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A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia

Nature Biotechnology volume 29, pages 1028–1033 (2011)Cite this article

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Abstract

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXaI16L) rendered partially inactive by a defect in the transition from zymogen to active protease1,2. Using mouse models of hemophilia, we show that FXaI16L has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients3. FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

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Figure 1: Effect of hFXaI16L on coagulation parameters.
Figure 2: Blood loss following tail-clipping.
Figure 3: Platelet and fibrin accumulation following laser-induced arteriole injury in WT mice and hFXaI16L-treated HB mice.

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Acknowledgements

This work was supported in part by US National Institutes of Health grant P01 HL-74124, Project 2; research funding from Pfizer (to R.M.C.); and the Judith Graham Pool Postdoctoral Research Fellowship, National Hemophilia Foundation (to L.I.). We would like to thank L. Albert and B. Carito (Pfizer), and A. Hannan (Children's Hospital of Philadelphia) for their technical assistance. We are also grateful to S. Krishnaswamy (Children's Hospital of Philadelphia/University of Pennsylvania) for useful suggestions and critical review of the manuscript.

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Authors and Affiliations

  1. Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

    Lacramioara Ivanciu, Raffaella Toso, Paris Margaritis, Giulia Pavani, Haein Kim, Alexander Schlachterman, Jian-Hua Liu, Valder R Arruda & Rodney M Camire

  2. The University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania, USA

    Paris Margaritis, Valder R Arruda & Rodney M Camire

  3. Pfizer Inc., Cambridge, Massachusetts, USA

    Valerie Clerin, Debra D Pittman, Rosalind Rose-Miranda, Kathleen M Shields, David V Erbe & James F Tobin

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  1. Lacramioara Ivanciu
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Contributions

L.I. designed, coordinated and performed experiments; R.T., P.M., G.P., H.K., A.S., J.-H.L. and K.M.S. performed experiments; R.R.-M. coordinated and performed experiments; V.C. designed and analyzed experiments; D.D.P. interpreted data; J.F.T., D.V.E., V.R.A. and R.M.C. designed experiments and analyzed data. L.I. and R.M.C. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Rodney M Camire.

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Competing interests

R.M.C. receives licensing fees and research funding from Pfizer. V.C., D.D.P., R.R.-M., K.M.S., D.V.E. and J.F.T. are employees of Pfizer, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1 and Supplementary Figures 1–8 (PDF 573 kb)

Supplementary Movie 1

Representative movie depicting thrombus formation following laser injury in a wild-type mouse (Balb/c). Brightfield images of the cremaster muscle along with accumulating platelets and fibrin are shown. Platelets (red) were detected by an Alexa555-labeled rat anti- CD41 F(ab)2 and fibrin (green) with Alexa488-labeled anti-fibrin antibody; areas of overlap are depicted by yellow. A 10 μm scale bar is shown in the lower left corner and a time stamp in the upper right corner. For convenience, the speed of the movie is increased by 5-fold. Further details of the methodology can be found in Methods. (MOV 4026 kb)

Supplementary Movie 2

Representative movie depicting thrombus formation following laser injury in an HB mouse (Balb/c) treated with FXaI16L (10 μg/kg) prior to laser injury. Platelets and fibrin accumulation were detected as described in Supplemental Movie 1 and in Methods. (MOV 5221 kb)

Supplementary Movie 3

Representative movie depicting thrombus formation following laser injury in an HB mouse (Balb/c) treated with FXaI16L (30 μg/kg). In this experiment, the animal was injured and monitored for approximately 3.5 min. After this observation period, FXaI16L was infused via a jugular vein cannulus and thrombus formation was immediately monitored. The animal was not repositioned or reinjured. Platelets and fibrin accumulation were detected as described in Supplemental Movie 1 and in Methods. (MOV 15529 kb)

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Ivanciu, L., Toso, R., Margaritis, P. et al. A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia. Nat Biotechnol 29, 1028–1033 (2011). https://doi.org/10.1038/nbt.1995

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