Reconstitution of CD39 in liposomes amplifies nucleoside triphosphate diphosphohydrolase activity and restores thromboregulatory properties Carolyn A.

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Reconstitution of CD39 in liposomes amplifies nucleoside triphosphate diphosphohydrolase activity and restores thromboregulatory properties Carolyn A. Haller, PhD, Wanxing Cui, MD, PhD, Jing Wen, MD, Simon C. Robson, MD, PhD, Elliot L. Chaikof, MD, PhD Journal of Vascular Surgery Volume 43, Issue 4, Pages 816-823 (April 2006) DOI: 10.1016/j.jvs.2005.11.057 Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 1 A, Coomassie staining of affinity-purified CD39 reveals one strong band migrating at approximately 70 kDa. B, Western immunoblot analysis confirms the expression of CD39 with a 70 kDa band recognized by BU61. C, The 54 kDa native CD39 product was observed following deglycosylation. Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 2 Substrate concentration dependence of adenosine diphosphatase (ADPase) activity by detergent solubilized CD39 (○) and liposome-incorporated CD39 (•). Values were normalized to rates obtained with 300 μM adenosine di phosphate (ADP). Data are average values from three independent experiments (standard errors <5%). Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 3 Inhibition of platelet aggregation by CD39 lipid vesicles. A, Platelets were activated with 10 μM adenosine diphosphate, final CD39 concentration is 2.1 × 10−7 M; (B) 5 μg/mL collagen, final CD39 concentration is 5.3 × 10−7 M; (C) 0.1 U/mL thrombin, final CD39 concentration is 1.1 × 10−6 M. Representative data sets are presented (n = 4). PRP, Platelet-rich plasma. Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 4 CD39 lipid vesicles promote platelet disaggregation. CD39 lipid vesicles (7.9 × 10−7 M final CD39 concentration) were added to plasma-rich platelets 1 minute after the addition of 5 μM adenosine diphosphate (ADP). Representative data set is presented (n = 4). Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 5 Platelet counts after thromboplastin injection. Results are mean platelet count ± SEM. White bar, control mice receiving empty vesicle injection 2 minutes before blood draw (n = 10). Black bar, mice receiving CD39 vesicle treatment 2 minutes before thromboplastin (65 μL/kg) injection (n = 10). Grey bar, mice receiving empty vesicle treatment 2 minutes before thromboplastin (65 μL/kg) injection (n = 10). TF, tissue factor. Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

Fig 6 A, Mortality after thromboplastin injection. Black bar, mice receiving CD39 liposomes (n = 12). Grey bar, mice receiving empty lipid vesicles (n = 15); P < .05. Representative pulmonary sections (×100) stained with hematoxylin and eosin from mice receiving thromboplastin (65 μL/kg) and treated either with (B) empty lipid vesicles or (C) CD39. Pulmonary thrombi (black arrows) were observed in empty lipid vesicle group, but were a far less frequent occurrence among mice treated with CD39 (P < .05). Journal of Vascular Surgery 2006 43, 816-823DOI: (10.1016/j.jvs.2005.11.057) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions