Turner and Joel Moake
Photo from Rice University
A pair of researchers solved a long-standing mystery about where factor VIII (FVIII) is made, stored, and deployed in the body, according to an article published in PLOS ONE.
The duo found that FVIII is synthesized in glomerular microvascular endothelial cells (GMVECs) and human umbilical vein endothelial cells (HUVECs).
FVIII and von Willebrand factor (VWF) are stored in Weibel-Palade bodies (WPBs) in both of these cell types. And stimulated GMVECs and HUVECs secrete cell-anchored, ultra-large VWF strings with FVIII bound to them.
The researchers said these findings may lead to improved therapy for hemophilia A.
“Great clinical advances have been made over the past 50 years, in spite of our lack of understanding of where FVIII was made and stored,” said study author Joel Moake, MD, of Rice University in Houston, Texas.
“Understanding how the body makes, stores, and deploys the protein will be increasingly important in the future as physicians look to develop gene therapies that could free patients from a reliance on regular injections of FVIII.”
The FVIII research in Dr Moake’s lab began in early 2014 and was based on a hunch by lead author Nancy Turner, who specializes in the study of endothelial cells.
“Endothelial cells are the gate-controllers for the blood system, and, to me, they make all the exciting proteins,” she said. “They’re right on the surface next to the blood, and they constantly interact with the plasma. Similar to circulating cells, they produce their own defensive proteins to fight infections.”
Though Turner had not previously studied FVIII, she had done extensive experiments on VWF, which is made in endothelial cells and stored in WPBs. And VWF and FVIII are often bound together.
Based on her prior work, Turner was intrigued by a pair of 2014 studies that examined FVIII in mouse endothelial cells and showed that FVIII was produced in the endothelial cells within the liver, and not in the liver cells.
“They stopped short of saying that FVIII was stored in endothelial cells, but they suggested the possibility,” she said. “One of the papers was very elegant, and I liked it a lot, but it made a statement that really bothered me.”
The paper explained that FVIII had been found in a half-dozen types of endothelial cell types, but never HUVECs, the type of endothelial cell found in large veins.
“HUVECs are the generic human endothelial cells that [researchers] use the first time they do anything,” Turner said. “They’re cheap. They’re easy to work with, and they’ve been the model for endothelial cells for, I don’t know, at least 50 years.”
“So far, everything I’ve ever looked at in endothelial cells has been consistent. The different types might have different amounts of something, but they’re very similar. There hasn’t been anything that was wholly different from one type to another. Not yet. So I thought, ‘OK, fine. I’m already doing gene-expression experiments. I might as well just throw FVIII in there and see.”
The experiments she was conducting involved both HUVECs and GMVECs, which are found in the smallest capillaries of the kidney. Turner was conducting an extensive analysis to see how protein production differed in the 2 varieties.
She examined the cells for FVIII messenger RNA and found that it was present, which meant it was possible the cells were making the protein. She then ordered a fluorescent antibody designed to detect FVIII.
“I did the experiment and looked, and not only was it there, but it was bright, easy to see, and it worked perfectly the first time,” Turner said. “I thought, ‘What have people been doing for 20 or 30 years? Why couldn’t they see this?’ And then I thought, ‘This was too easy. No one is going to believe me.’”
So Turner set about running controls to rule out mistakes. Were the antibodies interfering with one another? Was there any contamination? Was the microscope working correctly? Could the reading be a false-positive, an inadvertent result of another reaction she hadn’t expected?
“No. 1, I had to convince myself,” she said. “I am always skeptical if something is too easy. I have reviewed many, many papers, and I can always find what people do wrong.”
Once she had convinced herself, she and Dr Moake had to convince the paper’s referees. As she’d expected, they were skeptical. She said the bulk of the work over the past year involved doing a number of controls to remove any doubts about the findings.
In the end, the research confirmed that FVIII was made in both HUVECs and GMVECs. Moreover, Turner found that FVIII, like VWF, is both stored and secreted from WPBs.
Dr Moake said the discovery has clear implications for any future treatments that aim to repair genetic defects in patients with hemophilia A.
“Now that we recognize that FVIII is normally synthesized in endothelial cells and stored in Weibel-Palade bodies,” Dr Moake said, “those become the precise, most effective physiological targets for gene delivery.”
