Tomihisa Kawasaki

Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.

Antithrombotic effects of YM‐60828, a newly synthesized factor Xa inhibitor, in rat thrombosis models and its effects on bleeding time

The effects of YM‐60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic effects and its prolongation of template bleeding time in rats. YM‐60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus. In ex vivo studies, YM‐60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose‐dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time. In a venous thrombosis model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.0081 mg kg−1, 0.011 mg kg−1, 6.3 iu kg−1 and 4.7 iu kg−1, respectively. In an arterio‐venous shunt model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.010 mg kg−1, 0.011 mg kg−1, 10 iu kg−1 and 4.2  iu  kg−1, respectively. In bleeding time studies, all agents prolonged template bleeding time in a dose‐dependent manner. ED2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM‐60828, argatroban, heparin and dalteparin were 0.76 mg kg−1, 0.081 mg kg−1, 18 iu kg−1 and 25 iu kg−1, respectively. The ratio (ED2/ID50) of YM‐60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin. These data show that YM‐60828 can exert its antithrombotic effects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.

YM-60828, a novel factor Xa inhibitor: Separation of its antithrombotic effects from its prolongation of bleeding time

The antithrombotic effects of intravenous infusions of YM-60828 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-nap hthyl)methyl]sulfamoyl]acetic acid dihydrochloride), a novel factor Xa inhibitor, argatroban, heparin and dalteparin in an arterio-venous shunt model were studied in comparison with their effects on template bleeding time. In an arterio-venous shunt model, all agents exerted antithrombotic effects in a dose-dependent manner. ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.0087 mg/kg/h. 0.027 mg/kg/h, 22 IU/kg/h and 11 IU/kg/h, respectively. In bleeding time studies, all agents prolonged bleeding time in a dose-dependent manner. Doses (ED2) of YM-60828, argatroban, heparin and dalteparin, which caused 2-fold prolongation of bleeding time in the saline group, were 3.0 mg/kg/h, 0.25 mg/kg/h, 18 IU/kg/h and 26 IU/kg/h. respectively. The risk-benefit ratio (ED2/ID50) of YM-60828 was much greater than that of the other agents. These data suggest that the antithrombotic effect of YM-60828 is separate from its prolongation of bleeding time and that YM-60828 is much safer than conventional anticoagulant agents.

Relationship between the antithrombotic effect of YM-75466, a novel factor Xa inhibitor, and coagulation parameters in rats

The relationship between the antithrombotic effects of intravenous infusions of YM-75466 [N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naph thyl)methyl] sulfamoyl]acetic acid monomethanesulfonate), a novel factor Xa (FXa) inhibitor, and various coagulation parameters (prothrombin time, activated partial thromboplastin time, thrombin-antithrombin III complex (TAT), anti-FXa activity and anti-thrombin activity) in rats was studied and compared with results for heparin. In the arterio-venous shunt model, both agents exerted antithrombotic effects in a dose-dependent manner. Coagulation parameters were studied simultaneously with antithrombotic effects. YM-75466 did not prolong coagulation time even at the dose which exerted significant antithrombotic effects, while it decreased TAT level in plasma in a dose-dependent manner. YM-75466 exerted anti-FXa activity but not anti-thrombin activity. In contrast, heparin prolonged activated partial thromboplastin time in a dose-dependent manner and decreased TAT level in plasma with increasing inhibition of thrombus formation. Heparin exerted both anti-FXa and anti-thrombin activity in a dose-dependent manner. These results suggest that TAT is a suitable parameter for monitoring the antithrombotic effect of YM-75466 in the arterio-venous shunt model in rats and that YM-75466, unlike heparin, exerts its antithrombotic effect through specific inhibition of FXa without any effect on thrombin.

AKR-501 (YM477) a novel orally-active thrombopoietin receptor agonist.

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. We have searched for small molecule compounds that mimic the action of TPO by using human TPO receptor‐expressed in Ba/F3 cells, resulting in the discovery of AKR‐501 (YM477). AKR‐501 specifically targeted the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as rhTPO did. AKR‐501, however, was shown to be effective only in humans and chimpanzees with high species specificity. Therefore, we examined the in vivo platelet‐increasing effect of AKR‐501 in human platelet producing non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver CD34+ cells. Daily oral administration of AKR‐501 dose‐dependently increased the number of human platelets in these mice, with significance achieved at doses of 1 mg/kg and above. The peak unbound plasma concentrations of AKR‐501 after administration at 1 mg/kg in NOD/SCID mice were similar to those observed following administration of an active oral dose in human subjects. These results suggest that AKR‐501 is an orally‐active TPO receptor agonist that may be useful in the treatment of patients with thrombocytopenia.

Biological properties of a specific Gαq/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress

1 The effects of YM‐254890, a specific Gαq/11 inhibitor, on platelet functions, thrombus formation under high‐shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM‐254890 concentration dependently inhibited ADP‐induced intracellular Ca2+ elevation, with an IC50 value of 0.92±0.28 μM. 3 P‐selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM‐254890, with IC50 values of 0.51±0.02 and 0.16±0.08 μM, respectively. 4 YM‐254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP‐stimulated washed platelets. 5 YM‐254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM‐254890 attenuated ADP‐, collagen‐, TRAP‐, arachidonic acid‐ and U46619‐induced platelet aggregation with IC50 values of <1 μM, whereas it had no effect on phorbol 12‐myristate 13‐acetate‐, ristocetin‐, thapsigargin‐ or A23187‐induced platelet aggregation. 7 High‐shear stress‐induced platelet aggregation and platelet‐rich thrombus formation on a collagen surface under high‐shear flow conditions were concentration dependently inhibited by YM‐254890. 8 The antithrombotic effect of YM‐254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM‐254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM‐254890 is a useful tool for investigating Gαq/11‐coupled receptor signaling and the physiological roles of Gαq/11.

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.

AKR-501 (YM477) in combination with thrombopoietin enhances human megakaryocytopoiesis

OBJECTIVE AKR-501 (YM477) is an orally active thrombopoietin (TPO) receptor agonist that mimics the biological effect of TPO in vitro and in vivo. Here, we report that AKR-501 in combination with TPO has additive effect on megakaryocytopoiesis. MATERIALS AND METHODS Granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells were cultured with AKR-501, TPO, or a combination of the two in serum-free liquid culture system. The numbers of hematopoietic progenitor cells, megakaryocytic progenitor cells, and megakaryocytes were measured using flow cytometry. Further, the effect of AKR-501 on TPO binding to TPO receptor was examined. RESULTS Both AKR-501 and TPO alone increased the number of megakaryocytes, and the maximum activities of AKR-501 and TPO were similar. Interestingly, in the presence of TPO concentrations producing maximal stimulation, the addition of AKR-501 increased the number of megakaryocytes to about 200% of that generated with TPO only. In the time course experiment, the combination of AKR-501 and TPO augmented the numbers of hematopoietic progenitor cells and colony-forming unit in culture in the early stages. Thus, the combination of AKR-501 and TPO enhanced not only the differentiation into megakaryocytes, but also the expansion of human hematopoietic progenitor cells. Further, AKR-501 did not inhibit TPO binding to the TPO receptor. This result indicated the possibility that AKR-501 and TPO may act simultaneously on the TPO receptor, and this could be responsible for their additive effect of on megakaryocytopoiesis. CONCLUSIONS This study suggests that AKR-501 would be useful for the treatment of thrombocytopenia even at high plasma levels of endogenous TPO following chemotherapy.

Evaluation of a GPIIb/IIIa antagonist YM337 in a primate model of middle cerebral artery thrombosis.

We compared the antithrombotic effect of anti-GPIIb/IIIa antibody Fab fragment YM337 with that of a thromboxane A2 synthetase inhibitor, sodium ozagrel. With the monkeys under halothane anesthesia, the right middle cerebral artery was observed via a transorbital approach without cutting the dura mater. Photoillumination (wavelength 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg kg(-1)) was administered intravenously. The experimental drugs were intravenously injected 15 min before rose bengal injection and followed by continuous infusion for 3 h after dye injection. The thrombotic occlusion induced by this photochemical reaction in monkey middle cerebral artery was reproducible. YM337 significantly prolonged the time to first occlusion and the total time of arterial patency during the 3-h observation period after dye injection. In contrast, sodium ozagrel had no significant effect. YM337 but not sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation. However, while sodium ozagrel significantly inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Neurological deficit in the YM337-treated animals was significantly milder than that in the control group. The area of infarct in the YM337 treatment animals was smaller than that in the control group. The novel selective GPIIb/IIIa antagonist YM337 was effective in ameliorating the decrease in patency of the middle cerebral artery and reducing the area of cerebral infarction in monkeys.

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor.

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID₅₀ values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.