Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration.

Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIbα, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.

GPVI signaling is compromised in newly formed platelets after acute thrombocytopenia in mice.

At sites of vascular injury, exposed subendothelial collagens trigger platelet activation and thrombus formation by interacting with the immunoreceptor tyrosine-based activation motif (ITAM)-coupled glycoprotein VI (GPVI) on the platelet surface. Platelets are derived from the cytoplasm of megakaryocytes (MKs), which extend large proplatelets into bone marrow (BM) sinusoids that are then released into the bloodstream, where final platelet sizing and maturation occurs. The mechanisms that prevent activation of MKs and forming proplatelets in the collagen-rich BM environment remain largely elusive. Here, we demonstrate that newly formed young platelets (NFYPs) released after antibody-mediated thrombocytopenia in mice display a severe and highly selective signaling defect downstream of GPVI resulting in impaired collagen-dependent activation and thrombus formation in vitro and in vivo. The diminished GPVI signaling in NFYPs is linked to reduced phosphorylation of key downstream signaling proteins, including Syk, LAT, and phospholipase Cγ2, whereas the G protein-coupled receptor and C-type lectin-like receptor 2 signaling pathways remained unaffected. This GPVI signaling defect was overcome once the platelet counts were restored to normal in the circulation. Overall, these results indicate that the GPVI-ITAM signaling machinery in NFYPs after antibody-mediated thrombocytopenia only becomes fully functional in the blood circulation.

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

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 (ADAP; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice (Adap-/- ) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap-/- mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap-/- 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 ß1 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.

Blood collection, platelet isolation and measurement of platelet count and size in mice-a practical guide.

Inherited or acquired disorders of platelet production and function can result in thrombocytopenia and bleeding. Mouse models have proven useful for investigating the mechanisms that underlie these defects in humans. Precise methods for blood withdrawal, platelet isolation and measurement of platelet parameters are key for the generation of reproducible and conclusive data. Here, we provide three different protocols for mouse platelet isolation to encourage research knowledge transfer between experienced laboratories, while at the same time enabling less experienced researchers to implement a protocol that best suits their local expertise and equipment. We also address the issue that reported mouse platelet count and size vary considerably in the literature by investigating different factors that influence these important platelet parameters, namely: 1) genetic background and gender, 2) choice of analysis method (hematological analyzer or flow cytometry), 3) dilution of the blood sample and 4) choice of anticoagulant. The herein presented results and considerations may serve as a practical guide for both experienced and new researchers in the platelet field.

Differential Role of the RAC1-Binding Proteins FAM49b (CYRI-B) and CYFIP1 in Platelets.

Platelet function at vascular injury sites is tightly regulated through the actin cytoskeleton. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex (WRC) activates lamellipodia formation via ARP2/3, initiated by GTP-bound RAC1 interacting with the WRC subunit CYFIP1. The protein FAM49b (Family of Unknown Function 49b), also known as CYRI-B (CYFIP-Related RAC Interactor B), has been found to interact with activated RAC1, leading to the negative regulation of the WRC in mammalian cells. To investigate the role of FAM49b in platelet function, we studied platelet-specific Fam49b-/--, Cyfip1-/--, and Cyfip1/Fam49b-/--mice. Platelet counts and activation of Fam49b-/- mice were comparable to those of control mice. On fully fibrinogen-coated surfaces, Fam49b-/--platelets spread faster with an increased mean projected cell area than control platelets, whereas Cyfip1/Fam49b-/--platelets did not form lamellipodia, phenocopying the Cyfip1-/--platelets. However, Fam49b-/--platelets often assumed a polarized shape and were more prone to migrate on fibrinogen-coated surfaces. On 2D structured micropatterns, however, Fam49b-/--platelets displayed reduced spreading, whereas spreading of Cyfip1-/-- and Cyfip1/Fam49b-/--platelets was enhanced. In summary, FAM49b contributes to the regulation of morphology and migration of spread platelets, but to exert its inhibitory effect on actin polymerization, the functional WAVE complex must be present.

The WASH-complex subunit Strumpellin regulates integrin αIIbß3 trafficking in murine platelets.

The platelet specific integrin αIIbß3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbß3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbß3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbß3 surface expression. While exposure of the internal αIIbß3 pool after platelet activation was unaffected, the uptake of the αIIbß3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbß3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbß3 trafficking in murine platelets.

Absence of early platelet increment in healthy mice during decitabine treatment.

Treatment of myelodysplastic syndromes includes the administration of the hypomethylating agent decitabine. An early platelet response in decitabine-treated myelodysplastic syndrome patients is a predictor of overall survival. The effect of decitabine on megakaryocytes and the bone marrow, however, is understudied. We show that an early platelet increment was not detectable in healthy mice during decitabine treatment. Analyses of bone marrow sections revealed vessels with dilated lumina, decreased cellularity, but increased number of red blood cells and the presence of (pro)platelet-like particles. Taken together, decitabine treatment of healthy mice does not induce an early platelet increment, but affects the bone marrow.

Novel insights into mouse models of ectopic proplatelet release.

Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished ß1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.

BACKGROUND: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. OBJECTIVES: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). METHODS: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. RESULTS: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide. CONCLUSION: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

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

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.

The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis.

Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.

A novel body weight support system extension: initial concept and simulation study.

Body weight supported treadmill training is an approach to gait rehabilitation following a stroke or spinal cord injury. Although lateral control of balance is an important aspect of walking, many of the currently available body weight support systems have a fixed pulley configuration which can lead to lateral forces being developed in the supporting cables, interfering with the lateral balance task. In this paper, a novel extension for body weight support systems, used for treadmill walking, is presented which features a system of pulleys and trolleys. A model is developed for the device along with a basic feedback controller in order to enable simulation of the concept. The lateral forces induced by the novel system are greatly reduced in comparison to a fixed pulley system. This device has applications in balance training within gait rehabilitation programs.