Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice by David S. Paul, Caterina Casari, Congying Wu, Raymond Piatt,

Slides:

Advertisements

Similar presentations

Supplemental Material for Endothelial CXCR7 Regulates Breast Cancer Metastasis Amanda C. Stacer 1 Joseph Fenner 1 Stephen P. Cavnar 2 Annie Xiao 1 Shuang.

Advertisements

Red blood cells mediate the onset of thrombosis in the ferric chloride murine model by Justin D. Barr, Anil K. Chauhan, Gilbert V. Schaeffer, Jessica K.

Arterioscler Thromb Vasc Biol

Thymidine Phosphorylase Participates in Platelet Signaling and Promotes ThrombosisNovelty and Significance by Wei Li, Alba Gigante, Maria-Jesus Perez-Perez,

Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice by Sravya Kattula, James R. Byrnes,

by Miguel Vicente-Manzanares, Aranzazu Cruz-Adalia, Noa B

by Matt W. Goschnick, Lai-Man Lau, Janet L. Wee, Yong S. Liu, P

Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease by Alec A. Schmaier,

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models by Ronghua Meng, Jie Wu, Dawn C. Harper,

Vessel wall BAMBI contributes to hemostasis and thrombus stability

by Simon Stritt, Inga Birkholz, Sarah Beck, Simona Sorrentino, K

Necrotic platelets provide a procoagulant surface during thrombosis

The origin and function of platelet glycosyltransferases

A functional folate receptor is induced during macrophage activation and can be used to target drugs to activated macrophages by Wei Xia, Andrew R. Hilgenbrink,

Plasminogen promotes macrophage phagocytosis in mice

Angiotensin 1-7 and Mas decrease thrombosis in Bdkrb2−/− mice by increasing NO and prostacyclin to reduce platelet spreading and glycoprotein VI activation.

Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito by Shigeto Yoshida,

by Silke Huber, Reinhard Hoffmann, Femke Muskens, and David Voehringer

Alterations in platelet secretion differentially affect thrombosis and hemostasis by Smita Joshi, Meenakshi Banerjee, Jinchao Zhang, Akhil Kesaraju, Irina.

Liemin Au, Jeffrey P. Meisch, Lopa M. Das, Amy M. Binko, Rebecca S

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation by Kellie R. Machlus, Stephen K. Wu,

Rap1-GTP–interacting adaptor molecule (RIAM) is dispensable for platelet integrin activation and function in mice by Simon Stritt, Karen Wolf, Viola Lorenz,

Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy by Mark P. Chao, Chad Tang, Russell K. Pachynski,

Β1 integrin−mediated signals are required for platelet granule secretion and hemostasis in mouse by Tobias Petzold, Raphael Ruppert, Dharmendra Pandey,

Impaired activation of platelets lacking protein kinase C-θ isoform

Brain-derived microparticles induce systemic coagulation in a murine model of traumatic brain injury by Ye Tian, Breia Salsbery, Min Wang, Hengjie Yuan,

Volume 20, Issue 8, Pages (August 2017)

Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation by Shilei Chen, Changhong.

Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic.

Quinine-induced thrombocytopenia: drug-dependent GPIb/IX antibodies inhibit megakaryocyte and proplatelet production in vitro by José Perdomo, Feng Yan,

Cathepsin-B-dependent apoptosis triggered by antithymocyte globulins: a novel mechanism of T-cell depletion by Marie-Cécile Michallet, Frederic Saltel,

Role of sialic acid for platelet life span: exposure of β-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein.

Platelets can enhance vascular permeability

TLR5 signaling in murine bone marrow induces hematopoietic progenitor cell proliferation and aids survival from radiation by Benyue Zhang, Damilola Oyewole-Said,

Interaction of kindlin-3 and β2-integrins differentially regulates neutrophil recruitment and NET release in mice by Zhen Xu, Jiayi Cai, Juan Gao, Gilbert.

The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via β2 integrins (CD11/CD18)‏ by Ludwig T. Weckbach,

by Thomas D. Nightingale, Krupa Pattni, Alistair N. Hume, Miguel C

Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice by Kellie R. Machlus, Jessica C. Cardenas, Frank C.

Dasatinib enhances megakaryocyte differentiation but inhibits platelet formation by Alexandra Mazharian, Cedric Ghevaert, Lin Zhang, Steffen Massberg,

Volume 18, Issue 10, Pages (October 2010)

by Hairui Su, Chiao-Wang Sun, Szu-Mam Liu, Xin He, Hao Hu, Kevin M

Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis by Manasa K. Nayak, Nirav Dhanesha,

Mature murine megakaryocytes present antigen-MHC class I molecules to T cells and transfer them to platelets by Anne Zufferey, Edwin R. Speck, Kellie R.

Protein kinase C signaling dysfunction in von Willebrand disease (p

Enhancing functional platelet release in vivo from in vitro–grown megakaryocytes using small molecule inhibitors by Danuta Jarocha, Karen K. Vo, Randolph.

Inter-α inhibitor proteins maintain neutrophils in a resting state by regulating shape and reducing ROS production by Soe Soe Htwe, Hidenori Wake, Keyue.

Platelet-specific deletion of SNAP23 ablates granule secretion, substantially inhibiting arterial and venous thrombosis in mice by Christopher M. Williams,

Volume 20, Issue 4, Pages (July 2017)

by Silvia Mele, Stephen Devereux, Andrea G

Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation by Frederic Lagarrigue, Alexandre R. Gingras,

by Cheryl L. Maier, Amanda Mener, Seema R. Patel, Ryan P

Volume 3, Issue 5, Pages (November 2014)

Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice by Xuemei Fan, Conghui Wang, Panlai Shi, Wen Gao, Jianmin Gu, Yan.

Platelet HMGB1 is required for efficient bacterial clearance in intra-abdominal bacterial sepsis in mice by Hui Zhou, Meihong Deng, Yingjie Liu, Chenxuan.

by Hendra Setiadi, Tadayuki Yago, Zhenghui Liu, and Rodger P. McEver

Defective negative regulation of Toll-like receptor signaling leads to excessive TNF-α in myeloproliferative neoplasm by Hew Yeng Lai, Stefan A. Brooks,

ELMO1 deficiency enhances platelet function

by Megan S. Rost, Ilya Shestopalov, Yang Liu, Andy H. Vo, Catherine E

by Sarah K. Westbury, Kate Downes, Claire Burney, Maria L

by Defne Bayik, Debra Tross, Lydia A

TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice by Christopher W. Smith, Zaher Raslan, Lola Parfitt,

by Rodrigo Abreu, Frederick Quinn, and Pramod K. Giri

by Kelly E. Johnson, Julia R. Ceglowski, Harvey G. Roweth, Jodi A

Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies by Nadine Müller-Calleja, Svenja.

A specialized pathway for erythroid iron delivery through lysosomal trafficking of transferrin receptor 2 by Shadi Khalil, Maja Holy, Stephen Grado, Robert.

by Alyssa J. Moroi, Nicole M. Zwifelhofer, Matthew J. Riese, Debra K

Targeting CD47 in Sézary syndrome with SIRPαFc

Fig. 2 ODN enhances EV transfer between cells expressing TLR9.

Granulocyte colony-stimulating factor mobilizes dormant hematopoietic stem cells without proliferation in mice by Jeffrey M. Bernitz, Michael G. Daniel,

by Fabian C. Verbij, Nicoletta Sorvillo, Paul H. P

Presentation transcript:

Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice by David S. Paul, Caterina Casari, Congying Wu, Raymond Piatt, Swetha Pasala, Robert A. Campbell, Kathryn O. Poe, Dorsaf Ghalloussi, Robert H. Lee, Jeremy D. Rotty, Brian C. Cooley, Kellie R. Machlus, Joseph E. Italiano, Andrew S. Weyrich, James E. Bear, and Wolfgang Bergmeier BloodAdv Volume 1(18):1398-1408 August 8, 2017 © 2017 by The American Society of Hematology

David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Marked microthrombocytopenia in Arpc2fl/flPF4-Cre mice. Marked microthrombocytopenia in Arpc2fl/flPF4-Cre mice. (A) Schematic of gene targeting strategy. Exon 8 of the Arpc2 gene, flanked by LoxP sites, was excised by PF4 promoter-driven expression of Cre recombinase. (B) Representative immunoblot demonstrating knockdown of p34 and loss of Arp2 and Arp3. (C) Platelet count (Plts) (n = 6) and (D) size (n = 5) in peripheral blood of the indicated mice. (E) Representative scanning electron microscopy images of resting platelets. Arrows indicate abnormally small platelets in Arpc2fl/flPF4-Cre sample. (F) Percentage of reticulated platelets (n = 6-7). Blue bars, control; red bars, Arpc2fl/flPF4-Cre. Data presented as mean ± SEM, ***P < .001. ns, Not significant. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Premature platelet release into the bone marrow compartment in Arpc2fl/flPF4-Cre mice. Premature platelet release into the bone marrow compartment in Arpc2fl/flPF4-Cre mice. (A) Increased megakaryocyte density in representative images of bone marrow and spleen sections stained with hematoxylin and eosin. (B) Quantification of megakaryocyte density in indicated organs (8-13 random fields analyzed per organ; n = 3). (C) Percentage of proplatelet forming bone marrow megakaryocytes observed in culture (n = 7-8). (D) Representative flow cytometry dot plots showing increased number of platelet-sized CD42b+ particles in bone marrow from Arpc2fl/flPF4-Cre mice. (E) Quantification of CD42b+ particles in bone marrow (n = 6). (F) Representative confocal fluorescence images of bone marrow from the indicated mice. Green, megakaryocytes/platelets; red, endothelial cells/vasculature; blue, nuclei; blue bars, control; red bars, Arpc2fl/flPF4-Cre. Data presented as mean ± SEM, *P < .05, ***P < .001. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Arp2/3-deficient platelets are cleared from circulation at an increased rate. Arp2/3-deficient platelets are cleared from circulation at an increased rate. (A) Clearance of antibody-labeled platelets from circulation in control and Arpc2fl/flPF4-Cre mice (n = 5). (B) Clearance of antibody-labeled control and Arpc2fl/flPF4-Cre platelets cotransfused into wild-type recipient mice (n = 6). (C) Platelet count and (D) platelet size in the indicated mice at various time points following antibody-mediated depletion of circulating platelets (n = 7). Blue bars, control; red bars, Arpc2fl/flPF4-Cre. Data presented as mean ± SEM, *P < .05, **P < .01, ***P < .001. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Loss of Arp2/3 leads to macrophage-mediated clearance of platelets. Loss of Arp2/3 leads to macrophage-mediated clearance of platelets. (A) Binding of RCA-fluorescein isothiocyanate to platelets from mice treated (+neu) or not (−neu) with neuraminidase (n = 4-6). (B) Detection of platelet-associated antibodies with anti-mouse–IgG-488 (green bar, positive control). (C) Kinetics of phosphatidyl serine exposure in washed platelets incubated in the presence of 4 mM CaCl2 (n = 6). (D) Representative flow cytometry plot illustrating the interaction of RAW 264.7 macrophages and fluorescently labeled platelets. Changes in peripheral platelet count (E) and platelet size (F) in indicated mice following macrophage depletion by clodronate liposomes (lip) or treatment with PBS liposomes (n = 10). (G) Frequency analysis of circulating platelet size 72 hours after treatment with clodronate liposomes or PBS liposomes (mean distributions of 5-6 mice per group). Blue bars/lines, control; red bars/lines, Arpc2fl/flPF4-Cre. Data presented as mean ± standard deviation. *P < .05, ***P < .001. FSC-H, forward scatter height. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Altered actin dynamics in Arpc2fl/flPF4-Cre platelets. Altered actin dynamics in Arpc2fl/flPF4-Cre platelets. (A) Quantification of the surface area of unstimulated (resting) or stimulated platelets (500 μM Par4p, 750 ng/mL Cvx, or 10 μM adenosine 5′-diphosphate) spread on fibrinogen for 30 minutes (n = 5). (B) Representative immunofluorescent images of Cvx-stimulated platelets spread on a fibrinogen matrix and stained with phalloidin. (C) Representative scanning electron microscopy images of spread platelets on fibrinogen matrix from indicated mice, stimulated with Cvx (750 ng/mL). (D) Quantification of platelet F-actin (I), G-actin (II), and F-actin:G-actin ratio (III) at resting conditions or after stimulation with Par4p (500 μM) or Cvx (1 μg/mL) for 10 minutes before fixation (n = 5). (E) Representative transmission electron microscopy images of resting platelets from indicated mice; boxes indicate location of intracellular tubulin ring bundles. Scale bar, 200 nm. (B) Representative immunofluorescent images of tubulin rings in resting platelets from indicated mice. Blue bars, control; red bars, Arpc2fl/flPF4-Cre. Data presented as mean ± SEM. *P < .05, **P < .01, ***P < .001. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Mild platelet activation defect but intact hemostasis in Arpc2fl/flPF4-Cre mice. Mild platelet activation defect but intact hemostasis in Arpc2fl/flPF4-Cre mice. (A) Representative traces showing aggregation of platelet rich plasma stimulated with increasing doses of the indicated agonists. (B) Quantification of total clot area after platelet-mediated retraction (n = 3-5) and representative images of retracted clots. (C-D) Tail clip bleeding assay. Time to cessation of bleeding (C) and amount of blood loss (D) from severed tails of the indicated mice (n = 11-12). (E) Time to occlusion of carotid artery after induction of thrombosis by administration of FeCl3 solution to vessel (n = 8-9). (F) Kinetics of platelet accumulation and peak platelet density at injury site of saphenous vein after laser ablation (8 total injury sites per group; 3 mice per group). (G) Representative images of intradermal bleeding at sites of local inflammation induced by rPA reaction in platelet depleted and nondepleted Arpc2fl/flPF4-Cre mice (n = 3 mice). Blue/black bars/lines, control; red bars/lines, Arpc2fl/flPF4-Cre). Data presented as mean ± SEM. *P < .05. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology

Schematic representation of the role of Arp2/3 in platelet function and platelet homeostasis. David S. Paul et al. Blood Adv 2017;1:1398-1408 © 2017 by The American Society of Hematology