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Thromb Haemost 2011; 106(04): 665-674
DOI: 10.1160/TH11-04-0264
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Factor VIII activation by factor VIIa analog (V158D/E296V/M298Q) in tissue factor-independent mechanisms[*]

Kenichi Ogiwara
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara 634–8522, Japan
,
Keiji Nogami
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara 634–8522, Japan
,
Midori Shima
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara 634–8522, Japan
› Author Affiliations Financial support: This work was partly supported by grants for MEXT KAKENHI 21591370 and a special project award in Bayer Hemophilia Awards 2009.
Further Information

Publication History

Received: 26 April 2011

Accepted after major revision: 18 July 2011

Publication Date:
15 December 2017 (online)

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Summary

Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. The recombinant (r)FVIIa analog, NN1731 (V158D/E296V/ M298Q) containing a thrombin/FIXa-mimicking catalytic domain, is ~30-fold more effective on activated platelets without TF, but ~1.2-fold with TF, than rFVIIa for FX activation. We have recently demonstrated the FVIIa/TF-dependent FVIII activation in the early coagulation phase. We assessed the action of NN1731 on FVIII activation. NN1731/TF increased FVIII activity ~2.9-fold within 30 seconds, followed by rapid inactivation, and was slightly more active than rFVIIa/TF. NN1731-catalysed activation, however, was enhanced ~6-fold at 5 minutes (min), and its peak level persisted for ~30 min. NN1731/TF proteolysed FVIII at Arg740, Arg372, and Arg336, similar to rFVIIa/TF, but cleavage by NN1731 alone was much slower at Arg336 than at Arg740 and Arg372. The Km and Vmax for NN1731/TF-catalysed activation were ~1.8-fold lower and ~2.3-fold greater than rFVIIa/TF. The Km for NN1731 alone was ~1.3-fold lower than rFVIIa, whilst the Vmax was ~7.9-fold greater, indicating that the efficiency of FVIII activation by NN1731 and NN1731/TF was ~11- and ~4-fold greater, respectively, than equivalent reactions with rFVIIa. In SPR-based assays, NN1731 bound to FVIII and the heavy chain (Kd; 0.62 and 1.9 nM) with ~1.4- and ~3.1-fold higher affinity than rFVIIa, and the A2 domain contributed to this increase. Von Willebrand factor moderated NN1731-catalysed activation more significantly than NN1731/TF. In conclusion, NN1731 was a greater potential than rFVIIa in up-regulating FVIII activity, and the TF-independent FVIII activation might represent a potential extra mode of its enhanced haemostatic effect.

Keywords

Factor VIII(a) - factor VIIa-analog - activation - cleavage - tissue factor

Notes

* An account of this work was presented at the 50th annual meeting of the American Society of Hematology, December 7, 2008, San Francisco, CA, USA.


 

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