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Thromb Haemost 1984; 52(02): 117-120
DOI: 10.1055/s-0038-1661152
Original Article
Schattauer GmbH Stuttgart

Role of Endogenous Proteinase Inhibitors in the Regulation of the Blood Clotting System of the Horseshoe Crab, Limulus Polyphemus

Peter B Armstrong
1   The Marine Biological Laboratory, Woods Hole, MA, U.S.A.
2   The Department of Zoology, University of California, Davis, CA, U.S.A.
,
Jack Levin
1   The Marine Biological Laboratory, Woods Hole, MA, U.S.A.
3   The Departments of Laboratory Medicine and Medicine, University of California School of Medicine, San Francisco, CA, U.S.A.
,
James P Quigley
1   The Marine Biological Laboratory, Woods Hole, MA, U.S.A.
4   The Department of Microbiology and Immunology, Downstate Medical Center, State University of New York, Brooklyn, NY, U.S.A.
› Author Affiliations
Further Information

Publication History

Received 14 February 1984

Accepted 14 June 1984

Publication Date:
19 July 2018 (online)

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Summary

Blood clotting in Limulus is dependent on the activity of a proteinase which converts the zymogen, coagulogen, into a form that undergoes polymerization to form the clot. The abilities of a series of recently discovered endogenous proteinase inhibitors to inhibit this enzyme and thereby serve as potential regulators of its activity were explored. The blood plasma of Limulus contains a single inhibitor that is functionally and structurally homologous to vertebrate α2 macroglobulin. During exocytosis, the blood cells (amebocytes) release a series of inhibitors, including small quantities of the α2 macroglobulin homologue; a low molecular weight, acid-and heat-stable inhibitor; and an acid-labile activity. Of the three inhibitory activities, only the cell-released, acidlabile inhibitor is capable of inhibiting the clotting enzyme.

Keywords

Blood clotting - Limulus - Proteinase inhibitors - α2 macroglobulin - Clotting enzyme
 

  • References

  • 1 Bursey CR. Histological response to injury in the horseshoe crab, Limulus polyphemus. Can J Zool 1977; 55: 1158-1165
    CrossrefPubMedGoogle Scholar
  • 2 Copley AL. The clotting of Limulus blood. Fed Proc 1947; 6: 90-91 (Abstr)
    PubMedGoogle Scholar
  • 3 Gregoire C. Blood coagulation in arthropods. VI. A study by phase contrast microscopy of blood reactions in vitro in Onychophora and various groups of arthropods. Arch Biol (Liege) 1955; 66: 489-508
    PubMedGoogle Scholar
  • 4 Howell WH. Observations on the chemical composition and coagulation of the blood of Limulus polyphemus, Callinectes hastatus, and Cucumaria sp. Johns Hopkins University Circulars 1885; 5: 4-5
    PubMedGoogle Scholar
  • 5 Loeb L. Amebocyte tissue and amoeboid movement. Protoplasma 1928; 4: 596-625
    CrossrefPubMedGoogle Scholar
  • 6 Levin J. Blood coagulation and endotoxin in invertebrates. Fed Proc 1967; 26: 1707-1712
    PubMedGoogle Scholar
  • 7 Levin J, Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull Johns Hopkins Hospital 1964; 115: 265-274
    PubMedGoogle Scholar
  • 8 Levin J, Bang FB. A description of cellular coagulation in the Limulus. Bull Johns Hopkins Hospital 1964; 115: 337-345
    PubMedGoogle Scholar
  • 9 Armstrong PB, Rickies FR. Endotoxin-induced degranulation of the Limulus amebocyte. Exp Cell Res 1982; 140: 15-24
    CrossrefPubMedGoogle Scholar
  • 10 Dumont JM, Anderson E, Winner G. Some cytologic characteristics of the hemocytes of Limulus during clotting. J Morphol 1966; 119: 181-208
    CrossrefPubMedGoogle Scholar
  • 11 Omberg RL, Reese TS. Beginning of exocytosis captured by rapidfreezing of Limulus amebocytes. J Cell Biol 1981; 90: 40-54
    CrossrefPubMedGoogle Scholar
  • 12 Murer EH, Levin J, Holme R. Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab. J Cell Physiol 1975; 86: 533-542
    CrossrefPubMedGoogle Scholar
  • 13 Nakamura S, Levin J. Fractionation of Limulus amebocyte lysate. Characterization of activation of the proclotting enzyme by an endotoxin-mediated activator Biochim Biophys Acta 1982; 707: 217-225
    CrossrefPubMedGoogle Scholar
  • 14 Nakamura S, Levin J. Endotoxin-mediated Limulus proclotting enzyme activator and detection of a previously undescribed protease (protease N). Biochem Biophys Res Comm 1982; 108: 1619-1623
    CrossrefPubMedGoogle Scholar
  • 15 Nakamura S, Morita T, Harada-Suzuki T, Iwanaga S, Takahashi K, Niwa M. A clotting enzyme associated with the hemolymph coagula-tion system of a horseshoe crab (Tachypleus tridentatus): its purification and characterization. J Biochem (Tokyo) 1982; 92: 781-792
    CrossrefPubMedGoogle Scholar
  • 16 Ohki M, Nakamura T, Morita T, Iwanaga S. A new endotoxin sensitive factor associated with hemolymph coagulation system of horseshoe crab (Limulidae). FEBS Lett. 1980; 120: 217-220
    CrossrefPubMedGoogle Scholar
  • 17 Sullivan JD, Watson SW. Purification and properties of the clotting enzyme from Limulus lysate. Biochem Biophys Res Comm 1975; 66: 848-855
    CrossrefPubMedGoogle Scholar
  • 18 Nakamura S, Iwanaga S, Harada T, Niwa M. A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties J Biochem 1976; 80: 1011-1021
    CrossrefPubMedGoogle Scholar
  • 19 Tai JY, Seid RC, Huhn RD, Liu TY. Studies on Limulus amebocyte lysate II. Purification of the coagulogen and the mechanisms of clotting. J Biol Chem 1977; 252: 4773-4776
    PubMedGoogle Scholar
  • 20 Barrett AJ. α2 macroglobulin. Proteolytic Enzymes part C. In: Methods in Enzymology. Lorand L. (ed) Academic Press; New York: 1982. 80 737-754
    Google Scholar
  • 21 Starkey PM, Barrett AJ. α2 macroglobulin, a physiological regulator of proteinase activity. In: Proteinases in Mammalian Cells and Tissues. Barrett AJ. (ed) Elsevier North-Holland Biomedical Press; Amsterdam: 1977. pp 663-696
    Google Scholar
  • 22 Quigley JP, Amstrong PB. An endopeptidase inhibitor, similar to α2-macroglobulin, present in the plasma of an invertebrate, Limulus polyphemus. J Biol Chem 1983; 258: 7903-7906
    PubMedGoogle Scholar
  • 23 Quigley JP, Armstrong PB. An endopeptidase inhibitor found in Limulus plasma: an ancient form of α2-macroglobulin. Ann N Y Acad Sci 1983; 421: 119-124
    CrossrefPubMedGoogle Scholar
  • 24 Quigley JP, Armstrong PB. Characterization and isolation of a homologue of alpha-2-macroglobulin from the plasma of the horseshoe crab, Limulus. Biol Bull (Woods Hole) 1983; 165: 495 (Abstr.)
    PubMedGoogle Scholar
  • 25 Armstrong PB, Quigley JP, Levin J. A proteinase inhibitor released from the Limulus amebocyte during exocytosis. Biol Bull 1983; 165: 488 (Abstr.)
    PubMedGoogle Scholar
  • 26 Levin J, Bang FB. Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin. Thrombos Diathes Haemorrh 1968; 19: 186-197
    PubMedGoogle Scholar
  • 27 Young NS, Levin J, Prendergast RA. An invertebrate coagulation system activated by endotoxin: Evidence for enzymatic mediation. J Clin Invest 1972; 51: 1790-1797
    CrossrefPubMedGoogle Scholar
  • 28 Ganrot PO. Determination of α2-macroglobulin as trypsin-protein esterase. Clin Chim Acta 1966; 14: 493-501
    CrossrefPubMedGoogle Scholar
  • 29 Erlanger BF, Kokowski N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961; 95: 271-288
    CrossrefPubMedGoogle Scholar
  • 30 Soderhall K, Smith VJ. Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution. Devel Comp Immunol 1983; 7: 229-239
    CrossrefPubMedGoogle Scholar
  • 31 Heimburger N. Proteinase inhibitors of human plasma - their properties and control functions Cell Proliferation. In: Proteases and Biological Control Cold Spring Harbor Confer. Reich E, Rifkin DB, Shaw E. (ed) Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1975. 2 367-386
    Google Scholar
  • 32 Laskowski M, Kato I. Protein inhibitors of proteinases. Ann Rev Biochem 1980; 49: 593-626
    CrossrefPubMedGoogle Scholar
  • 33 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem 1983; 52: 655-709
    CrossrefPubMedGoogle Scholar
  • 34 Barrett AJ, Starkey PM. The interaction of α2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism Biochem J 1973; 133: 709-724
    CrossrefPubMedGoogle Scholar
  • 35 Sullivan JD, Watson SW. Factors affecting the sensitivity of Limulus lysate. Appl Microbiol 1974; 28: 1023-1026
    PubMedGoogle Scholar
  • 36 Tanaka S, Nakamura T, Morita T, Iwanaga S. Limulus anti-LPS factor: an anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system. Biochem Biophys Res Comm 1982; 105: 717-723
    CrossrefPubMedGoogle Scholar
  • 37 Cooper JF, Hochstein HD, Seligmann EB. The Limulus test for endotoxin (pyrogen) in radiopharmaceuticals and biologicals. Bull Parenteral Drug Assoc 1972; 26: 153-162
    PubMedGoogle Scholar
  • 38 Jorgensen JH, Smith RF. Preparation, sensitivity and specificity of Limulus lysate for endotoxin assay. Appl Microbiol 1973; 26: 43-48
    PubMedGoogle Scholar
  • 39 Lindberg RB, Inge WW, Mason AD, Pruitt BA. Natural variation in sensitivity of Limulus amebocyte lysate to endotoxin. Fed Proc 1972; 31: 791 (Abstr.)
    PubMedGoogle Scholar
  • 40 Tuede M, Baek LA. Evaluation of inter and intra batch differences in LAL and hemolymph from Limulus polyphemus. Acta Path Microbiol Immunol Scand Sect B 1983; 91: 9-15
    PubMedGoogle Scholar