Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice

Paul, David S.; Casari, Caterina; Wu, Ching Hsiang; Piatt, Raymond; Pasala, Swetha; Campbell, Robert A.; Poe, Kathryn O.; Ghalloussi, Dorsaf; Lee, Robert H.; Rotty, Jeremy D.; Cooley, Brian C.; Machlus, Kellie R.; Italiano, Joseph E.; Weyrich, Andrew S.; Bergmeier, Wolfgang

doi:10.1182/bloodadvances.2017006973

Cited by 38 publications

(49 citation statements)

References 38 publications

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“…While the data are generated using a single pharmacological inhibitor of formin function, it is well established that the FH2 domain is highly conserved across species and is absolutely required for the actin polymerization action of formin proteins. However, data from models of reduced Arp2/3 complex expression show that full spreading is lost, but platelets are still able to form filopodia, 26,27 presumably via the action of formins and the presence of preformed actin filaments. The observation that adding SMIFH2 to platelets that had already spread resulted in the loss of stress fibers and lamellipodia collapse (Figure 4) would seem to support the hypothesis that lamellipodia formation and persistence requires formin-mediated actin filaments.…”

Section: Discussionmentioning

confidence: 99%

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

Green

Zuidscherwoude

Alenazy

et al. 2020

Journal of Thrombosis and Haemostasis

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Background Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. Objectives Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. Methods Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. Results Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post‐translational modification. Conclusions These data therefore demonstrated an important role for formin‐mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross‐talk between the actin and tubulin cytoskeletons.

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Section: Discussionmentioning

confidence: 99%

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

Green

Zuidscherwoude

Alenazy

et al. 2020

Journal of Thrombosis and Haemostasis

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“…Of note, ectopic release of (pro)platelet-like particles was still detectable in ADAP-deficient mice after macrophage depletion and platelet recovery on day 5 post clodronate administration (supplemental Figure 14). Interestingly, mice lacking Wiskott-Aldrich syndrome protein (WASp), Arp2/3, or Profilin 1 (Pfn1), all of which display a thrombocytopenia, also show ectopic release of (pro)platelet-like particles into the BM compartment, 24,35,36 emphasizing the importance of F-actin organization in the terminal stages of platelet production. A multimolecular complex containing SLP-76, ADAP, Ena/VASP, Nck, and WASp has been identified in Raw264.7 macrophages, which mediates signaling between the FcRg receptor and the actin cytoskeleton.…”

Section: Discussionmentioning

confidence: 99%

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

Spindler

Eeuwijk

Schurr

et al. 2018

Blood

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Bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal-recessive small-platelet thrombocytopenia (CARST) was described; it is caused by mutations in the adhesion and degranulation-promoting adaptor protein (; synonym: ,) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice ( ) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole-sternum 3-dimensional confocal imaging and intravital 2-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the BM compartment. In addition, cultured BM-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP-deficient mice (PF4-cre) also produced fewer and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is an MK-intrinsic defect. Taken together, these results point to an as-yet-unidentified role of ADAP in the process of MK polarization and platelet biogenesis.

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“…6,7,9 Furthermore, studies in mice have associated loss of the actin-binding proteins tropomodulin 3, profilin 1, and Arp2/3 with thrombocytopenia. [10][11][12] However, the molecular mechanisms that orchestrate actin dynamics and mediate the crosstalk between actin and MT during platelet biogenesis are still incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

et al. 2020

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Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

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Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice

Cited by 38 publications

References 38 publications

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

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