Biology subjects

Mazharian, A.

Publications and source records attributed to Mazharian, A..

4 recordsLinked to original sources

bioRxiv2026

CRISPR/Cas9-mediated deletion of Shp1 and Shp2 reveals distinct roles in human megakaryopoiesis and proplatelet formation

The non-receptor protein-tyrosine phosphatases Shp1 (PTPN6) and Shp2 (PTPN11) play critical roles in hematopoietic signaling networks, yet their specific functions in human megakaryopoiesis and thrombopoiesis remain incompletely understood. While Shp2 is recognized in murine models as a positive regulator of thrombopoietin (Tpo)-mediated signaling through the Ras/MAPK and PI3K/AKT pathways, Shp1 has been implicated in RhoA-dependent cytoskeletal remodeling. However, the extent to which these roles translate to human megakaryocyte (MK) development and platelet production is not known. In this study, we systematically investigated the contributions of Shp1 and Shp2 to human MK development and function using CRISPR/Cas9-mediated gene deletion of PTPN6 and PTPN11 in CD34+ hematopoietic stem and progenitor cells (HSPCs), combined with pharmacological inhibition of Shp2 using the structurally-distinct allosteric inhibitors SHP099 and RMC-4550. Efficient gene editing of PTPN6 and PTPN11 resulted in efficient ablation of Shp1 and Shp2 in CD34+ HSPC-derived MKs. Genetic deletion or pharmacological inhibition of Shp2 markedly impaired MK proliferation, polyploidization, maturation, and proplatelet formation, whereas loss of Shp1 expression did not. Further, Shp2 inhibition significantly reduced platelet production in a 3-dimensional human bone marrow tissue model. Deletion and inhibition of Shp2 abrogated Tpo-induced ERK1/2 and AKT phosphorylation, confirming its essential role in Mpl receptor signaling. These findings demonstrate the distinct functional roles of Shp1 and Shp2 in MKs and establish Shp2 as a critical positive regulator of Mpl- mediated megakaryopoiesis and thrombopoiesis. Key PointsO_LIEfficient deletion of Shp1 and Shp2 in human CD34 progenitor cell-derived MKs using CRISPR/Cas9. C_LIO_LILoss of Shp2 expression impairs thrombopoietin-induced human MK maturation, proplatelet formation and Mpl signaling. C_LI

cell biology↗

bioRxiv2025

Synergistic effects of deleting the tyrosine phosphatases Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis in mice

The Src homology 2 (SH2) domain-containing non-transmembrane protein-tyrosine phosphatases 1 and 2 (Shp1 and Shp2) have been implicated in regulating signaling from a variety of receptors and cell types, including the thrombopoietin (Tpo) receptor Mpl in megakaryocytes (MKs) and platelets. We previously showed that deletion of Shp1 and Shp2 in the MK/platelet lineage in mice using the Pf4-Cre transgene/loxP system impairs megakaryopoiesis and thrombopoiesis. However, we also observed unexpected phenotypes including a motheaten-like phenotype in Shp1-deficient mice and severe myelofibrosis in mice lacking both phosphatases. To determine whether these were lineage-specific effects, we utilized the Gp1ba-Cre transgenic mouse to delete loxP-flanked Shp1 and Shp2 in mice. Bone marrow-derived MKs from these mice expressed approximately 20-25% of Shp1 and Shp2, whereas platelets contain 5-10% of each phosphatase compared with controls. Minor MK/platelet defects were observed in mice lacking either Shp1 or Shp2 alone, however mice lacking both Shp1 and Shp2 exhibited macrothrombocytopenia, mild bleeding following tail injury, and impaired GPVI-mediated platelet aggregation and Syk phosphorylation, associated with reduction GPVI and integrin 2 subunit expression. Reduced Shp1 and Shp2 expression resulting in a significant reduction in ploidy, a block in MK maturation and proplatelet-producing MKs. Tpo-mediated Ras/MAPK signaling was reduced in Shp1/2-deficient MKs. Treatment of MKs with structurally distinct Shp2 allosteric inhibitors recapitulated key aspects of the Shp2-deficient phenotype, including aberrant megakaryopoiesis and reduced Mpl signaling. Our study highlights the synergistic functions of Shp1 and Shp2 in the MK/platelet lineage, and identifies Shp2 as a potential therapeutic target in myeloproliferative neoplasms. Key PointsO_LIDeletion of Shp1 and Shp2 in the MK/platelet lineage in mice results in macrothrombocytopenia and minor effects on platelet function. C_LIO_LIDefects can be partially explained by reduced Mpl signaling and aberrant megakaryopoiesis in the absence of Shp2 activity. C_LI

cell biology↗

bioRxiv2024

G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

Key PointsO_LIBispecific cis-acting platelet receptor inhibitors (CAPRIs) mediate hetero-clustering of the ITIM receptor G6B with ITAM receptors. C_LIO_LIG6B-GPVI and G6B-CD32A CAPRIs specifically inhibit collagen- and immune complex-induced thrombus formation, respectively. C_LI Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR {gamma}-chain complex or Fc{gamma}RIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies.

cell biology↗

bioRxiv2019

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation, loss of which results in severe macrothrombocytopenia and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

biochemistry↗