Hemophilia Landscape Updates - February 2024
by Dr. David Clark
Hemophilia Carrier Screening
12/9/23 A group of physicians and researchers from Belgium pointed out that “Despite multiple awareness-raising initiatives, and in contrast with male persons with hemophilia (PwH), a considerable number of carriers and female PwH still go undiagnosed.” At the American Society of Hematology (ASH) meeting they presented the early findings of a carrier screening project.
They began by updating the family trees for all of the patients at their center and offering to genetically screen all of the females who hadn’t already been screened. In 228 families (180 A; 48 B), they found 900 females of which 454 were obligate carriers. An obligate carrier is a female whose father has/had hemophilia. She will be a carrier of her father’s mutated gene but may or may not have hemophilia herself. They also found 118 females who were not carriers, about 13%. This suggests that if you are a female in a hemophilia family, you only have about a 13% chance of not being a carrier. They also found eight women who should have been carriers based on the family trees but did not carry their “father’s” mutated gene and were actually not carriers. With so many subjects, genetic testing is still ongoing, so this is an interim report, but it still shows some concerning results.
Among the carriers of hemophilia B, 29.5% had factor IX levels below 40% of normal (considered the lower limit of normal internationally) and 41.0% had a factor level below 50% (the lower limit of normal in the U.S.). Note that women have been reported to bleed even with levels in the 60% range. In the hemophilia B-group, 1.3% of carriers had factor IX levels of 6-15%, 26.9% had levels of 16-39%, 24.4% had levels in the 40-60% range and the rest (about 47%) had levels above 60%. The proportions for hemophilia A were similar, plus there did not appear to be a significant difference between families with mild, moderate or severe hemophilia. The only significant difference is that the Bs had more instances of reproductive tract bleeding at 38.9% than the As at 5.7%.
The important finding here is that the average time to diagnosis for the females was seven years later than for the men included in the study as control subjects (members of the same families). Even for the women with factor levels below 40 or 50%, who actually do have hemophilia, the average age at diagnosis was 31.8 years for the Bs (25.8 for the As). (Note that Belgium has universal health care. The numbers in the U.S. may be worse.) The report concludes with the statement, “The carrier screening efforts that have been initiated at our center, and which should ideally be replicated across the hemophilia community globally, appear as a critical step towards providing equal access to hemophilia diagnosis and care to all potentially affected individuals, regardless of their gender.” [Krumb E et al., ASH abstract 288]
Is Inhibitor Development Affected by the Product Used?
11/20/23 Inhibitor development in hemophilia is still a mystery. There is a lot that we don’t know. One question that has come up over the years is whether the product used to treat the hemophilia patient affects inhibitor development. The answer has never been clear. Recently a large group of European and Canadian researchers decided to re-address that question. They looked at previously-untreated patients (PUPs – usually kids) with either hemophilia A or B at 56 European treatment centers and 23 Canadian centers. They found 312 subjects out of 1219 total hemophilia A severe PUPs who developed inhibitors, about 26%. For the As, inhibitor development was lower on plasma-derived factor VIII products; highest on standard half-life (SHL) products and intermediate on extended half-life (EHL) products. Inhibitor development rates also varied among the various SHL and EHL products.
For hemophilia B, the situation appears to be different. First, overall Bs had much lower inhibitor development rates, an average of 8% (14 of 173 study subjects). And, because there were many fewer subjects, the statistics were not able to show any significant difference among products. The results were that 11% (CI: 3-25%) developed an inhibitor on plasma-derived products, 8% (CI: 3-15%) on SHL products and 7% (CI: 1-22%) on EHL products.
Now, you might be thinking that’s the answer: plasma-derived is worse than SHL, which is worse than EHL. You would be wrong, and that’s why I included the confidence intervals (CI) with the numbers. In fact, this study doesn’t show any difference among the products.
The confidence intervals (CI) show the statistical ranges in which we believe that the result lies. For instance, with plasma-derived products the result is 11%, but we can only say with confidence that the actual number, if we included all possible subjects, is between 3% and 25%. To reduce the size of the confidence interval would require more subjects, but subjects with hemophilia B and an inhibitor are harder to find. Since the confidence intervals all overlap, we can’t say that the results are different.
Statistics are extremely important in science and medicine. Although the media hardly ever publish the statistics for the public to see, scientific publications usually do. Otherwise, we could be easily misled. Using the above example, for instance, I could say that 11% of hemophilia B PUPs develop inhibitors on plasma-derived products. My friendly-competitor in the next lab, however, could completely accurately tell me I was wrong – I only showed that the inhibitor rate is somewhere between 3% and 25%. That’s quite a difference, and I should be careful about staking my argument on the fact that I think the result is exactly 11%. Statistics show that 3%, 11%, 25% or anything in between are equally valid results from my data. (There goes my Nobel prize!)
Normally, we don’t show you the statistical results, but I’m showing them to you here, so you can get a better idea of how science actually works. We always look at the statistics before reporting findings in this newsletter, even if we don’t show them.
You don’t just do an experiment and get a result and that’s that. Usually, you do a number of experiments so you can get a statistical idea of how good your answer is. In medicine especially, we have to have good confidence in our results. We don’t just test one subject and assume that applies to all other patients. We have to test a number of subjects and average the results to get a good idea of what’s actually going on. The more subjects we test; the better our answer is.
So, the next time that your neighbor boasts that he’s getting 50 mpg from his new car, ask him what the confidence limits are on that number – maybe he had a tail wind? [Fischer K et al., Res Pract Thromb Haemost, online ahead of print 11/20/23]
Intradermal Injection Can Cause Inhibitor Development
11/4/23 Inhibitor development in hemophilia A can often be reversed by a method called immune tolerance induction (ITI) in which high factor VIII doses are given repeatedly over a period of time. However, ITI doesn’t work very well for hemophilia B, for unknown reasons. A group of US researchers wondered whether giving hemophilia B inhibitor patients factor IX injections into the skin (intradermal injections, ID) might work better. It didn’t, but their method may have given them an important insight into hemophilia B inhibitor development.
They found out that factor IX injection into the skin is actually a great way to give someone an inhibitor. Your skin is not just a simple covering for your body – it is a much more complex tissue that really protects you from a lot of outside dangers. It contains enzymes that can break down any foreign proteins that try to enter your body through the skin, plus a number of immune system components, including antibodies, that can fight off bacteria and viruses.
In hemophilia B mice, the researchers found that injection into the skin triggers inhibitor formation at about a 100 times lower dose than is typically needed to produce inhibitor formation by intravenous (IV) injection. Interestingly, however, they also found that ID factor injections seemed to keep the mice from developing the anaphylactic reactions that are often seen in B inhibitor patients. An anaphylactic reaction is a major allergic reaction that can be life-threatening.
So, what does this mean for inhibitor patients and also for products being developed for subcutaneous (SC) injection? Most B inhibitor patients probably did not acquire their inhibitors by ID contact, but it’s possible and needs more study. For the SC products being developed, it could be a concern? SC products are injected under the skin, not into it, but still in close proximity. As the authors point out, we have already seen cases in which manufacturers of currently-licensed EHL products have tried to develop an SC version. Development of two potential EHL-SC products was discontinued because of increased inhibitor development. This has been the case even with products that do not produce inhibitor formation when injected IV. This could be a can of worms that needs to be further explored. [Sherman A et al., Res Pract Thromb Haemost, online ahead of print 11/4/23]
Iron Overload in Hemophilia Joint Damage
12/6/23 We don’t know exactly what, on a molecular level, causes hemophilic joint damage. We know it’s caused by bleeding into the joints and there is evidence that iron from the hemoglobin in the blood is involved. In the last issue, we saw that a protein called YKL-40 is probably involved. Now another piece of the puzzle is becoming clear. A group from China has now shown that the excess iron from the blood can trigger macrophages to transform into an inflammatory form. Macrophages are a type of white blood cell, part of the immune system. When you bleed into a joint, macrophages are one of the types of immune cells that arrive at the bleeding site to clean up the mess. Chronic inflammation from repeated bleeds leads to damage to the synovial tissue around the joint, which is called synovitis. [Pang N et al., Haemophilia, online ahead of print 12/6/23]
Scramblases
8/25/23 Many of you have seen diagrams of the coagulation cascade (blood clotting system) and assumed that we therefore know everything about how blood clotting works. Not so fast! The diagrams that you see are only an approximation. There are many other molecules involved, probably many that haven’t even been identified yet, and many other processes. The diagrams give us a framework to think about clotting but aren’t the final answer, by far. We are still trying to understand the complex system that is blood clotting and still finding new components. Another relatively new group of compounds involved is the TMEM16 family of scramblases.
One of the major findings about clotting happened in the 1980s when it was discovered that the clotting reactions actually take place on surfaces, not in solution out in the bloodstream. The surfaces are the broken cell walls at the injury site and the cell walls (membranes) of activated platelets. This makes sense because you want the clotting reactions to be at the site of injury, not floating away in the bloodstream. Now we’re finding that those surfaces are not just passive but also participate in the clotting process. To explain this, we need to learn a little about cell membranes.
The cell wall or cell membrane is the covering of the cell. It is made of molecules called phospholipids. These are fatty molecules (lipids) that also contain phosphorus groups. They are long molecules that line up next to each other in a double layer to completely enclose the contents of the cell, as shown in the diagram below.
In this diagram[1], the image on the left shows a cutaway view of a cell surrounded by the cell membrane. Intracellular refers to inside the cell and extracellular refers to outside the cell. The blown-up image at the top shows the structure of the cell membrane, which is made of a phospholipid bilayer. The bilayer consists of two layers of phospholipid molecules lined up with their heads (round circles) facing the outside of the membrane and the tails (long sections) on the inside of the membrane. Because of the way the different parts of the phospholipid molecules attract each other, this forms a strong but flexible covering for the cell. The cell membrane also regulates which materials can pass in or out of the cell.
There are a number of different types of phospholipid molecules that make up the cell membrane. One of the most prevalent (about 15% of the total) is called phosphatidylserine (PS). It is procoagulant (promotes clotting) because it is the molecule to which many of the clotting factors bind during clotting. In the endothelial cells (ECs) that line the inside of the blood vessels, the PS molecules are all on the inside wall of the cell membrane where the blood can’t see them. The wall of the ECs that are in contact with the blood has no PS and is therefore anticoagulant – it impedes clotting.
When you have an injury the EC walls are broken open, which exposes the blood to the PS molecules on the inside of the cell wall. That provides a surface containing PS on which the clotting reactions can proceed. Tissue factor, which is a protein that is also on the inside wall of ECs, is also exposed and starts the clotting process by activating factor VII, which binds to the PS-containing surface.
Tissue factor and factor VII start the clotting process, but to really get enough clotting activity going requires activating the other clotting pathway, the pathway that includes factors VIII and IX. That pathway amplifies the clotting signal to produce enough fibrin to actually seal the blood vessel closed. That requires more PS-containing surface for the clotting reactions. That’s where the scramblases come in.
The recently-discovered scramblases scramble the arrangement of the phospholipid molecules in cell walls, bringing the PS to the outer surface of the injured cell and its neighboring intact cells. That provides more sites for the clotting factors to bind to while forming the clot. It also continues to localize the clotting reactions in the area of the injury.
Is the scramblase rearrangement really important? Apparently so, because when they looked at mice in which the scramblases had been inhibited, they saw that 50% of the clotting activity had also been inhibited. They also saw that fibrin (the protein that makes up the clot) did not stick to the EC surface. That means that the clot doesn’t stick to the injury site. Thus, this rearrangement that brings PS to the surface of the endothelial cells appears to be very important.
What does this mean for the average hemophilia patient? Probably not much, at least not right now, but as we continue to learn more about the clotting system, we potentially will be able to find better treatments for clotting and bleeding disorders. [Prouse T and Majumder R, J Thromb Haemost, online ahead of print 8/25/23]
[1] From Oregon State University at https://open.oregonstate.education/aandp/chapter/3-1-the-cell-membrane/, accessed 1/22/24.