Tetsushi Kawahara

Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator

We have identified cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the first time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C‐terminus, which had an intramolecular ring formed by an S–S bond and a possibly amidated C‐terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon–intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pufferfish AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body fluid regulation.

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor γ-chain complex

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Simultaneous targeting of CD14 and factor XIa by a fusion protein consisting of an anti-CD14 antibody and the modified second domain of bikunin improves survival in rabbit sepsis models

ABSTRACT Severe sepsis is a complex, multifactorial, and rapidly progressing disease characterized by excessive inflammation and coagulation following bacterial infection. To simultaneously suppress pro‐inflammatory and pro‐coagulant responses, we genetically engineered a novel fusion protein (MR1007) consisting of an anti‐CD14 antibody and the modified second domain of bikunin, and evaluated the potential of MR1007 as an anti‐sepsis agent. Suppressive effects of MR1007 on lipopolysaccharide (LPS)‐induced inflammatory responses were assessed using peripheral blood mononuclear cells or endothelial cells. Its inhibitory activity against the coagulation factor XIa was assessed using a purified enzyme and a chromogenic substrate. Anticoagulant activity was assessed using human or rabbit plasma. Anti‐inflammatory and anti‐coagulant effects and/or survival benefits were evaluated in an endotoxemia model and a cecal ligation and puncture model. MR1007 inhibited LPS‐induced cytokine production in peripheral blood mononuclear cells and endothelial cells, inhibited factor XIa, and exhibited anticoagulant activity. In an endotoxemia model, 0.3–3 mg/kg MR1007 suppressed pro‐inflammatory and pro‐coagulant responses in a dose‐dependent manner; at a dose of 3 mg/kg, the protein improved survival even when administered 8 h after the LPS injection. In addition, 10 mg/kg MR1007 administered 2 h post cecal ligation and puncture improved survival. However, MR1007 administered at doses up to 30 mg/kg did not increase ear bleeding time or bacterial counts in the cecal ligation and puncture model. Thus, simultaneous targeting of CD14 and factor XIa improves survival in the rabbit endotoxemia and sepsis models and represents a promising approach for the treatment of severe sepsis.