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S92 PULMONARY ENDOTHELIAL CELL SECRETION OF FACTOR VIII
1CL Shovlin, 2R Manning, 2G Angus, 1M Laffan, 1G Birdsey, 2FA Mauri. 1Imperial College London, London, UK, 2Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, UK
Introduction: Activated factor (F)VIII is responsible for sustained intravascular generation of thrombin via its role as a cofactor for FIXa in the coagulation cascade. Elevated plasma levels of FVIII are a strong predictor of recurrent venous thromboses/pulmonary emboli and are associated with thromboembolic pulmonary hypertension. The sources of plasma FVIII have remained elusive. Having demonstrated that FVIII levels are elevated in hereditary haemorrhagic telangiactasia associated with pulmonary arteriovenous malformations (Shovlin et al. Thromb Haemost 2007), we tested the hypothesis that human pulmonary artery endothelial cells (HPAEC) secrete FVIII and compared expression patterns with three other primary endothelial cell types under basal and stimulated conditions.
Methods: Primary human endothelial cells (Promocell) were cultured in individual wells of BD Falcon Culture Slides, fixed and permeablised with methanol. Wells were treated with one of four hybridoma-derived murine anti-human FVIII:Ag monoclonal antibodies and rabbit-raised anti-von Willebrand Factor (vWF), anti-calnexin or anti-COPII. Primary antibodies were detected with Alexa-Fluor labelled anti-rabbit or anti-mouse IgG. Stained cells covered with Vectashield were imaged on an LSM 510 Zeiss inverted fluorescence confocal microscope using sequential acquisition and LSM Image Browser software. Prior to immunohistochemistry of lung tissue blocks, antigen retrieval methods were optimised using paraffin-embedded formalin-fixed blocks derived from cultured HPAEC. FVIII secretion into conditioned media was quantified by ELISA (Immunobind, Axis-Shield) using manufacturer’s protocols.
Results: All four anti-FVIII antibodies to both heavy and light FVIII chains demonstrated significant reactivity in HPAEC. In contrast to reported retention of endogenous FVIII in hepatocyte endoplasmic reticulum (ER) (Becker et al. Thromb Haemost 2004), in HPAEC there was limited colocalisation with calnexin. Colocalisation was observed with …