Role of tumor suppressor p53 in megakaryopoiesis and platelet function

Pani A. Apostolidis; Donna S. Woulfe; Massiel Chavez; William M. Miller; Eleftherios T. Papoutsakis

doi:10.1016/j.exphem.2011.10.006

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

Pani A. Apostolidis, Donna S. Woulfe, Massiel Chavez, William M. Miller, Eleftherios T. Papoutsakis^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

Abstract

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53^-/- megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53^-/- marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53^+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53^-/- megakaryocytes and 31% greater apoptosis in p53^+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53^+/+ and p53^-/- mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53^-/- mice. Although their platelet counts were normal, p53^-/- mice exhibited significantly longer bleeding times and p53^-/- platelets were less sensitive than p53^+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

Original language	English (US)
Pages (from-to)	131-142.e4
Journal	Experimental Hematology
Volume	40
Issue number	2
DOIs	https://doi.org/10.1016/j.exphem.2011.10.006
State	Published - Feb 2012

Funding

P.A.A., D.S.W., W.M.M., and E.T.P. designed the study, wrote the manuscript and analyzed the data. P.A.A. and M.C. performed the research. This work was supported in part by funding to E.T.P. through the Delaware Biotechnology Institute and National Institutes of Health grant RO1 HL081241 to D.S.W. P.A.A. acknowledges personal funding through the Delaware Biotechnology Institute and the A.S. Onassis foundation. The authors acknowledge use of the Bioimaging Center (Dr. Jeffrey Kaplan) and the Center for Translational Cancer Research, both at the University of Delaware, and the Histotechnology Core Facility at the A. I. duPont Hospital for Children (Wilmington, DE, USA). Jinlin Jiang is acknowledged for help in image acquisition. The authors are thankful to Frank Warren, Sue Seta, Julie Mis, and Jillian Hash from the University of Delaware Office of Laboratory Animal Medicine for expert animal care and help with various procedures. P.A.A. wishes to thank Prof. J.D. Crispino for helpful suggestions and Drs. Stephan Lindsey and J.C. Kostyak for helpful discussions over the course of the research.

ASJC Scopus subject areas

Molecular Biology
Hematology
Genetics
Cell Biology
Cancer Research

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.exphem.2011.10.006

Cite this

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title = "Role of tumor suppressor p53 in megakaryopoiesis and platelet function",

abstract = "The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53-/- megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53-/- marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53-/- megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53-/- mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53-/- mice. Although their platelet counts were normal, p53-/- mice exhibited significantly longer bleeding times and p53-/- platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.",

author = "Apostolidis, {Pani A.} and Woulfe, {Donna S.} and Massiel Chavez and Miller, {William M.} and Papoutsakis, {Eleftherios T.}",

note = "Funding Information: P.A.A., D.S.W., W.M.M., and E.T.P. designed the study, wrote the manuscript and analyzed the data. P.A.A. and M.C. performed the research. This work was supported in part by funding to E.T.P. through the Delaware Biotechnology Institute and National Institutes of Health grant RO1 HL081241 to D.S.W. P.A.A. acknowledges personal funding through the Delaware Biotechnology Institute and the A.S. Onassis foundation. The authors acknowledge use of the Bioimaging Center (Dr. Jeffrey Kaplan) and the Center for Translational Cancer Research, both at the University of Delaware, and the Histotechnology Core Facility at the A. I. duPont Hospital for Children (Wilmington, DE, USA). Jinlin Jiang is acknowledged for help in image acquisition. The authors are thankful to Frank Warren, Sue Seta, Julie Mis, and Jillian Hash from the University of Delaware Office of Laboratory Animal Medicine for expert animal care and help with various procedures. P.A.A. wishes to thank Prof. J.D. Crispino for helpful suggestions and Drs. Stephan Lindsey and J.C. Kostyak for helpful discussions over the course of the research. ",

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AU - Chavez, Massiel

AU - Miller, William M.

AU - Papoutsakis, Eleftherios T.

N1 - Funding Information: P.A.A., D.S.W., W.M.M., and E.T.P. designed the study, wrote the manuscript and analyzed the data. P.A.A. and M.C. performed the research. This work was supported in part by funding to E.T.P. through the Delaware Biotechnology Institute and National Institutes of Health grant RO1 HL081241 to D.S.W. P.A.A. acknowledges personal funding through the Delaware Biotechnology Institute and the A.S. Onassis foundation. The authors acknowledge use of the Bioimaging Center (Dr. Jeffrey Kaplan) and the Center for Translational Cancer Research, both at the University of Delaware, and the Histotechnology Core Facility at the A. I. duPont Hospital for Children (Wilmington, DE, USA). Jinlin Jiang is acknowledged for help in image acquisition. The authors are thankful to Frank Warren, Sue Seta, Julie Mis, and Jillian Hash from the University of Delaware Office of Laboratory Animal Medicine for expert animal care and help with various procedures. P.A.A. wishes to thank Prof. J.D. Crispino for helpful suggestions and Drs. Stephan Lindsey and J.C. Kostyak for helpful discussions over the course of the research.

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N2 - The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53-/- megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53-/- marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53-/- megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53-/- mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53-/- mice. Although their platelet counts were normal, p53-/- mice exhibited significantly longer bleeding times and p53-/- platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

AB - The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53-/- megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53-/- marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53-/- megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53-/- mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53-/- mice. Although their platelet counts were normal, p53-/- mice exhibited significantly longer bleeding times and p53-/- platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

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Role of tumor suppressor p53 in megakaryopoiesis and platelet function

Abstract

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ASJC Scopus subject areas

UN SDGs

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