Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension

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.

Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

“…2,[7][8][9][10][11] Although it remains to be clarified whether endothelial cells from various vascular beds share this property, it is thought that nonhepatic endothelial cells might be a major source of FVIII secretion and contribute to the DDAVP-releasable pool in vivo. We wanted to know whether targeting FVIII expression to endothelial cells can reestablish a regulated releasable pool of FVIII and correct the hemophilia A phenotype.…”

“…[2][3][4][5] It has been proposed that synthesis of FVIII occurs in a subpopulation of endothelial cells. [6][7][8][9][10][11] Targeting FVIII expression to lung endothelial cells 12 or liver sinusoidal endothelial cells 13 has been shown to result in phenotypic correction in hemophilia A mice. In vitro studies performed in our laboratory have demonstrated that FVIII traffics to storage granules in a von Willebrand factor (VWF)-dependent manner and is coreleased with VWF by agonist stimulation.…”

Neutralizing capacity of inhibitors on FVIII is lower for natural FVIII/VWF complex than for isolated FVIII: in vitro comparative study in eleven different therapeutic FVIII concentrates

“…The most plausible explanation for this effect may lie in the way in which VWF and FVIII are naturally synthesized and released by endothelial cells. Both FVIII and ultra-large multimers of VWF (ULVWF) are packaged in the WeibelPalade bodies of select types of primary endothelial cells [5,6]. Upon secretion of the granules, strings of ULVWF anchored on the cell surface are cleaved by the metalloprotease ADAMTS13 into smaller FVIII/ VWF multimers [6,7], while pH-induced dissociation of VWF propeptide allows both FVIII to bind and FVIII/VWF multimers to adopt a globular conformation [8].…”

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

“…18 Several groups reported recently that subsets of endothelial cells exist that synthesize FVIII and store it with VWF in WPBs. [19][20][21][22] Previously, it was demonstrated that overexpression of FVIII in endothelial cells results in FVIII targeting to WPBs. [23][24][25][26] The targeting signal for FVIII sorting to WPBs is currently unclear, although the typical high-affinity interaction with VWF seems not required.…”