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  • 1
    ISSN: 1573-4943
    Keywords: limulus coagulation cascade ; coagulin gel ; bacterial endotoxins ; proclotting enzyme ; factor B, C, G, anti-LPS factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 → 3)-β-d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.
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  • 2
    ISSN: 1573-4943
    Keywords: prothrombin ; staphylocoagulase ; α-thrombin ; streptokinase ; Staphylococcus aureus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of α-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by α-chymotrypsin. Limited α-chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (Kd=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (Kd=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, α-chymotrypsin, and elastase.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The protein journal 11 (1992), S. 368-369 
    ISSN: 1573-4943
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0878
    Keywords: Clotting factors ; Antimicrobial substances ; Hemocytes ; Immunocytochemistry ; Tachypleus tridentatus (Chelicerata)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 μm in diameter) and less electron-dense than the D-granules (less than 0.6 μm in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.
    Type of Medium: Electronic Resource
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  • 5
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