Platelet endothelial aggregation receptor-1 (PEAR1) participates in platelet aggregation via sustaining αIIbβ3 activation. To investigate the role of PEAR1 in platelet formation, we monitored and ...manipulated PEAR1 expression in vitro in differentiating human CD34+ hematopoietic stem cells and in vivo in zebrafish embryos. PEAR1 expression rose during CD34+ cell differentiation up to megakaryocyte (MK) maturation. Two different lentiviral short hairpin knockdowns of PEAR1 did not affect erythropoiesis in CD34+ cells, but increased colony-forming unit MK cell numbers twofold vs control in clonogenic assays, without substantially modifying MK maturation. The PEAR1 knockdown resulted in a twofold reduction of the phosphatase and TENsin homolog (PTEN) phosphatase expression and modulated gene expression of several phosphatidylinositol 3-kinase (PI3K)-Akt and Notch pathway genes. In zebrafish, Pear1 expression increased progressively during the first 3 days of embryo development. Both ATG and splice-blocking PEAR1 morpholinos enhanced thrombopoiesis, without affecting erythropoiesis. Western blots of 3-day-old Pear1 knockdown zebrafish revealed elevated Akt phosphorylation, coupled to transcriptional downregulation of the PTEN isoform Ptena. Neutralization by morpholinos of Ptena, but not of Ptenb, phenocopied the Pear1 zebrafish knockdown and triggered enhanced Akt phosphorylation and thrombocyte formation. In summary, this is the first demonstration that PEAR1 influences the PI3K/PTEN pathway, a critical determinant of Akt phosphorylation, itself controlling megakaryopoiesis and thrombopoiesis.
• PEAR1 is a negative regulator of megakaryocyte proliferation in vitro and thrombocyte formation in vivo.• PEAR1 regulates the PI3K/PTEN pathway.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical ...manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.
•Adsorption of VWF type 2B mutants to platelets induces thrombocytopenia in VWD type 2B mice.•VWF/platelet complexes are phagocytosed by macrophages in liver and spleen.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel TUBB1 gene mutations that co‐segregated with TD in three distinct ...families leading to 1.1% of TUBB1 mutations in TD study cohort. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the β‐tubulin protein family. TUBB1 gene is expressed in the developing and adult thyroid in humans and mice. All three TUBB1 mutations lead to non‐functional α/β‐tubulin dimers that cannot be incorporated into microtubules. In mice, Tubb1 knock‐out disrupted microtubule integrity by preventing β1‐tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for β1‐tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin‐coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis.
Synopsis
Whole‐exome sequencing in a consanguineous family with congenital hypothyroidism (CH) revealed a novel homozygous mutation in TUBB1, encoding for a member of beta‐tubulins, essential for microtubule organisation. Until now, TUBB1 mutations were only known to be involved in platelet disorders.
Two more mutations were identified in two distinct families with thyroid dysgenesis (TD) and abnormal platelet physiology, reinforcing the link between TUBB1 mutations and thyroid disease.
TUBB1 is expressed in the developing and adult thyroid in humans and mice.
Tubb1−/− mice have large platelets and show hypothyroidism with early thyroid progenitors proliferation defects, altered thyroid migration and failure of thyroid hormone synthesis.
Platelet findings concerned basal activation and increased aggregation.
These results expand our knowledge on genetic background of CH and TD.
Whole‐exome sequencing in a consanguineous family with Congenital Hypothyroidism (CH) revealed a novel homozygous mutation in TUBB1, encoding for a member of beta‐tubulins, essential for microtubule organisation. Until now, TUBB1 mutations were only known to be involved in platelet disorders.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
OBJECTIVE—Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in ...platelets, the underlying mechanisms are not yet fully characterized.
APPROACH AND RESULTS—By studying a mouse model (cKO conditional knockout) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKO platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKO platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKO platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein.
CONCLUSIONS—Our results indicate that a kinesin-1–dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.
The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology ...and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low ...amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbβ3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic ...telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with ...nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management.
Plain language summary
A combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management?
Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia.
Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions.
We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist.
We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor.
The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management.
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