Research Description

Blood Coagulation in Health and Disease

WELCOME

Our research is aimed at understanding the development and function of blood platelets. These tiny, numerous cells initiate and co-ordinate blood clotting (i.e. hemostasis) at wound sites by adhering, aggregating and secreting a wide assortment of molecules. Platelets are also involved in the formation of arterial plaques and pathological clots such as the thrombi that cause heart attacks and strokes, major causes of mortality in the modern world. Many of our insights into the mechanisms of platelet development and function have come from studies of patients with inherited platelet disorders. Our particular interest is alpha granules, secretory vesicles that platelets use to transport and secrete several hundred different kinds of protein. We have identified several proteins and cellular pathways involved in the development of alpha granules in the cells that produce platelets, the bone marrow megakaryocytes.

Blood Platelets in Health and Disease

Each of us releases 100 billion platelets into our bloodstream every day. These tiny disk-shaped cells, only about 3 microns across, play vital roles as they patrol blood vessels for signs of injury. When they detect a breach they adhere, aggregate and activate formation of a clot that seals the wound and limits blood loss. Thus individuals lacking platelets or having dysfunctional cells experience abnormal bleeding. In addition to their key role in blood clotting, platelets are also involved in inflammation, immunity and wound healing, and in the formation of atherosclerotic plaques and pathological clots (i.e. thrombi) that trigger heart attacks and strokes. The many roles of platelets in health and disease are linked to their unique ability to store and release a wide range of biomolecules. These include a wide gamut of proteins stored within and released from secretory α‑granules, and studies of the development and function of these granules is a major focus of my research group.

High resolution laser fluorescence microscopy of a human platelet shows actin near the cell membrane and the internal calcium store system that surrounds abundant protein-carrying alpha granules.

Platelets and Megakaryocytes

Platelets are produced by megakaryocytes, cells that develop within the bone marrow until they reach enormous sizes of up to 100 microns across. At that point they generate extensions that protrude into blood vessels and shed platelets into circulation by the thousands. We study megakaryocyte development in a variety of different ways using cells derived from humans and mice. This allows us to monitor gene expression, protein localization and trafficking, as well as the development of alpha granules, using advanced methods such as high-resolution fluorescence and electron microscopy. The ability to manipulate megakaryocytes in vitro has allowed us to study their development in detail, and examine aspects such as the trafficking of proteins destined for alpha granules, some of which are synthesized by megakaryocytes, while others are taken up from the bloodstream.

Image of a cultured megakaryocyte (left, outer membrane in green) in the final stage of development when long extensions give rise to smaller anucleate bodies (right) that become platelets in vivo.

Hereditary Platelet Disorders: Bedside to Bench and Back Again

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.

Publications

View all publications on PubMed

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia.
Noetzli L, Lo RW, Lee-Sherick AB, Callaghan M, Noris P, Savoia A, Rajpurkar M, Jones K, Gowan K, Balduini C, Pecci A, Gnan C, De Rocco D, Doubek M, Li L, Lu L, Leung R, Landolt-Marticorena C, Hunger S, Heller P, Gutierrez-Hartmann A, Xiayuan L, Pluthero FG, Rowley JW, Weyrich AS, Kahr WH, Porter C, Di Paola J.
Nature Genetics. 2015 Nov 3; 47(5),535–538.  Read

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.
34. Begonja AJ, Pluthero FG, Suphamungmee W, Giannini S, Christensen H, Leung R, Lo RW, Nakamura F, Lehman W, Plomann M, Hoffmeister KM, Kahr WH, Hartwig JH, Falet H.
Blood. 2015 Jul 2;126(1):80-8.  Read

Platelet production: new players in the field.
Pluthero FG, Kahr WH.
Blood. 2016;127(7):797-9.  Read

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.
Kahr WH, Pluthero FG, Elkadri A, Warner N, Drobac M, Chen CH, Lo RW, Li L, Li R, Li Q, Thoeni C, Pan J, Leung G, Lara-Corrales I, Murchie R, Cutz E, Laxer RM, Upton J, Roifman CM, Yeung RS, Brumell JH, Muise AM.
Nature Communications. 2017 Apr 3;8:14816.  Read

NBEAL2 (Neurobeachin-Like 2) Is Required for Retention of Cargo Proteins by α-Granules During Their Production by Megakaryocytes.
Lo RW, Ling Li L, Leung R, Pluthero FG, Kahr WH.
Arteriosclerosis, Thrombosis, and Vascular Biology. 2018 Jul 26;38:2436-2447.   Read

The endoplasmic reticulum protein SEC22B interacts with NBEAL2 and is required for megakaryocyte α-granule biogenesis.
Lo RW, Li L, Pluthero FG, Leung R, Eto K, Kahr WHA
Blood. 2020 Aug 6;136(6):715-725.  Read

Gray platelet syndrome: NBEAL2 mutations are associated with pathology beyond megakaryocyte and platelet function defects
Pluthero FG, Kahr WHA
J Thromb Haemost 2021 Feb;19(2):318-322  Read

Platelet VPS16B is dependent on VPS33B expression, as determined in two siblings with arthrogryposis, renal dysfunction, and cholestasis syndrome
Penon-Portman M, Westbury SK, Li L, Pluthero FG, Liu RJY, Yao HL, Geng RSQ, Warner N, Muise A, .... Kahr WHA
J Thromb Haemost 2022 Jul;20(7):1712-1719 doi: 10.1111/jth.15711  Read

Evaluation of human platelet granules by structured illumination laser fluorescence microscopy
Pluthero FG, Kahr WHA
Platelets 2023 Dec;34(1):2157808  Read

The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B
Liu RJY, Al-Molieh Y, Chen SZ, Drobac M, Urban D, Chen CH, Yao HHY, Geng RSQ, Li L, Pluthero FG, Benlekbir S, Rubinstein JL, Kahr WHA
J Biol Chem 2023 Jun;299(6):104718. doi: 10.1016/j.jbc.2023.104718  Read