CK2b regulates thrombopoiesis and Ca21-Triggered platelet activation in arterial thrombosis

© 2017 by The American Society of Hematology. Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-) physiology has remained elusive. The present study explored the impact of the CK2 regulatory b-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β-/-) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-Triphosphate receptor-dependent intracellular calcium (Ca21) release. Presumably due to a blunted increase in the concentration of cytosolic Ca21, activation-dependent increases of a and dense-granule secretion and integrin aIIbb3 activation, and aggregation were abrogated in ck2β-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo wassignificantly blunted in ck2β-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2b-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2b as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy

NOX4 is an early initiator of neuropathic pain

Treatment of neuropathic pain remains challenging as the etiology is heterogeneous and pathomechanisms are incompletely understood. One possible mechanism is oxidative stress due to unphysiological reactive oxygen species (ROS) formation. The only know dedicated enzymatic source of ROS are NADPH oxidases of which the type 4 isoform (NOX4) has been suggested to be involved in the subacute and chronic phase of neuropathic pain. Here, we aim to translate this finding into a treatment strategy by examining the efficacy of the NOX1/4specific inhibitor GKT136901 using the chronic constriction injury (CCI) mouse model of neuropathic pain. Unexpectedly, post-nerve lesion treatment using GKT136901 was ineffective to reduce pain-related behavior after CCI. We therefore re-investigated the role of NOX4 using an independent KO mouse model. Early after CCI we found an increase in pro-inflammatory cytokines, ROS formation and the oxidative stress marker nitrotyrosine in the lesioned nerve together with an upregulated Nox4 gene expression. In NOX4 KO mice, mechanical allodynia was markedly reduced from day 4 after nerve injury as were all ROS related and acute biomarkers. In addition, we observed a reduction in the CCI-induced upregulation of pro-inflammatory cytokines in the sciatic nerve and dorsal root ganglia along with NOX4-deficiency. Thus, we conclude that NOX4 is involved in the development of neuropathic pain states by producing oxidative stress and subsequent cytokine dysregulation at the lesion site. This appears at very early stages immediately after nerve injury explaining ineffectiveness of post-acute pharmacological NOX inhibition. We suggest that future target validation of NOX4 should now focus on defining the possible therapeutic window in human neuropathic pain. (C) 2016 Elsevier Inc. All rights reserved.</p

TMEM16F-Mediated Platelet Membrane Phospholipid Scrambling Is Critical for Hemostasis and Thrombosis but not Thromboinflammation in Mice—Brief Report

Objective-It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. Approach and Results-We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. Conclusions-Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes

A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease-disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy

Možnosti a způsoby využití kalů a sedimentů z ČOV

V roce 2002 byly vyhodnoceny schopnosti hlavních producentů kalu využít kaly v zemědělství z hlediska technologie, byl zjišťován praktický dopad nakládání s kaly z komunálních ČOV ve vybraných zemích Evropy z hlediska hodnocení nebezpečných vlastností - zejména infekčnosti, provedeno orientační ekonomické posouzení celého procesu využívání a odstraňování čistírenských kalů v ČR, byly vybrány laboratoře pro provádění rozborů kalů, poloprovozně ověřena recyklace čistírenských kalů, navrženo hodnocení vlivu aplikace kalů z komunálních ČOV na zemědělskou půdu a rostlinnou produkci ve vztahu na půdní registr spolu s konkretizací potenciálních rizik s ohledem na plnění produkčních a ekologických funkcí půdy (návrh a rozpracování), proběhla příprava na budoucí úpravy vyhlášky podle výhledové novely Směrnice č. 86/278/EEC v ČR a hodnocení zejména zdravotního rizika při dalším využití kalů (kompostování, rekultivace, energetické využití) včetně stanovení mikrobiologických kritérií