Yoshitaka Hosaka

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.

Elevated plasma levels of soluble platelet glycoprotein VI (GPVI) in patients with thrombotic microangiopathy.

BACKGROUND Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets. This study examined the soluble platelet glycoprotein VI (sGPVI) levels in patients with TMA to evaluate the activation of platelets in thrombotic states. MATERIALS AND METHODS The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in patients with TMA. RESULTS The plasma levels of sGPVI were significantly higher in postoperative patients, patients with TMA and those with disseminated intravascular coagulation (DIC) than in those without thrombosis. The plasma levels of sGPVI were the highest in patients with TMA without markedly reduced ADAMTS13 and those were significantly reduced after plasma exchange. CONCLUSION The measurement of sGPVI level is therefore considered to be important for the diagnosis and evaluation of TMA.

Species Specificity of the Anticoagulant Activity of Human Urinary Soluble Thrombomodulin

The anticoagulant activities of human urinary soluble thrombomodulin (UTM) in blood taken from various species using several anticoagulant assay systems were compared; it was examined which coagulant assay system is appropriate for evaluation of the antithrombotic effects of UTM and how the species specificity of UTM is involved in the mechanisms of action of UTM. When anticoagulant activities were compared using activated partial thromboplastin time (APTT), thromboelastography (TEG), and thrombin generation test (TGT), the effect of UTM was found to be the strongest in humans among various species tested. Among the anticoagulant assays tested, TGT reflecting protein C (PC) activation by UTM, appeared to be more sensitive than APTT and TEG in detection of thrombomodulin activity. In the study of the mechanisms of action of UTM, UTM exhibited nearly the same antithrombin activity against human and rat thrombin; the rate of activation of human PC by thrombin/UTM complex was much higher than that of rat PC. Therefore, the species specificity of the anticoagulant activity of UTM may be attributable to thrombin/UTM-PC interaction, but not to UTM-thrombin interaction. From these results, we concluded that TGT reflecting PC activation by UTM will be a more useful assay than APTT and TEG for estimating the antithrombotic effects of UTM in humans. Furthermore, our findings suggest that UTM will exhibit more potent antithrombotic effects in humans than those in rats by strongly enhancing thrombin-catalyzed PC activation.

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.

Properties of soluble glycoprotein VI, a potential platelet activation biomarker

Abstract Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.

Effect of trapidil on effector functions of monocytes related to atherosclerotic plaque.

The infiltration and activation of inflammatory cells play an important role in the formation and stability of coronary atherosclerotic plaque in patients with acute coronary syndrome. In this study, we evaluated the effect of trapidil, an anti-platelet agent, on atheroma-related functions of human T cells and monocytes. Trapidil and anti-CD154 (CD40 ligand) antibody inhibited the increase of procoagulant activity in the mixed lymphocyte reaction; trapidil also suppressed the induction of tissue factor, monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 in the mixed lymphocyte reaction. Trapidil did not alter CD154 expression on isolated T cells, but it diminished CD40 expression on isolated monocytes and human monocytic leukemia THP-1 cells stimulated with interferon-gamma. Moreover, trapidil reduced MCP-1 production of isolated monocytes and THP-1 cells stimulated with interferon-gamma plus CD154-transfected cells. This effect was not seen with other tested anti-platelet agents and coronary vasodilators. In conclusion, trapidil directly acts on monocytes/macrophages to lower their susceptibility to CD154 on T cells.

A novel free fatty acid receptor 1 (GPR40/FFAR1) agonist, MR1704, enhances glucose‐dependent insulin secretion and improves glucose homeostasis in rats

Activation of G protein‐coupled receptor 40/Free fatty acid receptor 1 (GPR40/FFAR1), which is highly expressed in pancreatic β cells, is considered an important pharmacologic target for the treatment of type 2 diabetes mellitus. The aim of this study was to determine the effect of MR1704, a novel GPR40/FFAR1 agonist, on glucose homeostasis in rats. MR1704 is a highly potent and selective, orally bioavailable agonist with similar in vitro potencies among humans, mice, and rats. Treatment of rat islets with MR1704 increased glucose‐dependent insulin secretion. Augmentation of glucose‐dependent insulin secretion was abolished by adding a GPR40/FFAR1 antagonist. In mouse, insulinoma MIN6 cells, palmitic acid induced the activity of caspase 3/7 after a 72‐h exposure, while pharmacologically active concentrations of MR1704 did not. In an oral glucose tolerance test in normal Sprague‐Dawley rats, orally administered MR1704 (1–10 mg·kg−1) reduced plasma glucose excursion and enhanced insulin secretion, but MR1704 did not induce hypoglycemia, even at 300 mg·kg−1, in fasted Sprague‐Dawley rats. In addition, orally administered MR1704 reduced plasma glucose excursion and enhanced insulin secretion in diabetic Goto‐Kakizaki rats. Oral administration of MR1704 once daily to Goto‐Kakizaki rats reduced their blood glucose levels during a 5‐week treatment period without reducing pancreatic insulin content; as a result, hemoglobin A1C levels significantly decreased. These results suggest that MR1704 improves glucose homeostasis through glucose‐dependent insulin secretion with a low risk of hypoglycemia and pancreatic toxicity. MR1704 shows promise as a new, glucose‐lowering drug to treat type 2 diabetes mellitus.

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.

Discovery of novel aminobenzisoxazole derivatives as orally available factor IXa inhibitors.

Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.

Free fatty acid receptor 1 agonist, MR1704, lowers blood glucose levels in rats unresponsive to the sulfonylurea, glibenclamide

Preclinical Research & Development