Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis.

Recent studies have shown that integrin alpha v beta 3, a receptor for vitronectin, plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha v beta 3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin alpha v beta 3 are peptide antagonists that include the Arg-Gly-Asp (RGD) motif. We therefore reasoned that non-peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low-molecular-weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC(50), 0.83 microM). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S-180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in HUVECs was significantly reduced by thiolutin treatment, while those of other focal adhesion proteins including vinculin and focal adhesion kinase (FAK) were not. Metabolic labeling experiments showed that thiolutin enhanced degradation of paxillin in HUVECs. Protease inhibitors (MG115 and E64-D) decreased the rate of degradation of the paxillin induced by thiolutin and partially restored thiolutin-induced inhibition of HUVEC adhesion to vitronectin. Based on these findings, we concluded that thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces the paxillin level in HUVECs and suppresses tumor cell-induced angiogenesis in vivo.

Neutralization of DHG, a new depolymerized holothurian glycosaminoglycan, by protamine sulfate and platelet factor 4.

The neutralization of depolymerized holothurian glycosaminoglycan (DHG), unfractionated heparin (UFH), and low-molecular-weight heparin (LMWH) by protamine sulfate (PS) or platelet factor 4 (PF4) was studied. In in vitro studies, the prolongation of thrombin clotting time (TCT) by these glycosaminoglycans was completely neutralized by PS, whereas activated partial thromboplastin time (APTT) was relatively resistant to neutralization. In rats, prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT ex vivo. Heparin-cofactor-II-dependent antithrombin activity of DHG or UFH was neutralized by PF4 at a high concentration to the same extent; however, prolongation of the APTT by DHG was more resistant to neutralization by PF4 at a physiological plasma level than that by UFH. In conclusion, since the prolongation of bleeding time by DHG was neutralized by PS with concomitant normalization of TCT, and since PF4 neutralized the antithrombin activity of DHG, these proteins may be useful as antidotes for DHG to prevent bleeding in case of an overdose.

The anticoagulant and hemorrhagic effects of DHG, a new depolymerized holothurian glycosaminoglycan, on experimental hemodialysis in dogs.

We studied the use of depolymerized holothurian glycosaminoglycan (DHG) as an anticoagulant in experimental beagle-dog hemodialysis using a hollow-fiber dialyzer compared to that using unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), and nafamostat mesilate (FUT). Effectiveness was based on 5 h hemodialysis and no marked clot deposition in the extracorporeal circuit. At effective doses, UFH and LMWH significantly prolonged template bleeding time, in sharp contrast to FUT and DHG, which scarcely prolonged bleeding time during hemodialysis. DHG prolonged activated partial thromboplastin time (APTT) about 6 times that of normal plasma and prolonged thrombin clotting time (TCT) markedly; FUT showed marked APTT prolongation but hardly prolonged TCT in the hemodialysis circuit at the effective dose. The anticoagulant profile of DHG thus differs completely from that of FUT. These results suggest that DHG may be useful as anticoagulant for hemodialysis with low hemorrhagic risk.

DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin, in rats.

We characterized the antithrombotic, haemorrhagic, and ex vivo anticoagulant effects of a recently identified depolymerized holothurian glycosaminoglycan (DHG), and compared these effects with those of unfractionated heparin (UFH), low molecular weight heparin (LMWH), and dermatan sulfate (DS). In thrombin-induced venous thrombus formation in rats, DHG had a significant preventive effect at 0.3 mg/kg or more at 5 min after i.v., administration. UFH, LMWH, and DS also showed a significant antithrombotic effect at 0.3, 0.3, and 1 mg/kg, respectively, under the same experimental conditions. After rat tail transection, DHG, UFH, LMWH, and DS prolonged the bleeding time significantly at 10, 1, 1, and 10 mg/kg, respectively, at 5 min after i.v., injection. Therefore, DHG exerts its antithrombotic effect with less bleeding than UFH and LMWH in experimental animals. DHG prolonged the activated partial thromboplastin time in a dose-dependent manner at 0.3-3 mg/kg, at which dose an antithrombotic effect was exhibited without any significant haemorrhagic effect. All of these glycosaminoglycans prolonged thrombin clotting time markedly at their haemorrhagic doses.

Interaction of a new depolymerized holothurian glycosaminoglycan with proteins in human plasma.

In a rat template bleeding model, depolymerized holothurian glycosaminoglycan (DHG) prolonged bleeding time at 30 mg/kg i.v. but unfractionated heparin (UFH) had the same effect at 1 mg/kg i.v., indicating that DHG is much less bleeding than UFH. To characterize this difference, we examined the affinity of DHG for plasma proteins by means of a glycosaminoglycan-conjugated cellulofine column in comparison with that of UFH. The DHG column strongly bound factor V, factor IX, protein S, histidine-rich glycoprotein, platelet factor 4 (PF4), beta-thromboglobulin, von Willebrand factor, fibronectin, and heparin cofactor II, but did not bind fibrinogen, prothrombin, factor VII, protein C, antithrombin III (ATIII), plasminogen or alpha 2-plasmin inhibitor. The profile of protein binding to the UFH column was almost the same as that of the DHG column except that ATIII showed affinity for UFH. One of the reasons why DHG caused much less bleeding than UFH is thus suggested to be the differences in their affinity for ATIII in plasma.

Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin.

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII-depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors-independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors-independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.

Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Saccharomyces cerevisiae.

As a step to breed a Saccharomyces cerevisiae strain able to produce ethanol directly from cellulose, we combined cDNA for Aspergillus aculeatus FI-CMCase (FI-carboxymethyl cellulase) with the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae and used the resultant plasmid, pYEC91, to transform S. cerevisiae. The transformed cells produced active FI-CMCase within the cytoplasm. Western-blot analysis following SDS-polyacrylamide gel electrophoresis demonstrated that the cells contained a peptide having the same molecular mass and immunological identity as A. aculeatus FI-CMCase.

Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Escherichia coli.

FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells. The enzyme from E. coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A. aculeatus.