Abstract
BackgroundCoagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive.Methodology/Principal FindingsImmunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by rt-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab–reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 5′ exons within the int22h-1 repeat responsible for intron 22 inversions in 40% of severe haemophiliacs. A reciprocal relationship between the presence of short isoforms and full-length FVIII transcript suggested potential splice-switching mechanisms.Conclusions/SignificanceThe pulmonary endothelium is confirmed as a site of FVIII secretion, with evidence of synthesis, cell surface expression, and coexpression with vWF. There is complex alternate transcription initiation from the FVIII gene. These findings provide a framework for future research on the regulation and perturbation of FVIII synthesis, and of potential relevance to haemophilia, thromboses, and pulmonary hypertensive states.
Highlights
Coagulation cascade activation is essential for normal haemostasis [1]
There is complex alternate transcription initiation from the factor VIII (FVIII) gene. These findings provide a framework for future research on the regulation and perturbation of FVIII synthesis, and of potential relevance to haemophilia, thromboses, and pulmonary hypertensive states
FVIII Protein Expression in Lung Tissue Factor VIII expression by the pulmonary endothelium was assessed in situ, by comparing serial sections from the same donor blocks stained with the IgG1 isotype control mAb, CD31 mAb, or C5 mAb to FVIII
Summary
Coagulation cascade activation is essential for normal haemostasis [1]. Activated factor VIII (FVIIIa) is responsible for sustained intravascular generation of thrombin via its role as a cofactor for FIXa in the intrinsic Xase, with FVIIIa/FIXa responsible for most of the FXa produced by both extrinsic (tissue-factor initiated) and intrinsic coagulation cascades [2]. Elevated plasma levels of FVIII are unusual amongst general thrombotic risk factors, as they are a risk factor for venous thromboembolism, and associated with chronic thromboembolic pulmonary hypertension [9,10]. High levels of von Willebrand Factor (vWF), the glycoprotein with which FVIII circulates in a non-covalent complex [11], are observed in pulmonary hypertensive states [9,10]. The liver produces sufficient FVIII for normal plasma levels [12], with immunohistochemical evidence for stronger expression by hepatic sinusoidal endothelial cells than hepatocytes [13]. We hypothesised that pulmonary endothelial cells might be a source of plasma FVIII after demonstrating an age-independent association between elevated plasma FVIII levels and pulmonary arteriovenous malformations (AVMs) in hereditary haemorrhagic telangiectasia (HHT) [15]. We examined expression and processing of FVIII by a number of normal pulmonary and systemic endothelial types, in situ and in vitro, and compared to expression of von Willebrand Factor
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