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Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies

Nature Biotechnology volume 26, pages 114–119 (2008)Cite this article

Abstract

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbα)1,2,3,4,5. Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbα–VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbα. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbα or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

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Figure 1: Ex vivo analysis of human and mouse platelet interactions with VWF.
Figure 2: Structure and comparison of human and murine GPIbα–VWF-A1 interface.
Figure 3: Analysis of VWF gene transcription/translation, Factor VIII activity, and collagen binding.
Figure 4: Hemostasis and thrombosis.

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Acknowledgements

We thank David Andrews (University of Miami) for performing VWF multimer gel analysis and Jeffrey S. Jhang (Special Hematology and Coagulation Laboratory, Columbia University Medical Center) for performing factor VIII function analysis. We thank B. Coller (Rockefeller University, New York) for providing the function-blocking mAbs 6D1 and 7E3, and R. Montgomery (Blood Center of Wisconsin, Milwaukee) for the mAb AP-1. This research is supported by National Institutes of Health grants HL063244, HL073646, and CA119342.

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Authors and Affiliations

  1. Department of Pediatrics, Columbia University Medical Center, Irving Cancer Research Institute, 1130 St. Nicholas Ave., New York, 10032, NY, USA

    Jianchun Chen, Kui Tan, Hairu Zhou, Hsuan-Fu Lo & Thomas G Diacovo

  2. Department de Radiobiologie et Radiopathologie, Commissariat à l'Energie Atomique, DRR/LRGH, CEA-Evry, 2 rue Gaston Cré mieux, Evry Cedex, 91057, France

    Diana Tronik-Le Roux

  3. Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, 92037, CA, USA

    Robert C Liddington

  4. Department of Pathology, Columbia University Medical Center, Irving Cancer Research Institute, 1130 St. Nicholas Ave., New York, 10032, NY, USA

    Thomas G Diacovo

Authors
  1. Jianchun Chen
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  2. Kui Tan
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Contributions

J.C. generated the mutant VWF mice and performed experiments; K.T. performed Southern blot analysis and genotyping of mice; H.Z. bred, screened, maintained the animal colony and performed experiments; H.-F.L. sequenced genomic DNA, maintained the animal colony and performed experiments; D.T.-L.R. provided αIIb−/− animals; R.C.L. generated atomic models and contributed to the writing of the manuscript; T.G.D. cloned the VWF gene, performed experiments and wrote the manuscript.

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Correspondence to Thomas G Diacovo.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1 and Figures 1–3 (PDF 2259 kb)

Supplementary Video 1

Laser-induced thrombus formation in arterioles contained within the cremaster muscle of a VWFR1326H mutant mouse. (MOV 2435 kb)

The ability of human platelets to preferentially support thrombus formation in the VWFR1326H mutant mouse was monitored in vivo by using 2-channel confocal intravital microscopy. Purified human platelets were labeled with BCECF ex-vivo, and mouse platelets with rhodamine 6G by intravenous administration. Upon induction of laser damage to the arterial vessel wall (A), human (green), and not mouse platelets (red), rapidly adhered to the site of injury, forming a large thrombus composed mainly of human cells. Rhodamine-labeled white blood cells can be seen interacting with the vessel wall in a post-capillary venule (V) next to the arterial.

Supplementary Video 2

Laser-induced thrombus formation in arterioles contained within the cremaster muscle of a WT mouse. (MOV 2963 kb)

Simultaneous monitoring of human (green) and mouse (red) platelet behavior in an animal possessing WT VWF reveals the lack of ability of human platelets to significantly participate in thrombus formation.

Supplementary Video 3

Human platelets can form occlusive arterial thrombi in VWF1326R>H mice despite the absence of αIIbβ3 on murine platelets. (MOV 2065 kb)

Mutant VWF-A1 mice were bred with αIIb null animals to generate the following genotype: Homozygous VWF1326R>H / αIIb -/-. Purified human platelets, labeled with BCECF ex-vivo, were infused continuously into the animal through a catheter inserted in the femoral artery. Vascular injury was induced by subjecting the arteriole to 5 second of continuous laser light (sweeping motion across the entire diameter of the vessel) at an intensity just below that required to rupture the vessel wall.

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Chen, J., Tan, K., Zhou, H. et al. Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies. Nat Biotechnol 26, 114–119 (2008). https://doi.org/10.1038/nbt1373

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