223-234 28. Nilsson IM, Kirkwood TBL, Barrowcliffe TW (1979) In vivo recovery of factor VIII: a comparison of one-stage and twostage assay methods. Thromb Haemost 42:1230-1239 29. Nilsson IM, Vilhardt H, Holmberg L, Astedt B (1982) Association between factor VIII-related antigen and plasminogen activator. Acta Med Scand 211:105-112 30. Nilsson IM, Borge L, Gunnarsson M, Kristoffersson AC (1984) Factor VIII-related activities in concentrates. Stand J Haematol 33 [Suppl 41]: 157-172 31. Pasi K J, Williams MD, Enayat MS, Hili FGH (1990) Clinical and laboratory evaluation of the treatment of von Willebrand's disease patients with heat-treated factor VIII concentrate (BPL 8Y). Br J Haematol 75:228-233 32. Perkins HA (1967) Correction of the hemostatic defect in von Willebrand's disease. Blood 30:375-380 33. Rodhegiero F, Gill JC (1991) Guidelines for the treatment of yon Willebrand's disease with plasma derivatives. In: Lusher JM, Kessler CM (eds) Hemophilia and von Willebrand's disease in the 1990s. Elsevier Science Publishers, Amsterdam, pp 497-503 34. Ruggeri ZM, Zimmerman TS (1981) The complex multimeric composition of factor VIII/yon Willebrand factor. Blood 57: 1140-1143 35. Ruggeri ZM, Mannucci PM, Jeffcoate SL, Ingram GIC (1976) Immunoradiometric assay of factor VIII-related antigen, with observations in 32 patients with yon Willebrand's disease. Br J Haematol 33:221-232 36. Sakariassen KS, Bolhuis PA, Sixma JJ (1979) Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-von Willebrand factor bound to the subendothelium. Nature 279:636-638
259 37. Schimpf K, Mannucci PM, Kreutz W, et al. (1987) Absence of hepatitis after treatment with a pasteurized factor VIII concentrate in patients with haemophilia and no previous transfusions. N Engl J Med 316:918-922 38. Sixma J J, Sakariassen KS, Beese-Visser NH, Ottenhof-Rovers M, Bolhuis PA (1984) Adhesion of platelets to human artery subendothelium: effect of factor VIII-von Willebrand factor of various multimeric composition. Blood 63:128-139
39. Tuddenham EGD, Lane RS, Rotblat F, et al. (1982) Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human haemophilia A and von Willebrand's disease. Br J Haematol 52:259-267 40. Willebrand EA von (1926) Hereditar pseudohemofili. Finska L/ikars~illskapets Handlingar 67" 7-12 41. Zuzel M, Nilsson IM, Aberg M (1978) A method for measuring plasma ristocetin cofactor activity. Thromb Res 12:745-754
Annals of
Hematology 9 Springer-Verlag 1992
Original article Pharmacokinetics and hemostatic effect of different factor V l l l / v o n Willebrand factor concentrates in von Willebrand's disease type III S. L e t h a g e n , E. B e r n t o r p , a n d I . M . Nilsson
Department for CoagulationDisorders, Universityof Lund, Malm0 General Hospital, S-21401 Malm6, Sweden Received July 3, 1992/Accepted September 18, 1992
Summary. Four different plasma-derived concentrates composed of coagulation factor VIII (FVIII) and von Willebrand factor (vWF) of varying quality (Hemate-P, Behring; Profilate, Alpha; and FVIII-VHP-vWF, C.R.T.S Lille), or almost purified vWF (Facteur Willebrand, C.R.T.S Lille) and one recombinant FVIII concentrate (Recombinate, Baxter) were given, in doses of 30-60 IU VIII: C/kg or 70-110 IU RCof/kg, to five patients with von Willebrand's disease type III, in order to evaluate the role of the vWF in factor FVIII concentrates. All plasma concentrates except Profilate had a multimeric vWF pattern almost similar to that of normal plasma. Bleeding time (b.t.), VIII 9C, vWF: Ag, ristocetin cofactor activity, and multimeric pattern of the plasma-vWF were followed for 72 h. Both Duke b.t. and the multimeric pattern in plasma normalized after infusion of Hemate-P, FVIIIVHP-vWF, and Facteur Willebrand and, to a lesser extent, after Profilate. As expected, in response to Recombinate there was no effect on primary hemostasis, and the half-life of FVIII procoagulant activity (VIII:C) was very short. Normalization of the vWF is important not only for improving the primary hemostasis, but also for maintaining the plasma FVIII concentration on a high level, both by reducing the elimination rate of infused FVIII and via a secondary release of endogenous FVIII. If a prompt hemostatic effect is required, we recommend a concentrate containing both FVIII and all vWF multimers, but for prophylactic treatment, pure vWF may be used. Key words: von Willebrand's disease type III -
von
Willebrand factor - Factor VIII concentrates
Correspondence to: S. Lethagen,Department for CoagulationDisorders, University of Lund, Malmt3 General Hospital, S-21401 MalmO, Sweden
Introduction
Von Willebrand's disease (vWD) was first described by Erik von Willebrand [40] as an inherited bleeding disorder characterized by nose bleeding, menorrhagia, bleeding after tooth extractions and from wounds, and by a prolonged bleeding time despite a normal platelet count. In the 1950s, Nilsson and co-workers [25] showed that infusion of fraction I-O to patients with vWD both corrected the decreased FVIII concentration, the prolonged bleeding time, and the capillary bleeding, and induced a secondary increase in FVIII activity [26]. vWD was found to be caused by deficiency of the von Willebrand factor (vWF), which was present in plasma from both healthy individuals and patients with hemophilia A. In mild forms of vWD type I, where the vWF is qualitatively normal but decreased in concentration, treatment with 1-deamino-8-D-arginine vasopressin (DDAVP) is usually effective [18, 24]. As patients with vWD type III have no endogenous synthesis of vWF and manifest no response to DDAVP [29], they are dependent on replacement therapy in the event of severe bleeding. The highly purified commercial concentrates used in the treatment of hemophilia A have been found unsuitable for the treatment of vWD, as they have been shown to be ineffective in controlling the bleeding time in patients with vWD [7, 23], probably because they lack the high-molecularweight multimers of vWF [30], and they fail to support platelet adhesion to subendothelium in a perfusion chamber [38]. During recent years, several factor concentrates for use in vWD have been described. Hemate-P (Behring) has been reported to contain all multimers of the vWF [4] and to be efficient and safe for the treatment of vWD [1, 5, 37]. A French concentrate containing all vWF multimers and only a low concentration of FVIII [11, 21] and a British FVIII concentrate (8Y) [9, 31] have both been reported to shorten the prolonged bleeding time and to be hemostatically effective in vWD. The aim of the present study was to investigate the effect of factor concentrates of different composition and
254
Concentrates tested
q u a l i t y o f v W F a n d F V I I I o n h e m o s t a t i c variables in patients with v W D type III, a n d to evaluate the i m p o r t a n c e o f the presence o f the largest v W F m u l t i m e r s . A n o t h e r a i m was to evaluate the half-life o f V I I I : C after administ r a t i o n o f r e c o m b i n a n t F V I I I to patients with v W D type III, as c o m p a r e d with t h a t in severe h e m o p h i l i a A patients.
The following concentrates were studied: Hemate-P (Behring), prepared from cryoprecipitate by AI(OH)3 adsorption and glycin e precipitation, and virus inactivated with wet heat (60~ for 10 h), Profilate (Alpha) prepared from cryoprecipitate by AI(OH)3 adsorption, polyethylene glycol and glycine precipitation, and virus inactivated with solvent detergent inactivation (SDI); F VIII-VHPvWF and Facteur Willebrand (C.R.T.S. Lille), both prepared from cryoprecipitate by ion-exchange chromatography and affinity chromatography and virus inactivated with SDI [8] Recombinate (Baxter), a recombinant factor VIII concentrate manufactured with DNA technology using Chinese hamster ovary cells (CHO) and purified by ion-exchange and affinity chromatography.
Materials and methods Study population Five patients with vWD type III were included. Their clinical data are given in Table 1. In addition, three patients with severe hemophilia A, 25-39 years of age, were given recombinant FVIII.
Dosages FVIII concentrates were administered at dosages of 30-63 IU VIII : C/kg body wt. and Facteur Willebrand at dosages of 71ll0 IU Ristocetin cofactor activity/kg body wt. Each concentrate was given to three patients, except for Hemate-P, which was given to all five patients and served as a reference. Each patient received three concentrates, except for patient number 1, who received all five concentrates. Each patient was given about the same dosage of the different FVIII concentrates (Table 2). The patients were not bleeding and were not given any factor concentrates during a period of at least 1 week before receiving each study drug.
Table 1. Patient data Patient number
Sex
Age Body weight (years) (kg)
Baseline concentration of VIII:C vWF:Ag (IU/ml) (IU/ml)
1 2 3 4 5
Male Male Male Male Female
24 20 17 23 41
0.03 0.02 0.09 0.02 0.03
74 82 67 65 63
< < < <
1200 410
420 n.d. 480
0.40
0.63
0.63
2.23
1.53
0.87
2.02
1.78
0.98
150
> 1200
n.d.
0.37 0.70 0.75
0.07 0.73 0.62
0.03 0.72 0.56
0 1.23 2.36
0 0.54 0.65
0 0.37 0.29
0 0.59 0.37
0 0.27 0.25
> 1200 540 300
n.d. 600 1080
n.d. 600 > 1200
0.39
0.62
0.22
0.14
0 1.00 1.05 (10') 0.41
FVIII-VHP-vWF 2400 [30] Hemate-P 4000 [60] Profilate 4000 [60]
0.49
0.43
0.12
0.11
300
600
720
1.35 1.39
1.07 1:04
1.01 0.91
3.32 3.50
1.14 1.74
0.83 0.90
0.98 0.87
0.56 0.69
360 270
540 210
540 180
0.63
0.99
1.22
1.71
1.04
1.01
2.08 2.18 (10') 2.88
Facteur Willebrand 7360 [110] Hemate-P 2600 [40] Facteur Willebrand 4600 [71] Recombinate 2770 [43] Hemate-P 2500 [40] FVIII-VHP-vWF 3300 [52] Recombinate 4000 [63]
1.39
1.16
360
240
840
0.86 0.37
0.90 0.59
0.89 0.73
1.75 1.52
1.20 0.69
0.72 0.69
2.25 1.45
0.96 0.63
0.58 0.66
540 300
960 495
960 > 1200
0.29 1.30 1.12
0.04 1.11 1.16
0.03 0.79 0.85
0 2.55 1.57
0 1.05 0.53
0 0.56 0.31
0 1.92 1.59
0 0.78 0.63
0 0.50 0.33
> 1200 240 550
n.d. 480 1080
n.d. 480 > 1200
0.51
0.08
0.04
0
0
0
0
0
0
> 1200
n.d.
Concentrate Dosage IU[IU/kg] (VIII:C or RCof for Facteur Willebrand)
VIII: C IU/ml after 1h 12 h
Hemate-P 2960 [40] Profilate 3000 [40] FVIII-VHP-vWF 3300 [43] Facteur WiUebrand 5520 [74] Recombinate 2959 [40] Hemate-P 2500 [30] Profilate 2500 [30]
0.59 0.84 0.84
Duke bleeding time s after 10rain 12h 24h
n.d.
255
Assays
In vivo studies
Venou~ blood for coagulation analysis was collected into siliconeco~ted vacutainer tubes (Becton Dickinson) with a volume of 5 ml, containing 0.5 ml 0.129 M sodium citrate. Platelet-poor plasma (PPP) was obtained after centrifugation at 2000x g for 10 min. Plasma not immediately analyzed was frozen and kept at - 7 0 ~ until assessed. Platelet-rich plasma (PRP) was obtained after centrifugation at 275 x g for 10 min. Bleeding time was measured both with a Simplate-II device (normal range: 360-720 s) and according to Duke's method (norreal range: 60-300 s). Factor VIII clotting activity (VIII: C) was assayed with a chromogenic substrate method (Coatest Factor VIII, KabiVitrum) (normal range: 0.5-2.1 1U/ml) [17] and a one-stage clotting assay (normal range: 0.5-1.7 IU/ml) [28]. Von Willebrandfactor antigen (vWF:Ag) was measured with an IRMA using a monoclonal antibody against vWF (normal range: 0.5-2.0 IU/ml) [15, 35]. Standard plasma from 20 healthy subjects was calibrated against the 13th British standard (85/573) for analyses of VIII : C and the 1st British standard (85/644) for analyses of vWF:Ag. Results were expressed in IU/ml. The multimeric structure of vWF (multimeric sizing) in PPP was analyzed with sodium dodecyl sulfate agarose gel electrophoresis (1.9070 agarose concentration) as described elsewhere [34]. The multimeric bands were identified with a 125I-labeled mouse monoclonal antibody followed by autoradiography [15]. Ristocetin cofactor activity (RCof) was determined with a method described by Zuzel and co-workers [41] (normal range: 0.50-1.60 IU/ml) using formalin-fixed normal platelets and the patient's PPP. Platelets were fixed by incubating 1 part of PRP with 1 part of formalin in Tris-saline buffer (8 mg/ml formalin) for 18 h at 4~ The platelets were washed and resuspended in Trissaline buffer containing 0.15 M NaC1 and 0.1 M Tris, pH 7.4. Pharmacokinetics: The half-life of VIII : C was calculated as described by BjOrkman and co-workers [6]. Adverse events were checked for. All patients enrolled into the study gave their informed consent, and the study was approved by the Ethics Committee of the Medical Faculty, University of Lund.
P l a s m a c o n c e n t r a t i o n s o f V I I I : C, v W F : A g , R C o f a n d the p o s t - i n f u s i o n D u k e b l e e d i n g times are shown in Table 2. F i g u r e 2 shows the r e s p o n s e in v W F : A g , R C o f , V I I I - C a n d D u k e b l e e d i n g t i m e in p a t i e n t 1, w h o received all five concentrates. B o t h D u k e a n d S i m p l a t e II bleeding times exceeded 1200 s in all p a t i e n t s before t h e i n f u s i o n o f f a c t o r concentrates. D u k e b l e e d i n g t i m e s h o r t e n e d in response to all concentrates except R e c o m b i n a t e , w h i c h d i d n o t affect t h e b l e e d i n g time. W i t h t h e exception o f p a t i e n t 4, w h o m a n i f e s t e d a r e m a r k a b l y p o o r response to H e m a t e - P , t h e s h o r t e n i n g o f bleeding t i m e lasted l o n g e r after H e m a t e - P ( 2 4 - 3 6 h ) t h a n after F V I I I - V H P - v W F ( 4 - 2 4 h ) o r Facteur W i l l e b r a n d ( 8 - 2 4 h) (Table 2). A s can be seen f r o m Fig. 2, the s h o r t e n i n g o f b l e e d i n g t i m e after P r o f i l a t e was
Results In vitro studies T h e in vitro p r o p e r t i e s o f the different concentrates are given in Table 3. T h e m u l t i m e r i c v W F p a t t e r n was a l m o s t n o r m a l in vitro in H e m a t e - P , F V I I I - V H P - v W F , a n d Facteur W i l l e b r a n d , whereas P r o f i l a t e lacked the largest m u l timers (Fig. 1). N o v W F was detected in R e c o m b i n a t e .
Table 3. In vitro properties of the FVIII/vWF concentrates studied Concentrate
VIII : C (IU/ml)
Hemate-P Profilate FVIII-VHP-vWF Facteur Willebrand Recombinate
28 33 19 10 68
vWF : Ag IRMA (IU/ml)
RCof IU/ml
Multimeric pattern
69 108 40 68
56 105 43 43
Large Large Large Large
< 0.001
0
present absent present present
No vWF
Fig. 1. Multimeric structure of the vWF in normal plasma (NP) and in plasma from patient 1 with vWD type III before (B) and: 1, 4, 12, and 24 h after administration of HematexP, Facteur Willebrand, and Profilate and in vitro in the same three concentrates
256
VIII=C (IU/ml)
Vlll:C
(lU/rnl
1.0
1.0-
0.5
0.80.6-
vWF=Ag IRMA (IU/ml)
0.40.21.5
O" ....
Before 1
0.5
a,
13
i
12
//
,
24
//
3'6
,;,
48h
b
Fig. 3. Effect of administration on VIII 9C in plasma of three patients with vWD type III: patient 1 (40 IU/kg) (9 9 patient 4 ([] []), and patient 5 (A ~); and in three patients with severe hemophilia A: patients H1 (47 IU/kg) (o e), H2 (61 IU/kg) (m l), and H3 (35 IU/kg) (zx. zx)
Rcof (IU/ml)
1.5
0.5 Duke BT (see)
50OI Before
2 4
8
12
24
36
48
72 h
Fig. 2. Effect of administration of Hemate-P (9 9 F VIIIVHP-vWF ([] ~), Facteur Willebrand (& ... A), Profilate (e e), and Recombinate ( " B) on VIII:C, vWF:Ag (IRMA), and RCof in plasma and Duke bleeding time in patient 1, with vWD type III
of shorter duration, except when the dosage was large (60 IU VIII:C/kg; Table 2). The Simplate II bleeding time did not shorten after any of the concentrates (not shown). Except for Recombinate, which contains no vWF, all concentrates produced large increases in plasma vWF : Ag concentrations, peak levels usually occurring within the first hour (Fig. 2). Figure 1 shows the multimeric pattern of the vWF in plasma in patient 1: Almost all multimers appeared after Hemate-P, Facteur Willebrand, and FVIII-VHP-vWF, and were still detectable after 4 h, but after 12 h the largest multimers had disappeared. After Profilate the largest multimers were lacking. As expected, no vWF multimers were seen in patients receiving Recombinate (not shown), vWF was undetectable in platelets both before and after infusion of all concentrates (not shown), as demonstrated earlier [16]. RCof was increased by all concentrates except Recombinate, the highest levels being detected after Facteur Willebrand. Profilate produced increases in RCof similar to or slightly higher than those produced by Hemate-P, but in patient 2 the duration of the increase was very short
after Profilate. There was no correlation between the increase in RCof and the multimeric pattern of vWF in plasma. The plasma concentration of VIII:C was increased after all five concentrates and remained high for long periods after Hemate-P, FVIII-VHP-vWF, Facteur Willebrand, and Profilate (Table 2 and Fig. 2). Facteur Willebrand, which contains only small amounts of FVIII, induced the lowest initial increases in VIII:C (0.320.56 IU/ml after 10 min and 0.37-0.63 IU/ml after 1 h) but the VIII'C concentrations continued to increase for another 24-48 h (Fig. 2). In the vWD patients the duration of the V I I I ' C response after Recombinate was very short, the half-life being only 3 h 55 min, 5 h 4 min, or 5 h 55 min, as compared with 8 h 9 min, 10 h 1 min, or 11 h 7 min in the patients with severe hemophilia A (Fig. 3). No adverse events were seen after any of the concentrates.
Discussion
In vWD, the aim of treatment is to correct the defect in primary hemostasis, the prolonged bleeding time, and the FVIII deficiency. In mild vWD type I, this can be achieved by administration of DDAVP, but in vWD type III, where the vWF is completely absent and the FVIII concentration very low, plasma concentrates must be given. The first concentrates to become available were human fraction I - O (AHF-Kabi) [25] and cryoprecipitate [3, 32], both of which contain FVIII and the full complement of vWF multimers [4, 30] and have been found to promote platelet adhesion to the subendothelium [38]. Because of insufficient virus inactivation, AHF-Kabi is no longer produced, and the use of cryoprecipitate is questionable. Therefore, it is important to evaluate the efficacy of FVIII concentrates of higher purity and with a higher degree of virus safety for the treatment of patients with vWD type III. The aim of the present study was to investigate the effect of factor concentrates of different composition and
257 quality of vWF and FVIII on hemostatic variables in patients with vWD type III. Comparisons between concentrates should therefore be done primarily in individual patients. As Hemate-P was given to all five patients it may serve as a reference. The recovery of VIII 9C, vWF : Ag, and RCof differed markedly between the concentrates. The complex nature of the vWF protein complicates pharmacokinetic evaluation of FVIII/vWF concentrates in vWD, as the concentration of vWF.'Ag or the RCof in plasma does not necessarily reflect the biological effectiveness of the vWF, which is dependent on the multimeric structure of the vWF. The improvement of multimeric vWF pattern in plasma and shortening of bleeding time are important determinants of hemostatic capacity of concentrates and cannot be described with ordinary pharmacokinetic variables. It has been suggested that normal multimeric structure of the concentrates is a prerequisite for normalization of the bleeding time [4, 201. Studies with the annular perfusion chamber technique [2, 36] have shown one of the primary functions of vWF to be mediation of platelet interactions with the subendothelium, the largest vWF multimers being the most active [38]. Moreover, Federici and co-workers [10] found the largest vWF multimers to have stronger affinity than the smaller multimers for the platelet receptors glycoprotein Ib and I I b / I I I a . We found Hemate-P, FVIII-VHP-vWF, and Facteur Willebrand to have a multimeric vWF pattern in vitro almost similar to that of normal plasma and to temporarily normalize the multimeric vWF-pattern in plasma after intravenous administration. These three concentrates manifested lasting effects on Duke bleeding times and on the vWF and RCof concentrations in plasma, indicating that they may be clinically effective in connection with bleeding caused by impaired primary hemostasis in vWD, mainly bleeding from mucous membranes. Our findings are in agreement with those of earlier studies [5, 11]. Berntorp and Nilsson [5] found Hemate-P to temporarily correct the hemostatic defect in 11 patients with vWD (7/11 with type III) and to give satisfactory hemostasis in connection with childbirth and major surgery. Goudemand and co-workers [11] found Facteur Willebrand to temporarily correct the bleeding time, greatly improve the plasma vWF multimeric pattern, increase RCof, and give satisfactory hemostasis in connection with different bleeding episodes in 9 vWD patients (1/9 with type III). Profilate, on the other hand, shortened the Duke bleeding time despite the lack of the largest multimers and despite the fact that the multimeric structure in plasma did not normalize, though the effect on the bleeding time was of shorter duration than that of the other concentrates. The relatively short duration of Profilate's effect on bleeding times is probably due to its lack of the large multimers. The presence of the intermediate and small multimers of the vWF, which are also capable of mediating platelet adhesion, explains the fact that Profilate nevertheless shortened the bleeding time. Rodhegiero and co-workers [33] recently argued that normalization of the bleeding time is required only in cases of mucosal, especially gastrointestinal bleeding,
and that good postsurgical hemostasis had been obtained in several cases, even in connection with tooth extraction, despite the fact that the Ivy bleeding time was not corrected, provided that a sufficiently high FVIII concentration was obtained. We found, however, that none of the concentrates was able to shorten the Simplate II bleeding time, even if the multimeric pattern was normalized in plasma. The Duke bleeding time is a less sensitive test and is probably clinically more useful for evaluating the risk of excessive operative blood loss in patients with type III vWD [27]. Both Berntorp and Nilsson [5] and Goudemand and co-workers [11] used the Duke bleeding time method in their type-Ill patients. The amount and quality of platelet vWF are important determinants of primary hemostasis, bleeding time and severity of bleeding symptoms in vWD [12, 16, 19]. As known, platelets in vWD type III lack vWF. There was no uptake of vWF in platelets after any of the concentrates given in this study, which may explain why the Simplate II bleeding time does not normalize even if the vWF in plasma is normal. We found that Hemate-P, FVIII-VHP-vWF, Profilate, and Recombinate, which all contain FVIII, induced large initial FVIII increases due to their FVIII content. Facteur WiUebrand, which contains only small amounts of FVIII, induced a lower initial increase. It has long been known that administration of vWF to patients with vWD type III induces a secondary increase of FVIII in plasma [25], probably by enhancing secretion [13]. The secondary increase in FVIII was most evident after administration of Facteur Willebrand, as it contains only small amounts of FVIII, a rapid release of FVIII from endogenous reservoirs being seen already after 10 min, and the plasma concentrations of V I I I ' C peaked after 24-48 h, suggesting that the FVIII release continued, possibly until an optimal ratio between the amount of FVIII and vWF subunits in plasma had been established. Similar secondary FVIII increases were observed after administration of all vWF-containing concentrates (Fig. 2). A tendency was observed toward a faster drop in VIII:C concentrations after Profilate, which may have been due to a faster elimination rate of vWF. It is obvious that the half-life of FVIII is very short when the patient is devoid of vWF, as shown by the difference in response to the administration of Recombinate between the yon Willebrand patients and the hemophilia patients (Fig. 3). The normalization of plasma vWF is of importance not only for primary hemostasis, but also for maintaining the increased FVIII concentration at a high level for a long period of time. This is achieved both by a slower elimination rate of the infused FVIII due to binding to vWF [14, 39] and as a result of the vWF-induced release of endogenous FVIII [13, 25]. To sum up, several factor concentrates are now available which are suitable for treatment of patients with vWD. The present findings show that even if a FVIII concentrate lacking the largest multimers may have some hemostatic effect, it is advantageous to administer a concentrate consisting of all vWF multimers in order to achieve a more durable hemostatic effect. A pure vWF concentrate induces a long-lasting FVIII increase and
258 m a y therefore be used prophylactically, b u t in the event
o f bleeding o r as cover for surgery, w h e n a m o r e p r o m p t effect is necessary, a c o n c e n t r a t e also c o n t a i n i n g F V I I I s h o u l d be a d m i n i s t e r e d .
Acknowledgements. The investigation was supported by the Swedish Medical Research Council (grant no. 00087), the Medical Faculty, University of Lund, Malm6 General Hospital Research Foundations, and the Agnes and Nils Nilsson Foundation.
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