June 19 is World Sickle Cell Awareness Day. Sickle cell disease refers to a group of inherited red blood cell disorders. The Center for Disease Control and Prevention estimates that sickle cell disease affects 90,000 to 100,000 people in the United States, a majority of whom are African-American. Worldwide, it is estimated that 300,000 children are born each year with sickle cell diseases, though many go undiagnosed in developing countries. We recently spoke with Biophysical Society member Frank Ferrone, Drexel University, about the research his lab conducts related to sickle cell disease.
What is the connection between your research and sickle cell disease?
We have several projects underway that relate to sickle cell disease. Let me give two. In one, we are working to complete a device that can detect the presence of sickle cell disease, or sickle cell trait (and distinguish the two), using a drop of blood, in under a minute, at a cost of a few cents per test. And we’re almost there! In a more basic vein (sorry about that ) we are also exploring a radical hypothesis of ours that the structure of the sickle polymers that cause all the trouble in the disease is not understood properly—and if we are correct, our revision of this paradigm has implications for all pathological assembly diseases.
Why is your research important to those concerned about sickle cell disease?
In the US, children get tested, information gets shared (mostly), and emergency medicine is very good…and even here, sickle patients sometimes get misdiagnosed or fall through the cracks. In certain nations, sickle cell disease afflicts as many as 1 in 7 newborn children, and the resources simply don’t exist to conduct expensive tests, or to track the families whose children are affected. Thus an inexpensive and rapid test could be a godsend there. At the same time in the US, the NCAA dictates that student athletes should have their status known, since sickle trait poses a covert risk—kids can have no symptoms, and then under exertion experience tragic, even fatal, sickle-events. Now, we supposedly have medical records of everyone’s test—but that’s if you did get tested, and if you have the records. Imagine that instead of a kid having to wait for results of a test–an expensive one to boot–because his family can’t find his test results, that the school nurse could prick his finger and in a minute pronounce him good to go (or not).
As to the other project I mentioned, well there are currently no drugs that work by interfering with the polymeric structures that generate the pathology. Maybe if we understood the polymers better, that could change.
How did you get into this area of research?
For my PhD I did a project on normal hemoglobin, using laser photolysis to trigger a structural change, and following it with kinetic CD (it was a first). When looking around for a post-doc, I got invited to join Bill Eaton’s group at the NIH, using the photolysis trick to induce sickle cell hemoglobin polymerization. I knew nothing about sickle cell at the time, but the project sounded interesting, the group was exciting, and, as Bill explained it, “eventually everybody comes to visit the NIH” so the environment was immensely stimulating.
How long have you been working on it?
It’s now on 40 years! I carried the project with me to Drexel University’s Physics Department, where I’ve continued to work on this.
Do you receive public funding for this work? If so, from what agency?
While we did have many years of generous NIH support, at the moment we don’t have funding.
Have you had any surprise findings thus far?
Plenty! When I was transitioning to Drexel from the NIH, we came up with the idea that there were two kinds of nucleation that generated the sickle fibers, not just one like everybody thought, mainly because we had these experiments that couldn’t be explained any other way. That was a big surprise. And now it’s turned out to be fairly common in pathological assemblies.
We found that the second type of nucleation—onto the surface of polymers—came about because intermolecular contacts that stabilize the interior of polymers also appear on the polymer exterior! Anybody could have found this from the existing structures, but nobody thought to look. Even we bumped into it by accident.
When we employed our models to understand the assembly process, we got another big surprise. The ability of the hemoglobin molecules to oscillate about their equilibrium position in the polymer exercises a HUGE influence on the rate of the assembly, thanks to an effect known as vibrational entropy. Weak, “sloppy” bonds generate much faster nucleation—which we demonstrated experimentally.
And one of our most recent surprises was that the polymer formation gets hung up in a metastable state because of the extreme crowding. And in a red cell, that in turn leads to Brownian ratchet forces that can hold cells in narrow channels, like capillaries.
What is particularly interesting about the work from the perspective of other researchers?
Sickle cell is perhaps the “Granddaddy” of all the protein assembly diseases, which now include Alzheimer’s, prion diseases, Huntington’s, Parkinson’s… There is often a lot of hard biochemistry that goes into simply characterizing the assembly that is the root of these. Sickle hemoglobin can be prepared in large quantities, purified simply, and even reconstructed by site-directed mutagenesis, with the result that much more sophisticated physical questions can be posed and tested without a ridiculous overhead of construction and purification. Thus, as I mentioned, the double nucleation model, constructed for sickle cell polymerization, has been shown to operate on Aβ assembly. In addition, to do the analysis, we needed to invent a new mathematical approach, adopting perturbation methods to deal with intractable differential equations. This has been of great interest to others, too. Finally, for years it was our “burden” that all the standard kinetic and equilibrium equations we used had to be modified to account for the high concentration of hemoglobin in the red cells. I hated it! Fortunately, we succeeded in working it out, but now with the burgeoning interest in molecular crowding, it turns out that our work has a lot of applicability for another class of problems as well.
What is particularly interesting about the work from the perspective of the public?
Sickle cell disease has been known for over 100 years. It’s known as the first molecular disease. When people discuss gene therapy, sickle cell is invariably found in the list of targets. And yet there is but one drug. A solution to this long-standing problem generates interest. Moreover, there are about 240,000 new cases (i.e. sickle cell births) yearly in Africa. But the other part of the story, which maybe should be told to the public even more prominently, is that these assembly diseases are all connected in a fundamental way. Our analytical methods led to a novel result in poly-Q assembly. Our model was adopted for Aβ assembly. Answering basic questions well generates a tide that indeed floats all boats.
Do you have a cool image you want to share related to this research?
We have a movie! This was taken in our lab by Dr. Alexey Aprelev. We have a narrow micro-fluidic channel, 4 µm wide and 1.5 µm deep, and a red cell. In the movie the cell squeezes in and out just fine in response to externally oscillating the hydrostatic pressure. The hemoglobin inside the cell has CO bound, which can be removed by light very efficiently. Just as the cell is entering we turn on a laser, which you can see from the leaked light in the background. Our oscillatory pressure now does nothing! And most dramatically, note that the cell cannot exit either! This trapping is due to Brownian ratchet forces, as we published in a letter in Biophysical Journal in 2012. And once the laser is turned off, CO rebinds, and the cell once again can move in and out, recovering its deformability. Click here to view the video.