Much of the recent progress that has been made in understanding platelet production, structure and function has come from studies of hereditary conditions where one or more of these aspects are affected. Patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome have platelets that lack α-granules. In a series of studies we identified that the root cause of this defect is loss of one of two proteins: VPS33B and VPS16B, which we showed using yeast two-hybrid screens and mass spectrometry form a functional protein complex. Ongoing studies are extending this work in several directions, including determining the structure of the functional VPS33B/VPS16B complex. This work recently culminated in the discovery that the VPS33B/VPS16B complex represents the first bidirectional SEC/MUNC complex with the potential to bind up to 4 SNAREs simultaneously (see Liu et al. J Biol Chem 2023 Jun;299(6):104718).
In another project we used classical genetics and platelet mRNA expression analysis to reveal the cause of gray platelet syndrome (GPS), where patients have platelets containing some alpha granule components, but normal granules fail to form. We found that GPS is caused by loss of function of NBEAL2 (neurobeachin-like 2), a large protein with several potential functional domains about which little is known. We have explored NBEAL2 function using Nbeal2-knockout mice that recapitulate many aspects of GPS pathology. These studies have allowed us to observe that loss of NBEAL2 function leads to impaired megakaryocyte development and affects their ability to package and retain protein cargo into alpha granules. Current studies are focused on elucidating the cellular mechanisms whereby NBEAL2 facilitates maturation and stability of alpha granules.
A recent example where the expertise of my group and our collaboration with others proved highly productive involved solving a mystery concerning a patient with a puzzling set of symptoms involving platelets and the immune system. We discovered the root of these problems was loss of expression of ARPC1B, a component of the Arp2/3 complex that generates branched actin filaments in blood cells. This work received considerable media interest and was recognized by the inaugural Janet Rossant Research innovation Prize, and it has stimulated further exploration of ARPC1B deficiency by us and others. A novel technical aspect of this project was the generation of gene knockout human megakaryocyte precursor (imMKCL) cells using CRISPR/Cas9 gene editing, which were used to model megakaryocyte development in the absence of ARPC1B function.
Fluorescence (left) and scanning EM (right) imaging of normal platelets spreading on fibrinogen (top) and platelets from a patient lacking ARPC1B (bottom) show a striking difference in the ability of the deficient cells to spread and form adherent lamellipodia via actin filament branching.
