Collagen or Collagen-related Peptide Cause [Ca2+]i Elevation and Increased Tyrosine Phosphorylation in Human Megakaryocytes

Mountford, Joanne C.; Melford, Steven K.; Bunce, Christopher M.; Gibbins, Jonathan M.; Watson, Stephen P.

doi:10.1055/s-0037-1614345

Cited by 24 publications

(1 citation statement)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…They adhere to and spread on collagen and fibrinogen, they are known to aggregate, and they activate integrins and release granules in response to platelet agonists such as thrombin, collagen-related peptide (CRP), and adenosine 59-diphosphate (ADP). [7][8][9][10][11][12][13][14][15] Until recently, gene editing of CD34 1 -derived primary MKs has been challenging and inefficient, or it relied mostly on the use of viral transduction (ie, lentivirus) with sorting or drug selection. 16 Recent studies have overcome this limitation by using clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPRassociated protein 9 (Cas9) ribonucleoprotein (RNP) transfection to achieve highly efficient, nonviral gene editing in CD34 1 cells.…”

Section: Introductionmentioning

confidence: 99%

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

et al. 2021

View full text Add to dashboard Cite

Human anucleate platelets cannot be directly modified using traditional genetic approaches. Instead, studies of platelet gene function depend on alternative models. Megakaryocytes (the nucleated precursor to platelets) are the nearest cell to platelets in origin, structure, and function. However, achieving consistent genetic modifications in primary megakaryocytes has been challenging, and the functional effects of induced gene deletions on human megakaryocytes for even well-characterized platelet genes (eg, ITGA2B) are unknown. Here we present a rapid and systematic approach to screen genes for platelet functions in CD34+ cell-derived megakaryocytes called CRIMSON (CRISPR-edited megakaryocytes for rapid screening of platelet gene functions). By using CRISPR/Cas9, we achieved efficient nonviral gene editing of a panel of platelet genes in megakaryocytes without compromising megakaryopoiesis. Gene editing induced loss of protein in up to 95% of cells for platelet function genes GP6, RASGRP2, and ITGA2B; for the immune receptor component B2M; and for COMMD7, which was previously associated with cardiovascular disease and platelet function. Gene deletions affected several select responses to platelet agonists in megakaryocytes in a manner largely consistent with those expected for platelets. Deletion of B2M did not significantly affect platelet-like responses, whereas deletion of ITGA2B abolished agonist-induced integrin activation and spreading on fibrinogen without affecting the translocation of P-selectin. Deletion of GP6 abrogated responses to collagen receptor agonists but not thrombin. Deletion of RASGRP2 impaired functional responses to adenosine 5′-diphosphate (ADP), thrombin, and collagen receptor agonists. Deletion of COMMD7 significantly impaired multiple responses to platelet agonists. Together, our data recommend CRIMSON for rapid evaluation of platelet gene phenotype associations.

show abstract

Section: Introductionmentioning

confidence: 99%

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

et al. 2021

View full text Add to dashboard Cite

show abstract

The role of extracellular matrix stiffness in megakaryocyte and platelet development and function

Leiva

Léon

et al. 2018

American J Hematol

View full text Add to dashboard Cite

The extracellular matrix (ECM) is a key acellular structure in constant remodeling to provide tissue cohesion and rigidity. Deregulation of the balance between matrix deposition, degradation, and crosslinking results in fibrosis. Bone marrow fibrosis (BMF) is associated with several malignant and nonmalignant pathologies severely affecting blood cell production. BMF results from abnormal deposition of collagen fibers and enhanced lysyl oxidase-mediated ECM crosslinking within the marrow, thereby increasing marrow stiffness. Bone marrow stiffness has been recently recognized as an important regulator of blood cell development, notably by modifying the fate and differentiation process of hematopoietic or mesenchymal stem cells. This review surveys the different components of the ECM and their influence on stem cell development, with a focus on the impact of the ECM composition and stiffness on the megakaryocytic lineage in health and disease. Megakaryocyte maturation and the biogenesis of their progeny, the platelets, are thought to respond to environmental mechanical forces through a number of mechanosensors, including integrins and mechanosensitive ion channels, reviewed here.

show abstract

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Kamal

Green

Hearn

et al. 2018

Research and Practice in Thrombosis and Haemostasis

View full text Add to dashboard Cite

Essentials Intracellular calcium pathways regulate megakaryopoiesis but details are unclear.We examined effects of NMDAR‐mediated calcium influx on normal and leukemic cells in culture.NMDARs facilitated differentiation of normal but proliferation of leukemic megakaryocytes.NMDAR inhibitors induced differentiation of leukemic Meg‐01 cells. Background N‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts.ObjectivesThe aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells.MethodsMeg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls.ResultsThe most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves.Conclusions NMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

show abstract

Collagen or Collagen-related Peptide Cause [Ca2+]i Elevation and Increased Tyrosine Phosphorylation in Human Megakaryocytes

Cited by 24 publications

References 40 publications

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

The role of extracellular matrix stiffness in megakaryocyte and platelet development and function

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Contact Info

Product

Resources

About