Abstract:Glycoprotein Ib (GPIb), the receptor for von Willebrand factor, is a two-chain member constituent of the platelet/megakaryocytic lineage. Studies on its expression have been hampered by the difficulties in obtaining purified megakaryocytes in a sufficient number. We report a suspension liquid culture procedure that allowed isolation of more than 1 x 10(6) megakaryocytes with a purity ranging from 3% to 88% from the blood of patients with chronic myeloid leukemia, from fetal liver or from normal human bone marr… Show more
“…A significant reduction of YAP was observed in YAP-knockdown cells, and reduction of both YAP and TAZ was observed when a plasmid targeting both YAP and TAZ (shYAP/TAZ) was introduced into these cells ( Figure 1 A,B). CD41, which is a common marker for megakaryocyte and platelets, is expressed on the plasma membrane of mature megakaryocyte cells prior to platelet release [ 10 , 11 ]. Our previous work and other studies demonstrated that 20–40% of a population of MEG-01 cells is made up of CD41 + cells, and MEG-01 cells can produce platelet-like particles (PLPs) either spontaneously or upon treatment with platelet induction agents, such as valproic acid (VPA) [ 4 , 12 ].…”
Platelet transfusion is required for life-threatening thrombocytopenic bleeding, and single donor platelet concentrate is the ideal transfusion product. However, due to the inadequate number of donors that can donate a large volume of platelets, in vitro platelets production could be an alternative. We developed an in vitro production system designed to increase the platelet production yield from cultured cells. Previously, we reported that depletion of a Hippo pathway core kinase (LATS1/2) inhibited platelet production from cultured megakaryocytes. In this study, we further investigated the role of the Hippo pathway in megakaryocyte proliferation and platelet production by focusing on the role of its effector proteins (YAP and TAZ), which are down-stream targets of LATS1/2 kinase. We found that YAP plays an essential role in megakaryoblastic cell proliferation, maturation, and platelet production, while TAZ showed minor effect. Knockdown of YAP, either by genetic manipulation or pharmaceutical molecule, significantly increased caspase-3-mediated apoptosis in cultured megakaryocytes, and increased platelet production as opposed to overexpressing YAP. We, therefore, demonstrate a paradigm for the regulation of megakaryocyte development and platelet production via the Hippo signaling pathway, and suggest the potential use of an FDA-approved drug to induce higher platelet production in cultured cells.
“…A significant reduction of YAP was observed in YAP-knockdown cells, and reduction of both YAP and TAZ was observed when a plasmid targeting both YAP and TAZ (shYAP/TAZ) was introduced into these cells ( Figure 1 A,B). CD41, which is a common marker for megakaryocyte and platelets, is expressed on the plasma membrane of mature megakaryocyte cells prior to platelet release [ 10 , 11 ]. Our previous work and other studies demonstrated that 20–40% of a population of MEG-01 cells is made up of CD41 + cells, and MEG-01 cells can produce platelet-like particles (PLPs) either spontaneously or upon treatment with platelet induction agents, such as valproic acid (VPA) [ 4 , 12 ].…”
Platelet transfusion is required for life-threatening thrombocytopenic bleeding, and single donor platelet concentrate is the ideal transfusion product. However, due to the inadequate number of donors that can donate a large volume of platelets, in vitro platelets production could be an alternative. We developed an in vitro production system designed to increase the platelet production yield from cultured cells. Previously, we reported that depletion of a Hippo pathway core kinase (LATS1/2) inhibited platelet production from cultured megakaryocytes. In this study, we further investigated the role of the Hippo pathway in megakaryocyte proliferation and platelet production by focusing on the role of its effector proteins (YAP and TAZ), which are down-stream targets of LATS1/2 kinase. We found that YAP plays an essential role in megakaryoblastic cell proliferation, maturation, and platelet production, while TAZ showed minor effect. Knockdown of YAP, either by genetic manipulation or pharmaceutical molecule, significantly increased caspase-3-mediated apoptosis in cultured megakaryocytes, and increased platelet production as opposed to overexpressing YAP. We, therefore, demonstrate a paradigm for the regulation of megakaryocyte development and platelet production via the Hippo signaling pathway, and suggest the potential use of an FDA-approved drug to induce higher platelet production in cultured cells.
“…This receptor is normally expressed on the platelet plasma membrane at an average copy number of 25,000 per platelet. During thrombopoiesis GPIb is expressed as early as the megakaryoblast (4). Under normal physiologic conditions, resting platelets do not adhere to each other or to leukocytes and endothelial cells (5).…”
SummaryAntibodies directed against the glycoprotein (GP) Ib have been identified as the potential cause of various platelet disorders: Immune thrombocytopenic purpura (ITP) may be caused by such autoantibodies; Anti-thrombotic drugs targeting GPIb also induce thrombocytopenia. In order to elucidate the potential mechanism(s) of the anti-GPIb effects, we have examined by electron microscopy (EM) the effect of several antibodies directed against GPIb and GPIIb-IIIa on human culture megakaryocytes (MK). Virtually all antibodies to GPIb enhanced the interaction of newly formed platelets with MK when compared to other antibodies. These effects were retrieved when antibodies were tested on platelets. We conclude that antibodies to GPIb can potentially inhibit platelet release by MK, and can also induce homotypic platelet adhesion. These results may have implications in the pathophysiology of thrombocytopenia and platelet recovery in ITP, and shed light on the pathological effect of anti-GPIb antibodies used as antithrombotic drugs.
“…Upon stimulation with platelet agonists, megakaryocytes aggregate and start secretion, which is accompanied by Ca2+ oscillations [14]. Their plasma membrane has the same proportion of phospholipids and fatty acids [15, 161 and contains glycoproteins IbIIX [17] and a,,,P, [18, 191. The results reveal heterogeneity in P3 mobility both in resting megakaryocytes and during stimulation.…”
SummaryThe migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin αIIbβ3 (glycoprotein Ilb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the P3 subunit. The diffusion of P3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 X10'9 cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 μM) or α-thrombin (10 U/ml) at 22° C induced transient decreases in both parameters reducing D to 0.21 X 10‘9 cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, whereas cytochalasin D completely abolished the decrease in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile αIIbβ3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.
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