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1 Marine Biological Laboratory, Woods Hole, MA
2 University of California, Davis, CA
* Corresponding author: pbarmstrong{at}ucdavis.edu
The fibrillar blood clot functions as an important element of the innate immune system by its ability to entrap and immobilize bacteria that have entered the body via wounds, thereby preventing their systemic dissemination throughout the body of the injured host (1, 2). The coagulin clot of the horseshoe crab appears to play a similar role in protecting that animal from pathogenic attack. Bacteria entrapped in the coagulin clot are held so tightly as to abolish even thermal (Brownian) motion, and the clot synergizes with plasma in the killing of entrapped microbes (3). The present study investigates the proteins of the innate immune system of Limulus that bind to the fibrils of the coagulin clot, potentially supplementing the entrapment actions of the clot in two ways: first, by the lethality of clot-bound proteins for the entrapped microbes and second, by the ability of these proteins that decorate the clot fibrils to bind and inactivate the toxic products of entrapped microbes.
To establish the clot, the blood cells contained in 1 drop of blood collected under sterile conditions were dispersed in 2 ml of sterile 3% NaCl (Baxter Healthcare Corp., Deerfield, IL) in a 35-mm plastic petri dish (Falcon Cat # 35-1008). After 5 min to allow attachment of the cells to the dish surface, the saline was replaced with 50% or 100% sterile Limulus plasma. Under these conditions, the blood cells rapidly degranulated, releasing the coagulin blood-clotting system. A dense coagulin clot then formed above the monolayer of attached blood cells. After 0.5–2 h of washing with several changes of wash buffer, this was either fixed directly in 4% paraformaldehyde dissolved in 3% NaCl, 10 mM CaCl2 or was extracted with 0.5% Triton X-100 in the same buffer and then fixed in paraformaldehyde. All of the antibodies used for this report showed identical staining patterns for the two preparations. The various proteins used to prepare antibodies were purified as follows: coagulogen, the structural protein of the blood clot, as described by Srimal et al. (4); Limulus 
2-macroglobulin, as described by Armstrong et al. (5); the Limulus pentraxins, as described by Armstrong et al. (6); and hemocyanin, purified by ultracentrifugation (285,000 x g, 8 h) followed by gel filtration chromatography (Sephacryl S-300). Antibody production in rabbits and immunocytochemical staining utilized standard methods (7). The polyclonal antibodies were checked for specificity by Western blotting (8) and in some cases were affinity-purified on antigen-Sepharose affinity columns (7).
The fibrillar structure of the coagulin clot showed to advantage both by phase contrast microscopy and by immunocytochemical staining with anti-coagulogen antibodies (data not shown). The same fibrils also immunostained with antibodies prepared against highly purified preparations of Limulus 2-macroglobulin (data not shown), the Limulus pentraxins (Fig. 1), and hemocyanin (data not shown). Limulus 2-macroglobulin functions in the binding and clearance of proteases, including, presumably, the proteases of pathogenic microbes (9). The Limulus pentraxins show a potent cytolytic activity against foreign cells and may operate to assist in the cytolytic destruction of microbial invaders (6, 10). Hemocyanin is the respiratory protein in solution in the blood but additionally shows a phenoloxidase activity that potentially functions to kill microorganisms by the generation of oxygen radicals (11).
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2-macroglobulin may be covalent because it was not removed by treatment prior to fixation with boiling SDS-polyacrylamide gel sample buffer containing 2-mercaptoethanol. Most of the known ligand-recognition properties of the Limulus pentraxins are Ca+2-dependent (6). In contrast, binding to the coagulin clot is Ca+2-independent, because immunostaining is not diminished by treatment of the clot with a Ca+2-chelating agent, ethylenediaminetetraacetic acid (EDTA—0.1 M EDTA, 0.5 M NaCl, 10 mM Tris, pH 7.3). Most of the bound hemocyanin is removed by treatment of the clot with EDTA, but it is not certain whether this is simply a reflection of the dependence of the oligomeric structure of the hemocyanin molecule on Ca+2 (12) or a true Ca+2-dependent binding of hemocyanin to the coagulin fibrils. There are two potential sources for the clot-bound 2-macroglobulin: the plasma (13) and the 2-macroglobulin released from the secretory granules of the blood cells (14). We have not ruled out binding of plasma 2-macroglobulin, but secretory-granule-derived 2-macroglobulin does contribute to the clot-bound protein, because the clot fibrils produced by cells that degranulate in saline (0.5 M NaCl, 10 mM CaCl2) in the absence of plasma are decorated with 2-macroglobulin.
The fibrin fibers of the mammalian blood clot are known to bind a suite of proteins that assist in the functions of the clot. The blood clot of mammals binds fibronectin, which potentiates the immigration of wound-repair fibroblasts (15); FGF-2, which promotes proliferation of clot-associated endothelium (16, 17); and the serpins, plasmin activator inhibitor-2 (PAI-2) and 2-antiplasmin, which are presumed to protect the clot from proteolysis (18). However, we are not aware of any reports of agents of the immune system binding to the fibrin clot of mammals. Thus the observation that immune effector proteins bind to the Limulus clot suggests the novel idea that the clot is more than a passive entrapment device for invading microbes; it is potentially a delivery vehicle for proteins that are lethal to the entrapped microbes and proteins that inactivate toxic products of those microbes. Indeed, the clot synergizes with factors in the plasma in effecting the active killing of clot-entrapped microbes (3).
This research was supported by Grant No MCB-26771 from the National Science Foundation.
Literature Cited
This article has been cited by other articles:
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  Y. Matsuda, T. Osaki, T. Hashii, T. Koshiba, and S.-i. Kawabata A Cysteine-rich Protein from an Arthropod Stabilizes Clotting Mesh and Immobilizes Bacteria at Injury Sites J. Biol. Chem., November 16, 2007; 282(46): 33545 - 33552. [Abstract] [Full Text] [PDF]
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