Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To ...elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P‐selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live‐cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP‐selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.
Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro ...high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.
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•RKI-1447 identified in high-throughput screening as potent for SRSF2 mutant cells•RKI-1447 effective in xenograft model against primary human leukemia samples•The Inhibitor causes mitotic catastrophe, cytoskeleton reorganization, severe nuclear deformation•The results point to a new therapeutic approach for leukemias involving SRSF2 mutations
Molecular biology; Cell biology; Cancer
The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong ...cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.
Platelets play a vital role in the regulation of vascular integrity and hemostasis. Their diverse physiological activities depend on their tightly regulated adhesion to external surfaces, such as ...fibrin-based blood clots, and basement membranes that are exposed following blood vessel injury. Here, we describe the quantitative monitoring of the spatio-temporal dynamics of human platelets spreading on fibrinogen and type IV collagen, using advanced light- and electron microscopy techniques. We show that platelets sense the substrate before spreading on it, by extending filopodia that touch the adhesive ligand on the substrate, initially forming unstable 'sensing contacts', followed by the development of stable filopodial adhesions and spreading. In many of the platelets (60-75 %), filopodial spreading is followed by a steady increase in lamellipodial formation, which eventually leads to the full spreading of the platelets. Examination of microtubule dynamics in the early adhesion stages indicated that microtubules undergo major reorganization during platelet spreading on type IV collagen, manifested by their entry of one dominant MT extension into one filopodia during the initial adhesion stage. Moreover, we found that this microtubule-associated filopodia is located where the initial lamellipodium is formed, suggesting a role for microtubule extensions in platelet spreading. Notably, this association was observed only in platelets adhering to type IV collagen, but not to fibrinogen. Furthermore, we examined the molecular composition of the specific adhesion sites. Surprisingly, they revealed a universal pattern of distribution of the adhesome components vinculin, zyxin and actin. The above-mentioned substrate-specific differences prompted us to explore, in greater depth, the behavior of platelets on these two substrates. Further live-cell imaging, using interference reflection microscopy (IRM) optics, revealed major differences in temporal adhesion dynamics, between platelet attachment to fibrinogen (mainly via αIIbβ3) and collagen IV (mainly via GP-VI and α2β1) throughout the spreading process, from the early stages of adhesion, through filopodial and lamellipodial spreading, to the fully spread state. Utilizing the integrin activators manganese (Mn2+) and thrombin, we found that the apparent differences in platelet spreading dynamics on the two surfaces are attributable to differences in integrin activation.
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