09.08>> Snow White...    
Snow White and the Eighth Factor  

As the Queen stitched, snowflakes falling outside the window distracted her, and the needle suddenly pricked her finger. Three drops of bright red blood fell on the white snow outside! The Queen said to herself, ‘How nice it would be to have a pretty child, white as snow and rosy as the red blood.” Lo and behold, Snow White then was born. Of course, the Queen never thought about how that pinprick in her skin would be put together again. She just kept on stitching. All she knew was that within a few hours the pinprick would be gone. While she was embroidering, though, her body was working to make sure the tiny hole in the Queen’s hand was rapidly repaired.

Actually, many steps are involved in plugging-up even the tiniest of pinpricks in a finger. The initial plug, or blood clot, is initiated by platelets that circulate in the blood specifically for that purpose. Then there are many different proteins, called clotting factors, which contribute to stop the bleeding through the complex process of coagulation. These proteins include Factors I through XIII, protein C, protein S, von Willebrand Factor, and antithrombus III. They must work in a highly cooperative manner to do the job. To give you an idea of just how well-coordinated this process of coagulation is, look at the diagram below from the article on coagulation in Wikipedia:


While we all take blood clotting for granted, if any one of the critical coagulation factors is missing, the consequences can be deadly. For instance, deficiency of Factor VIII or IX leads to conditions known as Hemohilia A and B, respectively.  

Boys vs. Girls
Overall, hemophilia is rare (about 20,000 people in the US have the condition) but Hemophilia A is by far the most common type of the disease. It is estimated that one out of every five- to ten-thousand males in the US cannot make sufficient quantities of Factor VIII, which is crucial to the creation of a successful clot. Without Factor VIII, the body is unable to plug the hole fully, and as a result the person may keep bleeding indefinitely. There are different levels of severity depending on how much, or how little, Factor VIII the body is able to produce. In the most severe cases, even a pinprick could possibly prove deadly.

There are many reasons why sufficient amounts of Factor VIII may not be produced. The gene that makes Factor VIII is located on the X chromosome. Therefore, mutations in the gene encoding Factor VIII are inherited through the X chromosome and can be passed only from mother-to-child. If the child is female, she has two X chromosomes, so it does not matter if only one of the X chromosomes carries the Factor VIII mutation. In this situation, she will not suffer from hemophilia. She is known as a carrier of hemophilia because the affected chromosome can be passed on to her child. But if the child is male, he will inherit the X chromosome from his mother and the Y chromosome from the father, so the only copy of Factor VIII gene he receives will be the defective one from the hemophilia carrier mother. That is why only males suffer from the consequences of Hemophilia A.

People with Hemophilia A are unable to properly make blood clots due to the lack of Factor VIII. Even minor injuries may result in excessive bleeding and, without prompt medical attention and administration of Factor VIII, the person may die from blood loss. It is for this reason that hemophilia can be a dangerous disease if a person is unaware of it, and hemophiliacs need to carefully avoid trauma. While there are treatments to induce clotting in hemophiliacs, there is no known cure for Hemophilia A.

Of course, people with hemophilia can and do encounter injuries just like non-hemophiliacs: they break bones and require various types of surgery. A lot of work has been done over decades to understand the role of Factor VIII, including determining where in the body Factor VIII is made. Interestingly, it had been noted that hemophiliacs who received liver transplants were suddenly cured of their hemophilia.

The liver has several types of cells and it was unclear which one of these cells was most important for producing and releasing Factor VIII. This is where Dr. Sanjeev Gupta of the Albert Einstein College of Medicine in New York comes in. “These cases and evidence from previous studies suggested that something important was happening with Factor VIII and liver cells,” said Dr. Gupta, a Professor of Medicine and Pathology, and an expert in hepatology. “We decided to investigate.”

The Liver: There's More to It Than You Think
The liver is the largest internal organ in the human body and serves many important functions. It is responsible for metabolism, or processing of food you eat into energy you can use. The liver synthesizes many proteins, including those that circulate in the blood. It makes bile, which is secreted into the small intestine to help with digestion. The liver processes various chemicals, drugs and toxins to make them safe for the body. So overall, the liver is responsible for regulating blood sugar, amino acid and lipid levels, and synthesizing cholesterol, sugars and proteins, as well as removing drugs or toxins.

The liver is made up of many cell types. Two main types of liver cells are epithelial cells, or hepatocytes, and sinusoidal endothelial cells. Endothelial cells form the lining of the blood-filled spaces of the liver and possess highly specialized functions, including production of growth factors or cell signaling molecules, as well as scavenger functions required for incorporating specific substances.

This figure demonstrates transplanted liver sinusoidal endothelial cells (LSEC) after these cells integrated and proliferated in sinusoidal spaces of the mouse liver. Transplanted LSEC are in green color, which was produced by immunostaining for Green Fluorescent Protein (GFP) in transplanted LSEC. GFP protein will have been produced in only endothelial cells because its expression was regulated by the Tie-2 promoter, which is active in only endothelial cells. Native LSEC are in red color due to immunostaining for the CD31 marker of endothelial cells. On occasion, transplanted LSEC appear to be yellow due to overlapping green and red colors from GFP and CD31 expression in cells.

Dr. Gupta initially hypothesized that hepatocytes were making Factor VIII because of their wide variety of roles in the liver. To test this hypothesis, Dr. Gupta used a mouse model of Hemophilia A where the Factor VIII gene had been disrupted by DNA recombination methods or “knocked out ” from the genome. Liver cells in these animals were healthy except that they did not make Factor VIII. Dr. Gupta’s team then injected these hemophilia mice with normal hepatocytes to see if they would begin to produce Factor VIII. They did not.

So That Didn't Work. What Do We Do Now?

So, contrary to what Dr. Gupta had initially expected, transplantation of healthy hepatocytes did not have an effect on Factor VIII levels in hemophilia mice. “I was surprised,” said Dr. Gupta. “But I knew that hepatocytes were not the only option. It was possible that endothelial cells were making Factor VIII, or that Factor VIII was being produced by something different.” Dr. Gupta and his team then examined the capacity of cells other than hepatocytes (specifically, liver sinusoidal endothelial cells) and found that Factor VIII appeared in the blood of mice after transplantation of these cells. This was the first clue that transplantation of liver sinusoidal endothelial cells might be capable of correcting Hemophilia A.

In the next step, Dr. Gupta and his team determined whether liver endothelial cells could be replaced by transplanted cells. The effort was to develop suitable ways to have transplanted cells survive and proliferate in the liver, which was their natural home. This required a series of studies in mice with genetically marked cells from donor mouse livers. The goals of these studies were to identify transplanted cells and to create sufficient room in the recipient liver for transplanted cells to proliferate. Dr. Antonia Follenzi and other members of the team were successful in establishing that transplanted liver endothelial cells survived, proliferated, and functioned normally after the liver had been injured by a particular chemical. They then studied Factor VIII production from liver sinusoidal endothelial cells by injecting hemophiliac mice with endothelial cells from normal, healthy mice. “This time,” said Dr. Gupta, “large amounts of Factor VIII appeared in the blood stream and were maintained at high levels indefinitely.”  Moreover, hemophiliac mice acquired the capability to stop bleeding. In other words, transplanted liver endothelial cells were producing Factor VIII and mice treated with healthy cells had been cured of hemophilia.

“This is a huge finding,” remarked Dr. Gupta. We can now add Hemophilia A to the list of diseases that can be cured by liver cell therapy. This means that one day we will be able to inject people with Hemophilia A with healthy endothelial cells and restore the capacity of their livers to produce Factor VIII. This could be a lifesaver for many hemophiliacs. Perhaps one day hemophiliacs will be able, like the Queen, to go on stitching while that pinprick disappears.

This principle of replacing liver endothelial cells needs additional work before it can be used in clinical human medicine, although it represents a major advance in the area of tissue and organ transplantation

Dr. Gupta is Professor of Medicine and Pathology and Dr. Follenzi is Instructor of Pathology at Albert Einstein College of Medicine in New York. Their major area of research is liver-directed cell and gene therapy.

Dr. Sanjeez Gupta Dr. Sanjeev Gupta

Dr. Antonia Follenzi Dr. Antonia Follenzi

How Clotting Works How Clotting Works

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You Can Be A Star - Living with hemophilia
You Can Be A Star - Living with hemophilia


To Learn More

  • Follenzi, Antonia, et al. "Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice." Journal of Clinical Investigation. 2008; 118: 93.5-945

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Written by Rebecca Kranz with Andrea Gwosdow, Ph.D. Gwosdow Associates



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