Abstract

Abstract 3383

Introduction:

Anecdotal and published reports on the use of specific plasma-derived Factor VIII (FVIII)-von Willebrand Factor (vWF) concentrates in the treatment of congenital thrombotic thrombocytopenic purpura (TTP) led to the determination of the functional and antigenic content of ADAMTS13 in a number such concentrates commercially available in the US and/or the EU.

Deficiency or inhibition of ADAMTS13 is the putative cause of TTP. TTP is a rare disorder causing microvascular thrombosis resulting in low platelet counts. It affects 1 –5 patients per 1,000,000 population. The current treatment of this disorder consists of regular infusions of fresh frozen plasma (FFP) for inherited TTP and exchange plasmapheresis for the acquired version of the disorder. The possibility of utilizing FVIII-vWF concentrates that incorporate robust virucidal treatments in their manufacturing and the possible utilization of lower infusion volumes prompted the evaluation of these concentrates for ADAMTS13 content.

Methods:

For this analysis, we obtained 5 lots of Koate®-DVI, 2 lots of Human Bioplasma, and 1 lot of each of the following concentrates: Humate® P, Wilate®, Alphanate®, Emoclot® and Fractogel.® Following reconstitution of these concentrates in 10 mL of water for injection, we determined the concentration of ADAMTS13 antigen by an ELISA method previously described by Feys et al. (J Thromb Haemost. 2006; 4: 955–62) with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239). The lower limit of detection was 1% while the lower value of the normal range was 45%. The ADAMTS13 activity was measured using collagen binding (CBA) and FRET assays with minor modifications (Peyvandi et al. Haematologica 2008; 93: 232–239); in both assays, serially diluted normal human plasma (NHP) and plasma samples were diluted 1:10 in assay buffer and incubated 1:1 with vWF substrate in a final volume of 100 μl. In addition to the determination of ADAMTS13, we evaluated the amount of vWF antigen (vWF:Ag) using a commercial kit (Instrumentation Laboratory, IL US, Bedford, Massachusetts, USA) in these concentrates and the multimeric composition of this protein. Utilizing densitometry, we determined the percentage of ultra-large vWF multimers in each of these concentrate.

Results:

The analysis showed that among the tested concentrates, Koate®-DVI had the highest ratio of ADAMTS13/FVIII. This concentrated presented ratios of 9.1 ± 0.7% (Mean ± SD) and 8.4 ± 1.1% (Mean ± SD) per IU FVIII for ADAMTS13 activity and antigen respectively. The remainder of the concentrates contained only nominal amounts of ADAMTS13; only Alphanate® reached a ratio of 0.23% ADAMTS13 activity/IU FVIII. These data are in agreement with previous reports (Qorraj et al. Blood 2001, 116: 3677). A summary analysis data are shown in Table 1.

Factor VIII ConcentrateLot NumberFactor VIII Content (IU/mL)vWF:Ag (IU/mL)UL vWF Ratio (Normal Range 0.85 – 1.21)ADAMTS13 Activity (%)ADAMTS13 Antigen (%)
Koate-DVI (5 lots) 100 281±22* 0.39±0.04* 908±70* 842±12* 
Wilate A014A1814 90 130 0.49 12 13 
Humate P 30666911A 66 183 0.66 22 61 
Alphanate B2AC900111 100 165 0.09 230 387 
Emoclot 460804 100 54 0.16 22 58 
Human BioPlasma 8615 25 0.24 <3 No Sample 
Human BioPlasma 8617 100 24 0.61 <3 No Sample 
Fractogel 230/11 100 146 0.21 16 10 
Factor VIII ConcentrateLot NumberFactor VIII Content (IU/mL)vWF:Ag (IU/mL)UL vWF Ratio (Normal Range 0.85 – 1.21)ADAMTS13 Activity (%)ADAMTS13 Antigen (%)
Koate-DVI (5 lots) 100 281±22* 0.39±0.04* 908±70* 842±12* 
Wilate A014A1814 90 130 0.49 12 13 
Humate P 30666911A 66 183 0.66 22 61 
Alphanate B2AC900111 100 165 0.09 230 387 
Emoclot 460804 100 54 0.16 22 58 
Human BioPlasma 8615 25 0.24 <3 No Sample 
Human BioPlasma 8617 100 24 0.61 <3 No Sample 
Fractogel 230/11 100 146 0.21 16 10 
*

Mean ± SD.

Koate®-DVI displayed a ratio of vWF:Ag/FVIII of approximately 2.81, very similar to the ratio of 2.77 included in the single lot of Humate®-P. The ratio of ultra-large multimers to total multimers in Humate® P was higher, at 0.66 vs. 0.39 for Koate-DVI. However, the lower content of ultra-large multimers in Koate®-DVI may be advantageous in its possible utilization in the treatment of TTP.

Conclusion:

On the basis of these results, Koate-DVI may have a role to play in the management of congenital and, possibly, idiopathic TTP, especially on the basis of observations that FVIII accelerates the breakdown of vWF (Cao et al. PNAS 2008, 105: 7416–7421). Additional characterization, testing in pertinent animal models and pilot studies in humans would be required to determine the extent to which this concentrate can assist in the treatment of TTP.

Disclosures:

Guazzini:Kedrion S.p.A: Consultancy. Retzios:Kedrion Biopharma, Inc.: Consultancy.

Author notes

*

Asterisk with author names denotes non-ASH members.