Raised plasma oxidised LDL in acute cerebral infarction.

BACKGROUND: The association between oxidised low density lipoprotein (OxLDL) and cerebral infarction is suspected but not established. OBJECTIVES: To determine whether plasma OxLDL is a useful marker for monitoring oxidative stress in stroke patients. METHODS: Plasma OxLDL concentrations were determined in 56 stroke patients with cerebral infarction (n = 45) or cerebral haemorrhage (n = 11), and in 19 age matched controls, using a novel sandwich enzyme linked immunosorbent assay. RESULTS: Compared with the controls (0.130 (0.007) ng/ micro g LDL, mean (SEM)), OxLDL was significantly raised in patients with cerebral infarction (0.245 (0.022); p < 0.0001) but not in those with haemorrhage (0.179 (0.023)). Patients with cortical ischaemic infarcts (n = 22) had higher OxLDL levels than either the controls (p < 0.0001) or the patients with non-cortical ischaemic infarcts (n = 23) (p < 0.001). Increased OxLDL concentrations in patients with cortical infarcts persisted until the third day after stroke onset. The National Institutes of Health stroke scales in patients with cortical infarction were higher than in those with non-cortical infarction (p < 0.01). CONCLUSIONS: There is a significant association between raised plasma OxLDL and acute cerebral infarction, especially cortical infarction. Plasma OxLDL may reflect oxidative stress in stroke patients.

Effect of depolymerized holothurian glycosaminoglycan (DHG) on tissue factor pathway inhibitor: in vitro and in vivo studies.

Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicus Selenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor VIIa by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor VIIa inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitro activity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

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.

Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice.

A previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: > 0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED > 1 or > 3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.

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.