Shoji Takahashi

A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical applications

A conventional limulus test is not specific to endotoxin because of the presence in amebocyte lysate of a (1----3)-beta-D-glucan-sensitive factor. By fractionating coagulation enzymes in the lysate and recombining only those factors involved in endotoxin-induced coagulation, we have developed a new test specific to endotoxin. The recombined enzymes reacted only with endotoxin, and not with fungal polysaccharides. Conventional amebocyte lysate, on the other hand, reacted with both of them. A good linearity was obtained with this method between endotoxin concentration and absorbance with a sensitivity of 1 pg/ml of Escherichia coli 0111:B4 endotoxin. The regression lines for different types of endotoxins were parallel to one another. For the correct diagnosis of endotoxemia, this new test has a definite advantage over the one using whole amebocyte lysate.

Activation of a limulus coagulation factor G by (1 → 3)-g⊝d-glucans

Abstract Various oligo- and poly-saccharides differing in sugar compositions and types of linkage were examined for their ability to activate a limulus coagulation factor G, the first protease in an alternative coagulation cascade of the horseshoe crab, Tachypleus tridentatus , whose amebocytes have originally been known to contain a lipopolysacchaide(LPS)-driven pathway leading to the formationof coagulin gel. Linear and branched (1 → 3)-β- d -glucans and mixed linkage (1 → 3),(1 → 4)-β- d -glucans were found to exhibit the ability to activate factor G at concentrations of 10 −8 –10 −10 g/mL as assayed by amidolytic activity of the clotting enzyme. Laminaran oligosaccharides, laminaran dextrins (number-average mol. wt. ≦ 5800), and other polysaccharides including carboxymethylcellulose, amylose, starch, d -fructans, α- l -arabinan, β- d -xylans, (1 → 3)-β- d -galactan, water-soluble chitin derivatives, chondroitin, chondroitin sulfates, hyaluronan, keratan sulfate, heparins, heparan sulfate, and LPSs were virtually inactive as activators even at a concentration of 10 −7 g/mL. The activating ability increased with increasing number-average mol. wt. (6800–216 000) of linear (1 → 3)-β- d -glucans. The apparent activating ability of gyrophoran, nigeran, and yeast α- d -mannan was largely abolished by digestion with a highly purified Arthrobacter luteus endo-(1 → 3)-β- d -glucanase, which provided supportive evidence for the activation to be ascribed to contaminating (1 → 3)-β- d -glucan(s). Possible participation of ordered structures of (1 → 3)-β- d -glucans in the activation of factor G is discussed.

Inhibition of high-molecular-weight-(1 → 3)-β-d-glucan-dependent activation of a limulus coagulation factor G by laminaran oligosaccharides and curdlan degradation products

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.