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Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

University of Birmingham

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

Deep vein thrombosis (DVT) with its major complication, pulmonary embolism, is a global health problem. Mechanisms of DVT remain incompletely understood. Platelets play a role in DVT, but the impact of specific platelet receptors remains unclear. Platelet C-type lectin-like receptor 2 (CLEC-2) is known to maintain the physiological state of blood vasculature under inflammatory conditions. DVT is a thromboinflammatory disorder developing largely as sterile inflammation in the vessel wall. We hypothesized therefore that CLEC-2 might play a role in DVT. Here, using a murine DVT model of inferior vena cava (IVC) stenosis, we demonstrate that mice with general inducible deletion of CLEC-2 or platelet-specific deficiency in CLEC-2 are protected against DVT. No phenotype in the complete stasis model was observed. Transfusion of wild-type platelets into platelet-specific CLEC-2 knockout mice restored thrombosis. Deficiency in CLEC-2 as well as inhibition of podoplanin, a ligand of CLEC-2, was associated with reduced platelet accumulation at the IVC wall after 6 hours of stenosis. Podoplanin was expressed in the IVC wall, where it was localized in the vicinity of the abluminal side of the endothelium. The level of podoplanin in the IVC increased after 48 hours of stenosis to a substantially higher extent in mice with a thrombus vs those without a thrombus. Treatment of animals with an anti-podoplanin neutralizing antibody resulted in development of smaller thrombi. Thus, we propose a novel mechanism of DVT, whereby CLEC-2 and upregulation of podoplanin expression in the venous wall trigger thrombus formation.

Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis.

Deep vein thrombosis (DVT) and its complication pulmonary embolism have high morbidity reducing quality of life and leading to death. Cellular mechanisms of DVT initiation remain poorly understood.

Holly Payne

Institute of Cardiovascular Sciences,
College of Medical and Dental Sciences,
Institute of Cardiovascular Sciences, College of Medical and Dental Sciences

University of Birmingham

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Holly Payne

Institute of Cardiovascular Sciences,College of Medical and Dental Sciences,Institute of Cardiovascular Sciences, College of Medical and Dental Sciences

University of Birmingham

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Institute of Cardiovascular Sciences,
College of Medical and Dental Sciences,
Institute of Cardiovascular Sciences, College of Medical and Dental Sciences