The Influence of Recombinant von Willebrand Factor of Different Multimer Sizes on the Activity of Factor VIII in Thrombin Generation and Chromogenic Assays S. Knappe, S. Till, F. Scheiflinger, and M. Dockal, Baxter Innovations GmbH, Vienna, Austria
Introduction
Objective
Results (cont.)
Von Willebrand factor (VWF) is a multimeric plasma glycoprotein with multiple physiological functions. VWF mediates between platelet surface receptors and the extracellular matrix component collagen during primary hemostasis. In addition, it binds and stabilizes procoagulant factor VIII (FVIII) in plasma. FVIII/VWF complex formation prevents FVIII from interaction with lower affinity binding partners such as FIXa, phospholipids and clearance receptors (1). FVIII is also protected from enzymatic (in)activation when bound to VWF. Thus, FVIII survival in the circulation depends on its “chaperone” VWF.
We investigated the effect of recombinant VWF (rVWF; BAX 111) and fractions thereof on the procoagulant activity of recombinant FVIII (rFVIII; ADVATE) in the absence of platelets in two assay systems.
Fractionated rVWF with reduced Mw had a smaller effect on FVIII procoagulant activity than BAX 111 containing ultra high Mw multimers (Figure 2). Thus, rVWF size-dependently protects or stabilizes FVIII in plasma in the absence of VWF’s platelet-mediated effects. The effect of rVWF and rVWF fractions was also assessed in a chromogenic FVIII activity assay (Figure 3). As in CAT, a differential activity increase was observed. Full rFVIII recovery was only achieved with larger rVWF multimers and at concentrations higher than ~0.2 IU/mL.
1 pM TF
16.7 pM FXIa
Phospholipids
4 µM (PPP LOW reagent)
4 µM (PC80/PS20)
rFVIII (mIU/mL)
1000
1000
1000
50
50
50
VWF (mIU/mL)
1000
200
20
50
10
1
1:1
1:0.2
1:0.02
1:1
1:0.2
1:0.02
~ 1:50
~ 1:10
~ 1:1
~ 1:50
~ 1:10
~ 1:1
Ratio rFVIII:VWF (IU/IU) Ratio rFVIII:VWF (moles monomers)
References:
Presented at the
(1) Terraube et al. Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance. Haemophilia 2010;16(1):3-13. th (2) Hemker et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33:415. (3) Turecek et al. Development of a plasma-and albumin-free recombinant von Willebrand factor. Haemostaseologie 2009; Suppl 1:S32-8.
Normal plasma 1 IU/mL rFVIII+ 1 IU/mL rVW rVWF (IU/mL) 0.2 IU/mL r
60 40
FVIII/VWF-def. plasma
20
0.02 IU/mL 1 w/o rVWF 0.2 FVIII/VWF-d plasma 0.02 Normal pla
0
80 70
0
10
20
30
40
50
75
rVWF BAX 111 HMW rVWF
65
MMW rVWF LMW rVWF
55 45
60
0,0 0,2 0,4 0,6 0,8 1,0 1,2
Thrombin generation assays were established to study the effects of rVWF on rFVIII activity independent of platelet-mediated effects. rVWF supports the procoagulant activity of FVIII. The effect is optimally supported by VWF of high Mw such as BAX 111 and is concentrationdependent. This novel assay system will serve to better characterize FVIII/VWF complexes for development of hemophilia or VWD therapies.
60 SSC Meeting of the International Society on Thrombosis and Haemostasis
10
20
30
40
50
60
rVWF (mIU/mL)
(A) Effect of rVWF BAX 111 and fractions with decreasing Mw on the procoagulant activity of rFVIII in the TF-triggered CAT (Table 1; n=2). Thrombin generation decreases with lower multimer size and lower rVWF concentration. (B) A similar trend is observed in the CAT triggered by FXIa (Table 1; n=2).
Figure 3: Effect of rVWF fractions on chromogenic FVIII activity
A
0,25
1,2
B
0,20
The authors of this presentation make the following disclosure of financial or personal relationships with commercial entities that may have a direct or indirect interest in the subject matter: All authors are full time employees of Baxter Innovations GmbH, Vienna, Austria.
0
rVWF (IU/mL)
(A) Thrombin (nM) over time in FVIII/VWF-depleted plasma incubated with rFVIII/rVWF (BAX 111) at different ratios. (B) Peak thrombin (nM) plotted against rVWF concentrations. Thrombin generation nearly reached the activity of a normal plasma control (grey line).
Disclosures:
70
rVWF (IU/mL)
40
Time (min)
90
50
50
60
110
0,0 0,2 0,4 0,6 0,8 1,0 1,2
30
0
85
130 Peak Thrombin (nM)
Peak Thrombin (nM)
B
80
B
95
rFVIII (IU/mL)
FXIa-triggered CAT
A
dOD405/min
TF-triggered CAT Trigger
A
Conclusions
Table 1: Overview of tested CAT conditions
Figure 2: Effect of rVWF fractions on TF- or FXIa-triggered thrombin generation
Figure 1: Effect of rVWF on TF-triggered thrombin generation Peak Thrombin (nM)
The activity of a rFVIII (ADVATE) was determined using calibrated automated thrombography (CAT) (2) with different coagulation triggers and a chromogenic assay in FVIII/VWF-double deficient platelet-poor plasma. rFVIII was pre-incubated with rVWF (3), or different rVWF multimer fractions with high (HMW), medium (MMW) and low (LMW) molecular weight (Mw). rVWF fractions were obtained by size-exclusion chromatography. rFVIII/VWF mixtures (Table 1) were added to FVIII/VWF-depleted plasma (Precision BioLogic). Plasma was treated with corn trypsin inhibitor (final conc. 41.3 µg/mL; Haematologic Technologies) to inhibit undesired contact activation through FXIIa. Thrombin generation was triggered with 1 pM tissue factor (TF) and 4 µM phospholipids (PPP LOW; Thrombinoscope) or 16.7 pM FXIa (Enzyme Research Laboratories) and 4 µM phospholipids PL80/20 (80% phosphatidylcholine/20% phosphatidylserine; Avanti). Samples were recalcified by FluCa reagent containing the substrate and CaCl2 (Thrombinoscope). Fluorescence was measured in a Fluoroskan Ascent® plate reader (ThermoScientific; filters 390 nm excitation and 460 nm emission) at 37°C for 90 min. All measurements were performed in duplicate in two independent experiments. In the TFtriggered CAT, rFVIII and VWF were analyzed at near physiologic concentrations. In the FXIatriggered CAT, rFVIII and VWF concentrations were reduced to gain sensitivity. Chromogenic activity was determined using the TECHNOCHROM® FVIII:C kit.
Thrombin generation in FVIII/VWF-deficient plasma was only partially restored by supplementation with 1 IU/mL FVIII. Addition of rVWF further increased thrombin formation reaching a normal plasma control (Figure 1). The activity increase was dependent on rVWF concentration, reaching saturation at about 0.4 IU/mL rVWF, which corresponds to a 20-fold molar excess of rVWF monomers.
Thrombin (nM)
Methods
Results
0,15 0,10
Plasma std. rFVIII - rVWF rFVIII + rVWF
0,05
1,0 0,8 0,6 0,4
0,00 0,0
0,5
1,0
1,5
2,0
FVIII (IU/mL)
rVWF BAX 111 HMW rVWF MMW rVWF LMW rVWF 0,0 0,2 0,4 0,6 0,8 1,0 1,2 rVWF (IU/mL)
(A) FVIII activity was measured in FVIII/VWF-deficient plasma in the presence and absence of 1 IU/mL rVWF (BAX 111). Only in the presence of rVWF rFVIII retains full activity. (B) Activity of 1 IU/mL rFVIII mixed with rVWF fractions (n=2).
Presented at the 8th Bari International Conference October 3-5, 2014; Bari, Italy