Today's article under review, from 2007, comes to us from the National Amyloidosis Centre in London. (You may notice some amyloidosis superstars among the list of authors.) The findings in this article are what make fibrinogen amyloidosis different from all other types of amyloidosis (as far as I am aware.)
Authors: Glenys A. Tennent, Stephen O. Brenna, Arie J. Stangou, John O'Grady, Philip N. Hawkins, and Mark B. Pepys (National Amyloidosis Centre, London, UK; Christchurch Hospital, Christchurch, New Zealand; King's College Hospital, Londin, UK)
Journal: Blood (2007)
Abstract:
Hereditary systemic amyloidosis caused by fibrinogen Aα-chain gene mutations is an autosomal dominant condition with variable penetrance, usually of late onset, and typically presents with nephropathy leading to renal failure. Amyloid deposits often develop rapidly in transplanted kidneys, and concomitant orthotopic liver transplantation has lately been performed in several patients with the hope of halting amyloid deposition. Fibrinogen is produced in vitro by hepatocytes but also by other human cell types, and although the liver is the source of plasma fibrinogen in vivo in rats, this is not known in humans. Transplantation of livers expressing wild-type fibrinogen into patients with variant fibrinogen amyloidosis provides a unique opportunity to establish the source of human plasma fibrinogen. We therefore characterized plasma fibrinogen Aα-chain allotypes by electrospray ionization mass spectrometry mapping of tryptic digests before and after liver transplantation. Before liver transplantation, fibrinogen amyloidosis patients with the Glu526Val Aα-chain variant had approximately equal proportions of peptide with the wild-type sequence TFPGFFSPMLGEFVSETESR, and with the amyloidogenic variant sequence TFPGFFSPMLGEFVSVTESR, as expected for individuals heterozygous for the mutation. After transplantation, only the wild-type sequence was detected, and the liver is thus the source of at least 98% of the circulation fibrinogen.
Here is a link to the PDF if you would like to follow along: http://bloodjournal.hematologylibrary.org/content/109/5/1971.full.pdf
The article starts with some basic info about fibrinogen. You may already know that fibrinogen is a protein that is important in the formation blood clots, and there are many fibrinogen mutations known to cause blood clotting disorders. But here are some bits of information to keep in the back of your mind when someone asks you "what is fibrinogen?"
The next few paragraphs of the article describe in detail the laboratory procedures used to isolate fibrinogen and do other analysis of the various blood samples. I will not pretend to come close to understanding any of that, so we will move on to the results.
And here are the results, summarized in one simple chart:
Don't worry if you do not have a clue what that chart means. I didn't either the first time I saw it, and it took a little digging into the article and a few other places to understand it enough to know what is important for this discussion. But I think I can explain it without getting too technical.
First, this chart displays something called a tryptic map for each of six different blood samples, represented by the letters A through F. Think of a tryptic map as a way of indicating the various components a substance is made of. In this case the substance is the fibrinogen A alpha chains extracted from a blood sample. The horizontal scale along the bottom of each tryptic map, as noted in the not-very-helpful caption, runs from 1060 to 1150. Those numbers have something to do with atomic weights (m/z is mass-to-charge ratio), and we care about what shows up at 1118 and 1133. Wild-type fibrinogen shows up at 1133, and variant fibrinogen (for the Glu526Val mutation) shows up at 1118. So we see in the map for row A there is a large spike at 1133, and there is basically nothing at 1118. Now compare that to row A, which has shorter spikes at both 1133 and 1118, both of which seem to be at about the 50% level on the vertical scale.
Now that we know what these charts are showing, and we know to focus on what appears at 1118 and 1133, we can see the big picture a little better once we know who these blood samples were taken from, and when. Here is that info:
A: Healthy person without the Glu526Val mutation
B: Case 1, 29 days before transplant
C: Case 1, 46 days after transplant
D: Case 1, 137 days after transplant
E: Case 2, immediately before transplant
F: Case 2, 92 days after transplant
Now we can see what these charts are telling us. As we would expect, the person without the mutation (Row A) has no variant fibrinogen. There is only a spike at 1133. Rows B and E, on the other hand, both show patients with the mutation before transplant. They each have approximately equal amounts of variant and wild-type fibrinogen (spikes at 1118 and 1133.)
When we look at the rows representing blood drawn after transplant (Rows C, D and F), we only see spikes at 1133 again, indicating those patients have no circulating variant fibrinogen. Although it is not shown here, the article also mentions that no variant fibrinogen was detected in Case 3, the patient who had a liver transplant 7 years prior.
So that is really the main point of this article, that fibrinogen appears to be produced solely in the liver. If there is any variant fibrinogen produced outside the liver, it is essentially undetectable. The article concludes with this: ". . . our present results thus formally validate the rationale for use of liver transplantation in treatment of hereditary fibrinogen amyloidosis."
I want to discuss one more thing about the chart from the article, which sheds some light on the terminology sometimes used when referring to this mutation. At the bottom of the figure above, below chart A, there are two lines, each starting with "T-62." T-62 is a peptide, and peptides are building blocks of proteins. In this case, T-62 is a building block of the fibrinogen protein molecule. The building blocks of peptides are amino acids, and what is shown on those two rows are the amino acid sequences for the variant and wild-type versions of this particular peptide, which is the one affected by the Glu526Val mutation. You will notice there is only one letter different between the two, and it is underlined. In wild-type fibrinogen it is "E" and in variant fibrinogen it is "V." So, what do E and V represent? Actually, E and V are the same thing as Glu and Val in Glu526Val. In case you do not remember from my basic DNA lesson in the post from March 20, 2013, Glu is the three-letter abbreviation for the amino acid Glutamic Acid, and Val is the three-letter abbreviation for the amino acid Valine. So E is the single-letter abbreviation for Glutamic Acid (the letter G is assigned to Glycine), and V is the single-letter abbreviation for Valine. That is why this mutation is sometimes written as E526V.
This article gives us some good, solid data to explain why liver transplantation is so successful in the treatment of fibrinogen amyloidosis. It's a little on the technical side, but science has to be that way sometimes. And if you thought this article was technical, just wait until the next one.
=====
Citation:
(1) Tennent GA, Brennan SO, Stangou AJ, O'Grady J, Hawkins PN, Pepys MB. Human plasma fibrinogen is synthesized in the liver. Blood 2007; 109: 1971-1974.
The article starts with some basic info about fibrinogen. You may already know that fibrinogen is a protein that is important in the formation blood clots, and there are many fibrinogen mutations known to cause blood clotting disorders. But here are some bits of information to keep in the back of your mind when someone asks you "what is fibrinogen?"
- The typical concentration of fibrinogen in the blood is 2 to 4 grams per liter. Assuming an average adult has a total blood volume of approximately 5 liters, that average adult would have roughly 10 to 20 grams of fibrinogen circulating in their blood under normal conditions.
- The half-life of fibrinogen is approximately four days. That means that if you could track all of the fibrinogen molecules that your body manufactured on a specific day, half of that fibrinogen would be gone four days later. (In case you are curious, the half life of white blood cells is about 7 hours, and the half life of red blood cells is about 50 days.)
- A fibrinogen molecule consists of three chains, usually written in the medical literature as Aα, Bβ, and γ. Those symbols, if they appeared correctly wherever you are reading this, are alpha, beta, and gamma, the first three letters of the Greek alphabet. (That will likely be the last time I use the symbol for alpha instead of just writing "alpha," since I have to copy it over from Word.) The mutations that cause amyloidosis affect the A alpha chain, which is why the journal articles often refer to it as "fibrinogen A alpha chain" or "fibrinogen alpha chain" amyloidosis. Those are the same thing as "fibrinogen amyloidosis." There are mutations that affect the fibrinogen beta or gamma chains, but none of those are known to cause amyloidosis. In the future, if a mutation of the beta or gamma chain is discovered to cause amyloidosis, then there will be a need to differentiate between fibrinogen alpha chain amyloidosis and fibrinogen beta or gamma chain amyloidosis.
At the time this article was written, fibrinogen was known to be produced by liver cells in vitro (outside the body, such as in a test tube or petri dish), but it was unknown to what extent fibrinogen is produced by other types of cells in vivo (within the body). Other proteins are known to be produced as needed by other types of cells, and the concentration of fibrinogen is known to increase in response to injury, infection or inflammation, so the authors of this article set out to determine whether or not a significant amount of fibrinogen was produced other than in the liver.
The article then gives an overview of current treatments for fibrinogen amyloidosis, which is kidney or combined liver and kidney transplants. When just a kidney transplant is done, the transplanted kidney is usually affected by amyloid deposits, whereas when a combined liver and kidney transplant is done, patients have shown improvement due to a lack of buildup of additional amyloid deposits and regression of the existing deposits. The article states that the first liver transplants for fibrinogen amyloidosis were done on the presumption that the liver is the source of the variant (mutant) fibrinogen, but that has never been investigated until now.
Before continuing I need to define some terms. When a person has the mutation for fibrinogen amyloidosis (and they are heterozygous, meaning they have one normal copy of the gene and one mutated copy), half of the fibrinogen produced will be normal, and the other half will be mutated instead of normal. The medical term most often used to refer to the mutated fibrinogen (or any other protein) is "variant," and the term most often used to refer to the normal fibrinogen (or any other protein) is "wild-type." I had no idea what the term "wild-type" meant the first time I heard it, and I was really surprised when I first learned that it simply means normal. But if you think of "wild-type" as the type most often occurring in nature, or "in the wild," it starts to make some sense. So this article review will frequently refer to variant fibrinogen and wild-type fibrinogen.
The last item discussed in the article before describing the actual analysis that was performed was the use of liver transplantation to treat transthyretin amyloidosis (ATTR). Those patients may improve after a liver transplant, but with many of the ATTR mutations, amyloid deposits can still build up for two reasons. First, transthyretin is also produced outside the liver, and second, wild-type transthyretin can still deposit as amyloid. (That was discussed at the familial amyloidosis meeting in October. They believe that the amyloid deposits initially formed by the variant transthyretin act as sort of a scaffold that wild-type transthyretin continues to build on, even without additional variant transthyretin.) The good news for those of us with fibrinogen amyloidosis is that there is no evidence that wild-type fibrinogen can form amyloid deposits.
Ok, now for the data analysis in the article. They analyzed blood taken at various times from five unrelated patients, all of whom had the Glu526Val mutation for fibrinogen amyloidosis. They also analyzed blood taken from healthy subjects of the same ages for comparison. Here is a description of each patient and when blood was taken from the ones who had received transplants:
Case 1: Male. Combined liver and kidney transplant in 2004. Blood was drawn 29 days before transplant, 46 days after, and 137 days after
Case 2: Male. Combined liver and kidney transplant in 2004. Blood was drawn immediately before the transplant and 92 days after.
Case 3: Female. Combined liver and kidney transplant approximately 1999. Blood was drawn 7 years after transplant.
Case 4: Male, waiting on transplant.
Case 5: Female, waiting on transplant.
The article then gives an overview of current treatments for fibrinogen amyloidosis, which is kidney or combined liver and kidney transplants. When just a kidney transplant is done, the transplanted kidney is usually affected by amyloid deposits, whereas when a combined liver and kidney transplant is done, patients have shown improvement due to a lack of buildup of additional amyloid deposits and regression of the existing deposits. The article states that the first liver transplants for fibrinogen amyloidosis were done on the presumption that the liver is the source of the variant (mutant) fibrinogen, but that has never been investigated until now.
Before continuing I need to define some terms. When a person has the mutation for fibrinogen amyloidosis (and they are heterozygous, meaning they have one normal copy of the gene and one mutated copy), half of the fibrinogen produced will be normal, and the other half will be mutated instead of normal. The medical term most often used to refer to the mutated fibrinogen (or any other protein) is "variant," and the term most often used to refer to the normal fibrinogen (or any other protein) is "wild-type." I had no idea what the term "wild-type" meant the first time I heard it, and I was really surprised when I first learned that it simply means normal. But if you think of "wild-type" as the type most often occurring in nature, or "in the wild," it starts to make some sense. So this article review will frequently refer to variant fibrinogen and wild-type fibrinogen.
The last item discussed in the article before describing the actual analysis that was performed was the use of liver transplantation to treat transthyretin amyloidosis (ATTR). Those patients may improve after a liver transplant, but with many of the ATTR mutations, amyloid deposits can still build up for two reasons. First, transthyretin is also produced outside the liver, and second, wild-type transthyretin can still deposit as amyloid. (That was discussed at the familial amyloidosis meeting in October. They believe that the amyloid deposits initially formed by the variant transthyretin act as sort of a scaffold that wild-type transthyretin continues to build on, even without additional variant transthyretin.) The good news for those of us with fibrinogen amyloidosis is that there is no evidence that wild-type fibrinogen can form amyloid deposits.
Ok, now for the data analysis in the article. They analyzed blood taken at various times from five unrelated patients, all of whom had the Glu526Val mutation for fibrinogen amyloidosis. They also analyzed blood taken from healthy subjects of the same ages for comparison. Here is a description of each patient and when blood was taken from the ones who had received transplants:
Case 1: Male. Combined liver and kidney transplant in 2004. Blood was drawn 29 days before transplant, 46 days after, and 137 days after
Case 2: Male. Combined liver and kidney transplant in 2004. Blood was drawn immediately before the transplant and 92 days after.
Case 3: Female. Combined liver and kidney transplant approximately 1999. Blood was drawn 7 years after transplant.
Case 4: Male, waiting on transplant.
Case 5: Female, waiting on transplant.
The next few paragraphs of the article describe in detail the laboratory procedures used to isolate fibrinogen and do other analysis of the various blood samples. I will not pretend to come close to understanding any of that, so we will move on to the results.
And here are the results, summarized in one simple chart:
 |
| Representative ESIMS tryptic maps of purified A alpha chains recorded in the m/z range 1060-1150. |
Don't worry if you do not have a clue what that chart means. I didn't either the first time I saw it, and it took a little digging into the article and a few other places to understand it enough to know what is important for this discussion. But I think I can explain it without getting too technical.
First, this chart displays something called a tryptic map for each of six different blood samples, represented by the letters A through F. Think of a tryptic map as a way of indicating the various components a substance is made of. In this case the substance is the fibrinogen A alpha chains extracted from a blood sample. The horizontal scale along the bottom of each tryptic map, as noted in the not-very-helpful caption, runs from 1060 to 1150. Those numbers have something to do with atomic weights (m/z is mass-to-charge ratio), and we care about what shows up at 1118 and 1133. Wild-type fibrinogen shows up at 1133, and variant fibrinogen (for the Glu526Val mutation) shows up at 1118. So we see in the map for row A there is a large spike at 1133, and there is basically nothing at 1118. Now compare that to row A, which has shorter spikes at both 1133 and 1118, both of which seem to be at about the 50% level on the vertical scale.
Now that we know what these charts are showing, and we know to focus on what appears at 1118 and 1133, we can see the big picture a little better once we know who these blood samples were taken from, and when. Here is that info:
A: Healthy person without the Glu526Val mutation
B: Case 1, 29 days before transplant
C: Case 1, 46 days after transplant
D: Case 1, 137 days after transplant
E: Case 2, immediately before transplant
F: Case 2, 92 days after transplant
Now we can see what these charts are telling us. As we would expect, the person without the mutation (Row A) has no variant fibrinogen. There is only a spike at 1133. Rows B and E, on the other hand, both show patients with the mutation before transplant. They each have approximately equal amounts of variant and wild-type fibrinogen (spikes at 1118 and 1133.)
When we look at the rows representing blood drawn after transplant (Rows C, D and F), we only see spikes at 1133 again, indicating those patients have no circulating variant fibrinogen. Although it is not shown here, the article also mentions that no variant fibrinogen was detected in Case 3, the patient who had a liver transplant 7 years prior.
So that is really the main point of this article, that fibrinogen appears to be produced solely in the liver. If there is any variant fibrinogen produced outside the liver, it is essentially undetectable. The article concludes with this: ". . . our present results thus formally validate the rationale for use of liver transplantation in treatment of hereditary fibrinogen amyloidosis."
I want to discuss one more thing about the chart from the article, which sheds some light on the terminology sometimes used when referring to this mutation. At the bottom of the figure above, below chart A, there are two lines, each starting with "T-62." T-62 is a peptide, and peptides are building blocks of proteins. In this case, T-62 is a building block of the fibrinogen protein molecule. The building blocks of peptides are amino acids, and what is shown on those two rows are the amino acid sequences for the variant and wild-type versions of this particular peptide, which is the one affected by the Glu526Val mutation. You will notice there is only one letter different between the two, and it is underlined. In wild-type fibrinogen it is "E" and in variant fibrinogen it is "V." So, what do E and V represent? Actually, E and V are the same thing as Glu and Val in Glu526Val. In case you do not remember from my basic DNA lesson in the post from March 20, 2013, Glu is the three-letter abbreviation for the amino acid Glutamic Acid, and Val is the three-letter abbreviation for the amino acid Valine. So E is the single-letter abbreviation for Glutamic Acid (the letter G is assigned to Glycine), and V is the single-letter abbreviation for Valine. That is why this mutation is sometimes written as E526V.
This article gives us some good, solid data to explain why liver transplantation is so successful in the treatment of fibrinogen amyloidosis. It's a little on the technical side, but science has to be that way sometimes. And if you thought this article was technical, just wait until the next one.
=====
Citation:
(1) Tennent GA, Brennan SO, Stangou AJ, O'Grady J, Hawkins PN, Pepys MB. Human plasma fibrinogen is synthesized in the liver. Blood 2007; 109: 1971-1974.
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