Regulatory components of the alternative complement pathway in endothelial cell cytoplasm, factor H and factor I, are not packaged in Weibel-Palade bodies.

Nancy A Turner,Sarah E Sartain,Shiu-Ki Hui,Joel L Moake

doi:10.1371/journal.pone.0121994

Abstract

It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF —- but not factor H.

Highlights

Von Willebrand factor (VWF) is a large multimeric protein composed of monomeric subunits (~250 kDa) linked by disulfide bonds into large polymers
[8] These proteins include the components of the alternative complement pathway, including the negative complement regulatory proteins, factor (F) H and FI. (FH and FI are functionally related control proteins.) In our initial investigation of the release of complement components from human umbilical vein endothelial cells (HUVECs), we did not detect organelle storage of any complement protein, including FH and FI, prior to release from HUVECs. [8] A more recent article made the contradictory contention that FH is stored in HUVEC Weible-Palade bodies (WPBs) and is co-secreted with VWF during cell stimulation
Using immunofluorescent microscopy and non-overlapping spectral secondary antibody pairs, FH was detected throughout the HUVEC cytoplasm (Fig. 1A and 1D), whereas only VWF was detected in HUVEC WPBs (Fig. 1B and 1E)

Summary

Introduction

Von Willebrand factor (VWF) is a large multimeric protein composed of monomeric subunits (~250 kDa) linked by disulfide bonds into large polymers. VWF multimers are synthesized in human vascular endothelial cells (ECs) and in megakaryocytes. [1] The multimers are stored in the Weible-Palade bodies (WPBs) of endothelial cells, as well as in the alpha granules of megakaryocytes and their derivative circulating cells, platelets. [2] (WPBs and alpha granules are analogous organelles with similar contents.) When stimulated, ECs secrete ultra-large (UL), hyper-adhesive VWF multimers that initiate platelet adhesion. We recently demonstrated that human umbilical vein endothelial cells (HUVECs) express the mRNA for complement components, and synthesize and release the associated proteins. [8] A more recent article made the contradictory contention that FH is stored in HUVEC WPBs and is co-secreted with VWF during cell stimulation. Conducted additional image analysis to determine the precise endothelial cell location of FI

Methods

Results

Conclusion