PMID- 38330198 OWN - NLM STAT- MEDLINE DCOM- 20240401 LR - 20240413 IS - 2473-9537 (Electronic) IS - 2473-9529 (Print) IS - 2473-9529 (Linking) VI - 8 IP - 7 DP - 2024 Apr 9 TI - Altered platelet-megakaryocyte endocytosis and trafficking of albumin and fibrinogen in RUNX1 haplodeficiency. PG - 1699-1714 LID - 10.1182/bloodadvances.2023011098 [doi] AB - Platelet alpha-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and alpha-granule deficiencies in RUNX1-haplodeficiency. CI - (c) 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. FAU - Del Carpio-Cano, Fabiola AU - Del Carpio-Cano F AD - Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. FAU - Mao, Guangfen AU - Mao G AD - Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. FAU - Goldfinger, Lawrence E AU - Goldfinger LE AUID- ORCID: 0000-0003-0525-6008 AD - Division of Hematology, Department of Medicine, Cardeza Foundation for Hematologic Research, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA. FAU - Wurtzel, Jeremy AU - Wurtzel J AD - Division of Hematology, Department of Medicine, Cardeza Foundation for Hematologic Research, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA. FAU - Guan, Liying AU - Guan L AUID- ORCID: 0000-0002-7470-564X AD - Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. FAU - Alam, Mohammad Afaque AU - Alam MA AUID- ORCID: 0000-0001-5801-2512 AD - Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. FAU - Lee, Kiwon AU - Lee K AUID- ORCID: 0000-0003-1340-3602 AD - Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA. AD - Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Seoul, Korea. FAU - Poncz, Mortimer AU - Poncz M AUID- ORCID: 0000-0001-7237-3613 AD - Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA. FAU - Rao, A Koneti AU - Rao AK AUID- ORCID: 0000-0002-3078-7778 AD - Sol Sherry Thrombosis Research Center, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. AD - Department of Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA. LA - eng GR - R01 HL159006/HL/NHLBI NIH HHS/United States PT - Journal Article PL - United States TA - Blood Adv JT - Blood advances JID - 101698425 RN - 0 (Caveolin 1) RN - 9001-32-5 (Fibrinogen) RN - 0 (Core Binding Factor Alpha 2 Subunit) RN - 0 (Hemostatics) RN - 0 (Albumins) RN - 0 (Immunoglobulin G) RN - 0 (IFITM3 protein, human) RN - 0 (Membrane Proteins) RN - 0 (RNA-Binding Proteins) RN - 0 (RUNX1 protein, human) RN - Platelet Disorder, Familial, with Associated Myeloid Malignancy SB - IM UOF - medRxiv. 2023 Oct 23;:. PMID: 37961544 MH - Humans MH - Megakaryocytes/metabolism MH - Caveolin 1/metabolism MH - Fibrinogen/metabolism MH - Core Binding Factor Alpha 2 Subunit/genetics/metabolism MH - Endocytosis MH - *Hemostatics MH - *Leukemia, Erythroblastic, Acute MH - Albumins/metabolism MH - Immunoglobulin G MH - Membrane Proteins/metabolism MH - RNA-Binding Proteins/metabolism MH - *Blood Platelet Disorders MH - *Leukemia, Myeloid, Acute MH - *Blood Coagulation Disorders, Inherited PMC - PMC10997914 COIS- Conflict-of-interest disclosure: The authors declare no competing financial interests. EDAT- 2024/02/08 18:42 MHDA- 2024/04/01 06:43 PMCR- 2024/02/10 CRDT- 2024/02/08 15:13 PHST- 2024/01/29 00:00 [accepted] PHST- 2023/06/30 00:00 [received] PHST- 2024/04/01 06:43 [medline] PHST- 2024/02/08 18:42 [pubmed] PHST- 2024/02/08 15:13 [entrez] PHST- 2024/02/10 00:00 [pmc-release] AID - 514869 [pii] AID - 10.1182/bloodadvances.2023011098 [doi] PST - ppublish SO - Blood Adv. 2024 Apr 9;8(7):1699-1714. doi: 10.1182/bloodadvances.2023011098.