The Decorated Clot: Binding Of Agents Of The Innate Immune System To The Fibrils Of The Limulus Blood Clot

This is an article about a research supported by the National Science Foundation that aims to determine the involvement of proteins in the immune system that allow it to be bound to the fibrils in a fibrillar blood clot. For a brief foundation on the fibrillar blood clot, this is a significant element in the body’s complex immune system and aids the process by trapping bacteria that enter the body. It renders the bacteria still to keep from spreading further into the body systems of the immune compromised person. In the horseshoe crab, scientists found that it contained a similar mechanism called the coagulin clot that does more or less the same functions and activities of the fibrillar blood clot. When viewed under phase contrast microscopes, the coagulin clot appears to be so tightly packed and combines with plasma in order to lyze the trapped pathogenic microbes. In instituting the clot, scientists placed one drop of blood onto a solution of sterile 3 percent sodium chloride with a volume of 2 ml. Five minutes were allowed for the cells to adhere to the surface of the Petri dish. The solution was then changed to 50 or 300 percent sterile Limulus plasma. The blood cells were said to quickly degranulate, producing and activating the coagulin blood clotting system. A coagulin clot was observed to form on top of the layer of the blood cells. Several more processes were done to this specimen, including washing with a wash buffer and fixed in a solution of paraformaldehyde, sodium chloride and calcium chloride. The proteins that were utilized to set up the antibodies were purified in a complex purification system. Phase contrast microscopes were able to show the fibrillar structure of the clot clearly. The specimen was further enhanced with immunocytochemical staining. An image defining the structures of the blood clots are provided in the article. The figures show how the Limulus pentraxins are bound to the fibrils in the blood clot in the specimen. Another image shows the immunostain accompanied with an antibody as seen against the Limulus pentraxins. Another image projects the dark bodies, as viewed under the phase contrast microscopes, as the blood cell nuclei as they adhere to the surface of the culture medium. In the research, normal rabbit serum was also used in place of a specific antibody, and the fibrils formed were easily distinguished under phase contrast microscopy.

The blood of mammals are known to contain fibrin fibers that allow for the formation of a number of proteins to assist with the function of a blood clot. Fibronectin is the blood clot that initiates the fibroblasts that repairs wounds and encourages propagation of clot related endothelium. The serpins are believed to be plasmin activators that assume the role of protector of the clot from proteolysis or the breakdown of proteins or peptides into simpler, less complex molecules. The fact that the immune effector proteins latches to the Limulus clot paves the way to the idea that the clot does much more than trapping invasive microbes. It could deliver the potentially lethal proteins necessary to inactivate the toxic properties and kill the microbes as well.

ORIGINAL TEXT: 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).

View larger version (152K): [in this window] [in a new window] Figure 1. Binding of the Limulus pentraxins to fibrils of the Limulus blood clot in specimens not extracted with Triton X-100. The fibrils of the clot produced by monolayers of Limulus blood cells are visible by phase contrast microscopy (A, C) and immunostain with an antibody against the Limulus pentraxins (B). The arrows in A and B indicate the same fibril visible by phase contrast microscopy (A) and immunofluorescence (B). The darkish bodies seen by phase contrast microscopy (A, C) are the nuclei of the blood cells attached to the culture surface. When normal rabbit serum replaces specific antibody, the fibrils that are clearly visible by phase contrast microscopy (C) fail to stain (D). B and D were photographed under identical conditions and were manipulated identically in Photoshop, so are directly comparable.

The binding of 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.