Affiliations: Institut Curie, France
Journal reference: https://dx.doi.org/10.1182%2Fblood-2018-07-861427
Summary: Our lab explored the reasons why platelets can be too large and in low numbers in certain blood diseases called macrothrombocytopenia that usually cause lots of bleeding. We also pinpoint a potential treatment to treat such platelet defects.
Introduction: a very rare set of blood diseases called macrothrombocytopenia
Platelets, also called thrombocytes, are a component of blood that react to bleeding by clumping and initiating a blood clot. Their size is crucial for their production and activity: if they are too big, they cannot be produced in sufficient numbers, which leads to a rare condition called macrothrombocytopenia (only a few hundred affections in the world).
Multiple mechanisms regulate platelet size, most of which revolve around cytoskeleton activity, i.e. the inner structure that maintains cell shape (exactly like the skeleton for the human body). Many of these mechanisms were discovered by studying macrothrombocytopenia patients, but the small number of subjects makes it very difficult to characterize the condition in detail. To solve this problem, our lab generated a special type of stem cell called induced pluripotent stem cells (iPSCs), in which we modify blood cells isolated directly from a patient. These special cells can be propagated almost indefinitely, eliminating the need to extract fresh cells from patients’ blood every time an experiment is performed.
We focus our attention on a specific macrothrombocytopenia caused by genetic mutations in a gene called FLNA (Filamin A) on the X chromosome. All patients affected by these mutations are women and have both macroplatelets and normal size platelets in their blood. This is the consequence of a phenomenon called X inactivation: as women carry two X chromosomes, their cells randomly shut down one of the two chromosomes. As a result, the active X can be the one with a normal copy of the FLNA gene in some cells and the one with the mutated gene in other cells.
Method: We used a special kind of cell generated in our lab to explain the defect in platelets in the presence of a mutation called FLNA
Megakaryocytes are cells responsible for the production of platelets. Using the special stem cells generated in our lab, we were able to produce large numbers of megakaryocytes.
We realised two things:
1) Megakaryocytes with mutated FLNA produced lower numbers of proplatelets, the precursors of platelets (from which platelets are formed)
2) Megakaryocytes with mutated FLNA were not able to produce FLNA protein
We hypothesised that the defect in proplatelet numbers was due to a defect in the megakaryocyte skeleton and its interaction with their microenvironment (i.e. surrounding cells and molecules). To test this hypothesis, we seeded normal and mutated megakaryocytes on microscopy slides and we covered the slides with proteins normally found in the bone marrow (like megakaryocytes).
Results: We might have found a promising lead to treat macrothrombocytopenia in the presence of FLNA mutation
Results showed that mutated megakaryocytes interacting with bone marrow proteins were constantly contracted and their cytoskeleton formed a lot of stress fibres.
Stress fibres are abnormal structures typically associated with increased activity of a protein called RhoA and its partner ROCK1/2. We thus decided to further explore this couple of proteins in normal and mutated megakaryocytes.
We observed that RhoA activity was higher in mutated megakaryocytes than in normal ones. This difference was due to the absence of FLNA protein in FLNA mutated cells, as RhoA and FLNA proteins normally interact.
We decided to block the activity of ROCK1/2 (RhoA’s partner protein) with a small molecule. Results were drastic: it restored the number of proplatelets in mutated megakaryocytes, hinting at a possible treatment for macrothrombocytopenia.
In conclusion, FLNA protein is essential for the correct activity of the cytoskeleton during platelet production. In its absence, platelets are produced in much lower numbers and are of the wrong size. This can be reversed using a small molecule able to shut down the activity of the protein ROCK1/2, potentially highlighting a treatment for those patients.