Sub-Cellular Localization of Metalloproteinases in Megakaryocytes

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that play essential roles as the mediator of matrix degradation and remodeling during organogenesis, wound healing and angiogenesis. Although MMPs were originally identified as matrixin proteases that act in the extracellular matrix, more recent research has identified members of the MMP family in unusual locations within the cells, exerting distinct functions in addition to their established role as extracellular proteases. During thrombopoiesis, megakaryocytes (Mks) sort MMPs to nascent platelets through pseudopodial-like structure known as proplatelets. Previous studies identified gelatinases, MMP-2 and MMP-9, as a novel regulator system of Mks and the platelet function. In this work we have exploited a sensitive immunoassay to detect and quantify multiple MMP proteins and their localization, in conditioned medium and sub-cellular fractions of primary human CD34⁺-derived Mks. We provide evidence that Mks express other MMPs in addition to gelatinases MMP-2 and MMP-9, peculiar isoforms of MMP-9 and MMPs with a novel nuclear compartmentalization

Thrombopoietin/TGF-β1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis

Extracellular matrix (ECM) components initiate crucial biochemical and biomechanical cues that are required for bone marrow homeostasis. In our research, we prove that a peri‐cellular matrix composed primarily of type III and type IV collagens, and fibronectin surrounds human megakaryocytes in the bone marrow. The data we collected support the hypothesis that bone marrow megakaryocytes possess a complete mechanism to synthesize the ECM components, and that thrombopoietin is a pivotal regulator of this new function inducing transforming growth factor‐β1 (TGF‐β1) release and consequent activation of the downstream pathways, both in vitro and in vivo. This activation results in a dose dependent increase of ECM component synthesis by megakaryocytes, which is reverted upon incubation with JAK and TGF‐β1 receptor specific inhibitors. These data are pivotal for understanding the central role of megakaryocytes in creating their own regulatory niche within the bone marrow environmen

Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype