Key Points• Uremic solute IS increases platelet activity via activation of ROS/p38MAPK signaling.• Klotho counteracts ISinduced thrombosis by restraining platelet hyperactivity.Thrombosis is a common complication of chronic kidney disease (CKD), but the causes and mechanisms of CKD-associated thrombosis are not well clarified. Here, we show that platelet activity is remarkably enhanced in CKD mice, with increase of serum indoxyl sulfate (IS), a typical uremic toxin, which cannot be effectively cleared by routine dialysis. Ex vivo and in vitro experiments reveal that IS displays a distinct ability to enhance platelet activities, including elevated response to collagen and thrombin, increases in plateletderived microparticles, and platelet-monocyte aggregates. The flow chamber assay and carotid artery thrombosis model demonstrate that IS-induced platelet hyperactivity contributes to thrombus formation. Further investigations disclose that reactive oxygen species (ROS)-mediated p38MAPK signaling plays a key role in IS-induced platelet hyperactivity. Moreover, we show that Klotho, which is expressed dominantly in the kidneys, has the capacity to counteract IS-induced platelet hyperactivity by inhibiting ROS/p38MAPK signaling, whereas Klotho reduction may aggravate the effect of IS on platelet activation in CKD and klotho 1/2 mice. Finally, we demonstrate that Klotho protein treatment can protect against IS-induced thrombosis and atherosclerosis in apoE 2/2 mice. Our findings uncover the mechanism of platelet hyperactivity induced by IS and provide new insights into the pathogenesis and treatment of CKD-associated thrombosis.

Key Points NE and EPI promote megakaryocyte adhesion, migration, and proplatelet formation via α2-adrenoceptor-ERK1/2 signaling. Sympathetic stimulation enhances platelet production, which may facilitate recovery of thrombocytopenia or aggravate atherosclerosis.

Quiescence maintenance is an important property of hematopoietic stem cells (HSCs), whereas the regulatory factors and underlying mechanisms involved in HSC quiescence maintenance are not fully uncovered. Here, we show that steroid receptor coactivator 3 (SRC-3) is highly expressed in HSCs, and SRC-3-deficient HSCs are less quiescent and more proliferative, resulting in increased sensitivity to chemotherapy and irradiation. Moreover, the long-term reconstituting ability of HSCs is markedly impaired in the absence of SRC-3, and SRC-3 knockout (SRC-3) mice exhibit a significant disruption of hematopoietic stem and progenitor cell homeostasis. Further investigations show that SRC-3 deficiency leads to enhanced mitochondrial metabolism, accompanied by overproduction of reactive oxygen species (ROS) in HSCs. Notably, the downstream target genes of peroxisome proliferator-activated receptor-coactivators 1α (PGC-1α) involved in the regulation of mitochondrial metabolism are significantly upregulated in SRC-3-deficient HSCs. Meanwhile, a significant decrease in the expression of histone acetyltransferase GCN5 accompanied by downregulation of PGC-1α acetylation is observed in SRC-3-null HSCs. Conversely, overexpression of GCN5 can inhibit SRC-3 deficiency-induced mitochondrial metabolism enhancement and ROS overproduction, thereby evidently rescuing the impairment of HSCs in SRC-3 mice. Collectively, our findings demonstrate that SRC-3 plays an important role in HSC quiescence maintenance by regulating mitochondrial metabolism.

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Oxidized human defensin 5 (HD5 OX ), a Paneth cell-secreted antibacterial peptide with three characteristic disulfide bonds, protects the host from invasion by morbigenous microbes in the small intestine. HD5 OX can be reduced by thioredoxin (Trx) in vitro, while the biochemical properties of the reduced linear peptide, HD5 RED , remain unclear. Here, we first confirm that HD5 RED does exist in vivo. Furthermore, we reveal that the recruitment of HD5 RED to the outer membrane of Gram-negative bacteria and to the anionic lipid A is lower than that of HD5 OX , and HD5 RED is less efficient in penetrating bacterial outer and inner membranes and inducing membrane depolarization, which confers an attenuated antibacterial activity to HD5 RED . However, due to its higher structural flexibility, the binding of HD5 RED to bacterial lipopolysaccharide (LPS) is markedly stronger than that of HD5 OX . Consequently, HD5 RED is more effective in suppressing the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated macrophages by blocking the interaction between LPS and LPS-binding protein, thus suggesting that HD5 RED might act as a scavenger to neutralize LPS in the gut. This study provides insights into the antibacterial and immunoregulatory effects of HD5 RED and expands the known repertoire of the enteric defensins.Defensins are small (2-5 kDa), Cys-rich, endogenously produced, amphiphilic peptides that serve a fundamental role in the first line of host immune defence against invading microbes 1 . Based on their sequence homology and disulfide pairings, human defensins are classified into two major subfamilies, α and β 2,3 , which are primarily discovered in neutrophils and epithelia, respectively 4,5 .Oxidized human defensin 5 (HD5 OX ), a 32-residue α defensin, is stored in the secretory granules of Paneth cells in the small intestine as a propeptide (E20-R94) and is processed by anionic and/or meso isoforms of trypsin during secretion or shortly thereafter at a cleavage site between R62 and A63 6 . Because HD5 OX exhibits the widest spectrum of antibacterial activity among the α defensins 7 , it is a promising candidate that may be used to target pathogens that are clinically resistant to traditional antibiotics 8 . We have previously prepared bioactive HD5 OX using Pichia pastoris and designed a more potent antibiotic peptide based on its active region 9,10 . Recently, the thioredoxin (Trx) system was found to catalyze the reduction of intestinal defensins 11,12 . Notably, the antibacterial activity of human β defensin (HBD) 1, a ubiquitously expressed peptide in human epithelia, is enhanced upon disulfide reduction 13 . Based on the abundance of HD5 OX in the intestine 14 , the impact of disulfide reduction on its antibacterial activity warrants investigation.Human defensins are also noted for their immunoregulatory properties. For instance, HBDs have been shown to recruit immature dendritic cells, memory T cells, macrophages, and monocytes via G i -protein-coupled receptors 15,16 , and human neutrophil...

Key Points hGH has a distinct capacity to promote the differentiation, especially the terminal differentiation of human primary megakaryocytes. hGH exerts a complementary and synergistic effect with c-Mpl ligands on thrombopoiesis.

It is known that insulin-like growth factor-1 (IGF-1) also functions as a hematopoietic factor, although its direct effect on thrombopoiesis remains unclear. In this study, we show that IGF-1 is able to promote CD34 cell differentiation toward megakaryocytes (MKs), as well as the facilitation of proplatelet formation (PPF) and platelet production from cultured MKs. The in vivo study demonstrates that IGF-1 administration accelerates platelet recovery in mice after 6.0 Gy of irradiation and in mice that received bone marrow transplantation following 10.0 Gy of lethal irradiation. Subsequent investigations reveal that extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt activation mediate the effect of IGF-1 on thrombopoiesis. Notably, Akt activation induced by IGF-1 is more apparent than that of ERK1/2, compared with that of thrombopoietin (TPO) treatment. Moreover, the effect of IGF-1 on thrombopoiesis is independent of TPO signaling because IGF-1 treatment can also lead to a significant increase of platelet counts in homozygous TPO receptor mutant mice. Further analysis indicates that the activation of Akt triggered by IGF-1 requires the assistance of steroid receptor coactivator-3 (SRC-3). Therefore, our data reveal a distinct role of IGF-1 in regulating thrombopoiesis, providing new insights into TPO-independent regulation of platelet generation.

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