Jiro Hirose

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.

Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.

Protective effect of recombinant neutrophil elastase inhibitor (R-020) on sepsis-induced organ injury in rat

Severe inflammatory responses after major surgeries, trauma, and infection develop multiple organ dysfunction. In the mechanisms of the pathogenesis of these responses, activated neutrophils are thought to be important in terms of their ability to produce various kinds of proteinases, which can degrade various proteins constructing human tissues. Among their proteinases, neutrophil elastase is the strongest serine proteinase secreted from activated neutrophils. Thus, we examined in this study the inhibitory effect and therapeutic efficacy of newly produced recombinant human Kunitz-type proteinase inhibitor (R-020), which coded the second domain of human urinary trypsin inhibitor. R-020 was effective in significantly improving the survival rate after induction of the rat lethal peritonitis model (cecal ligation and punctureinduced septic shock model). We suggest that various serine proteinases are implicated in the pathogenesis of neutrophil-related multiple organ failure and that recombinant human Kunitz-type proteinase inhibitor might be effective in the treatment of these kinds of organ dysfunction.

The new sepsis marker, sCD14-ST, induction mechanism in the rabbit sepsis models

Soluble CD14 subtype (sCD14-ST) is a fragment of CD14 and is markedly increased in sepsis patients. We developed a new immunoassay to detect sCD14-ST and evaluated the efficacy of this marker for diagnosis of sepsis. For developing the strategies of sCD14-ST as a sepsis diagnostic marker, the induction mechanism must be known.

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.