Platelet Granule Formation Gives Insight Into Rare Disease

If you take a normal platelet and examine it under an electron microscope, you will see a bunch of black dots. The specks may not seem like much at first, but cell biologists at The Children’s Hospital of Philadelphia Research Institute speculate that these dense granules hold the key to unlocking the mechanisms behind a rare disease called Hermansky-Pudlak syndrome (HPS) and other forms of bleeding disease.

HPS is estimated to affect one in 500,000 to one in 1,000,000 individuals worldwide. In certain places, like Puerto Rico, it is much more prevalent — about one in 1,800 individuals. People with the disease have a tendency to bruise and difficulties with blood clotting, which can be deadly under certain circumstances such a pregnancy, major surgery, or dental surgery.

“Those dense granules don’t get made in a set of patients, and the consequence of that is the patients bleed too much, and they’re not able to make blood clots efficiently,” said Michael S. Marks, PhD, who received a grant in May from the National Heart, Lung, and Blood Institute to better understand how platelet dense granules form and why their creation is disrupted in HPS.

Dense granules are lysosome-related organelles (LROs) within platelets that act as a storage compartment for small molecules such as calcium, adenosine diphosphate, and serotonin. These molecules are released when platelets begin to accumulate at sites of blood vessel damage.

“We know that there are important things stored in those granules, but we have no idea how they get there,” Dr. Marks said. “This is the first time anybody’s taken an approach to try and understand how dense granules are put together and how those molecules get stored in them.”

HPS can be difficult to diagnose because it affects multiple organs, and the symptoms can be variable and nonspecific. Mutations in the genes associated with HPS prevent the formation of LROs or impair their performance in platelets, pigment cells, and lung cells. Subsequently, in addition to excessive bleeding, a main feature of the disease is oculocutaneous albinism, which causes abnormally light coloring of the skin, hair, and eyes. By the time people with HPS reach their 30s, a lung defect called pulmonary fibrosis appears that rapidly worsens, and the lung scarring often is fatal.

“Getting a good diagnosis early would be really important,” Dr. Marks said. “That would be the most immediate impact of this grant.”

The study aims to achieve a better understanding of two dense granule integral platelet membrane proteins that Dr. Marks’ team at CHOP and a collaborating group in Colorado recently identified — SLC35D3 and VMAT2 — which may act as vehicles to import dense granule contents. The investigators suspect that this delivery is impaired in HPS, and the dense granules do not form completely. Necessary machinery could be missing at a crucial time when a dense granule’s membrane coalesces from other membranes in megakaryocytes, which are platelets’ precursor cells that reside in bone marrow. It appears that this occurs during a late stage of differentiation of platelets from megakaryocytes.

In order to test this hypothesis, scientists in Dr. Marks’ lab will collaborate with another group of experts in megakaryocyte and platelet formation from the lab of Mortimer Poncz, MD, division chief of hematology at CHOP. They will take megakaryocytes from mice and then modify them by adding fluorescent proteins that hopefully will allow the investigators to visualize dense granule formation using live cell imaging. The next step will be to put the megakaryocytes back into the mice where they will produce platelets, and then take the platelets out to analyze them.

Dr. Marks also will compare the same mutations in human cells by using megakaryocytes created from stem cells of patients with HPS. Deborah L. French, PhD, a specialist in making induced pluripotent stem cells, will assist with this part of the project. Dr. French is director of the Human Stem-Cell Vector Core within the Center for Cellular and Molecular Therapeutics at CHOP.

The investigators will determine if SLC35D3 and VMAT2 live on dense granule membranes and whether or not they are expressed on platelet membranes in both the human and mouse HPS models. What they discover could guide new diagnostic approaches.

“If we’re right, then we could make antibodies to those proteins,” Dr. Marks said. “Then it should be a very simple test to look for exposure of that protein when you activate platelets.”

Such a diagnostic test would be helpful for identifying HPS as well as other forms of bleeding disease due to mutations in genes that encode proteins on these dense granules. As researchers learn more about the proteins’ structure and the jobs that they perform, this knowledge could be applied to designing drug therapies that either enhance the proteins’ activity, as would be the case in HPS, or decrease the proteins’ activity, which could potentially modulate diseases that cause too much blood clotting.

“Ultimately, maybe, we’ll be able to come up with some kind of treatment,” Dr. Marks said. “That will depend on whether we’re right with some of our guesses about the proteins that are on dense granules and whether we can characterize them further and identify steps that would be downstream from them.”

Dr. Marks also is looking forward to working on a future study with Susan Guttentag, MD, that recently received grant funding. That investigation will focus on the formation of another LRO, the lamellar body, in lung epithelial cells. Dr. Guttentag and colleagues have shown that HPS models interfere with this process which underlies the lung fibrosis in HPS.