Coagulin

Last updated
Coagulin
Identifiers
SymbolCoagulin
Pfam PF02035
InterPro IPR000275
SCOP2 d1aoca_ / SCOPe / SUPFAM
Available protein structures:
Pfam   structures / ECOD  
PDB RCSB PDB; PDBe; PDBj
PDBsum structure summary
Limulus polyphemus horseshoe crabs use coagulin to form gel clots Atlantic horseshoe crab (Limulus polyphemus).jpg
Limulus polyphemus horseshoe crabs use coagulin to form gel clots

Coagulin is a gel-forming protein of hemolymph that hinders the spread of bacterial and fungal invaders by immobilizing them. It is produced in the coagulogen form before being cleaved into the active form through a serine proteinase cascade. [1] [2] [3] It has been most extensively studied in horseshoe crabs. It has also been produced by other organisms, such as Bacillus coagulans I4 in a plasmid location. [4] In human medicine, coagulation of coagulin is the basis of detection of bacterial endotoxin through the Limulus amebocyte lysate test for parenteral medications.

Contents

Structure

Coagulogen contains a single 175-residue polypeptide chain that is cleaved after Arg-18 and Arg-46 by a Limulus clotting enzyme contained in the granular hemocyte cells of the hemolymph. A pathway is initiated in which ultimately the limulus clotting enzyme cleaves coagulogen to coagulin. Cleavage releases two chains of coagulin, chains A and B, covalently linked by two disulfide bonds, together with the peptide C. [1] [2] [5] The A-B fold wraps around the helical peptide C, forming a compact structure. [6] The approximate mass of coagulin is 3-4 kDA by SDS-PAGE. [4] Gel formation results from interlinking of coagulin molecules. [1] Before interlinking the coagulin monomers, peptide C is cleaved from coagulogen. Removal of peptide C exposes an extended hydrophobic cove of the newly cleaved molecule, allowing interaction with a second molecule’s hydrophobic edge. [6] [7] The full-length structure of a coagulogen is known ( PDB: 1AOC ​); it shares the same cystine-knot cytokine superfamily (fold) as neurotrophins, with several cystines conserved.

Coagulation

Hemolymph coagulation is a part of the invertebrate immune response. Factors within the hemolymph are activated and initiate a pathway where insoluble clots are formed in order to prevent leakage of bodily fluids and immobilized microbes from infecting the organism. [8] This is crucial as invertebrate organisms do not have adaptive immune systems comparable to the one in the mammalian immune system. [7] In crustaceans, hemolymph coagulation depends on the transglutaminase-mediated cross-linking of specific plasma-clotting proteins, but without the proteolytic cascade. [9]

In horseshoe crabs, the proteolytic coagulation cascade is triggered by lipopolysaccharides and beta-1,3-glucans. There are two types of hemocytes within the horseshoe crab hemolymph: granular and nongranular. The granular hemocytes are activated by bacterial endotoxins lipopolysaccharides (LPS) that are found on the surface of Gram-negative bacteria. “...LPS comprises approximately 70% of the outer membranes of gram-negative bacteria.” [10] They are also activated by beta-1,3-glucans that are found on the cell walls of yeast and some fungi. [5] [7]

In the LPS-activated pathway, LPS activates zymogen factor C. It is autocatalytically converted into the activated form factor C. The active factor C converts inactive factor B into active factor B. Active factor B converts the proclotting enzyme into the clotting enzyme. [5] The clotting enzyme cleaves coagulogen into coagulin, resulting in noncovalent coagulin homopolymers through head-to-tail interaction.

In the beta-1,3-glucan activated pathway, there are slight differences. Beta-1,3-glucan activates zymogen factor G. It is autocatalytically converted into the activated form factor G. From here, the pathway converges into the LPS-activated pathway. The active factor G converts the proclotting enzyme into the clotting enzyme to cleave coagulogen into coagulin. [5] In both pathways, gel formation occurs when the final enzyme transglutaminase cross-links coagulin. [7]

However, horseshoe crab transglutaminase does not cross-link coagulins intermolecularly. Recently, coagulins were discovered to be cross-linked on hemocyte cell surface proteins called proxins. This indicates that a cross-linking reaction at the final stage of hemolymph coagulation is an important innate immune system of horseshoe crabs. [9]

In comparison, mammalian blood coagulation differs from hemolymph coagulation. Mammalian blood coagulation is largely dependent on platelets and fibrin, whereas hemolymph does not contain platelets or fibrin but hemocytes.[ citation needed ] Mammalian blood coagulation is based on the proteolytically induced polymerization of fibrinogens. There are two pathways (Tissue factor and Contact) that result in thrombin converting fibrinogen to fibrin. Fibrin monomers noncovalently interact with each other and polymerize to form the blood clot. [11] Fibrin and coagulin are analogous to each other. Similarities between mammalian blood coagulation and hemolymph coagulation include gel formation, TGase, and serve as a part of wound healing. [7] However, the clot formed in hemolymph coagulation is softer than the mammalian fibrin clot. [5] [7]

Uses

Limulus amebocyte lysate test

Limulus amebocyte lysate is found only in horseshoe crabs, specifically the Limulus polyphemus species. In the presence of bacterial endotoxins (LPS) and beta-1,3-glucans, it initiates the coagulation pathways . It is employed as an FDA-approved assay method to test sterility of medical instruments and injectable drugs, such as in the pharmaceutical industry. [12]

Since the 1970s, the Limulus amebocyte lysate test has been used to test for endotoxins in human blood samples. The original method (Limulus gelation test) involved qualitatively looking for coagulin gel formation. After a one hour incubation, if the sample was coagulated, it formed a solid clot that was positive for endotoxins. If the sample was not coagulated, it would be liquid and was negative for endotoxins. [10] However, the technique was limited by its sensitivity. [13]

Today, the Limulus test is one hundred times more sensitive and uses a chromogenic method of detection. [5] When coagulogen is cleaved by the clotting enzyme, coagulin is produced. However the clotting enzyme also produces a chromogenic end product known as pNA. pNA (Boc-Leu-Gly-Arg-p-nitroanilide) is the chromogenic product that emits a yellow color. [14] [15] The concentration of endotoxins in a sample can be calculated by measuring the absorbance of released pNA at 405 nm. [5] [15]

Evolution

Coagulin is found in the four species of horseshoe crabs: Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, and Carcinoscorpius rotundicauda. They are deemed “living fossils” as they have been around for 445-500 million years with little significant change compared to their ancestors. [7] [12] The coagulin precursor, coagulogen, has a mutation rate of 1.2 x 10−9 per amino acid per year as compared to its mammalian analog, fibrinogen, with a mutation rate of 8.3 x 10−9. [7] It is contained in hemocytes, a type of phagocyte. There are different types of phagocytes and are found in all invertebrate groups (as either hemocytes, amoebocytes, or coelomocytes). Comparing vertebrae and Limulus polyphemus coagulation systems, none of the cascade proteins (including coagulogen) share a common protein domain with two exceptions, Hemolectin and TGase. While the two systems are functionally similar, the coagulation proteins “have different evolutionary histories.” [16]

See also

Related Research Articles

<span class="mw-page-title-main">Thrombus</span> Blood clot

A thrombus, colloquially called a blood clot, is the final product of the blood coagulation step in hemostasis. There are two components to a thrombus: aggregated platelets and red blood cells that form a plug, and a mesh of cross-linked fibrin protein. The substance making up a thrombus is sometimes called cruor. A thrombus is a healthy response to injury intended to stop and prevent further bleeding, but can be harmful in thrombosis, when a clot obstructs blood flow through healthy blood vessels in the circulatory system.

<span class="mw-page-title-main">Platelet</span> Component of blood aiding in coagulation

Platelets or thrombocytes are a component of blood whose function is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot. Platelets have no cell nucleus; they are fragments of cytoplasm derived from the megakaryocytes of the bone marrow or lung, which then enter the circulation. Platelets are found only in mammals, whereas in other vertebrates, thrombocytes circulate as intact mononuclear cells.

<span class="mw-page-title-main">Coagulation</span> Process of formation of blood clots

Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. It results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The process of coagulation involves activation, adhesion and aggregation of platelets, as well as deposition and maturation of fibrin.

<span class="mw-page-title-main">Disseminated intravascular coagulation</span> Medical condition where blood clots block small blood vessels

Disseminated intravascular coagulation (DIC) is a condition in which blood clots form throughout the body, blocking small blood vessels. Symptoms may include chest pain, shortness of breath, leg pain, problems speaking, or problems moving parts of the body. As clotting factors and platelets are used up, bleeding may occur. This may include blood in the urine, blood in the stool, or bleeding into the skin. Complications may include organ failure.

<span class="mw-page-title-main">Thrombin</span> Enzyme involved in blood coagulation in humans

Thrombin is a serine protease, an enzyme that, in humans, is encoded by the F2 gene.

<span class="mw-page-title-main">Atlantic horseshoe crab</span> Species of arthropod

The Atlantic horseshoe crab, also known as the American horseshoe crab, is a species of horseshoe crab, a kind of marine and brackish chelicerate arthropod. It is found in the Gulf of Mexico and along the Atlantic coast of North America. The main area of annual migration is Delaware Bay along the South Jersey Delaware Bayshore.

<span class="mw-page-title-main">Lipopolysaccharide</span> Class of molecules found in the outer membrane of Gram-negative bacteria

Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a polysaccharide that are bacterial toxins. They are composed of an O-antigen, an outer core, and an inner core all joined by covalent bonds, and are found in the bacterial capsule, the outermost membrane of cell envelope of Gram-negative bacteria, such as E. coli and Salmonella. Today, the term endotoxin is often used synonymously with LPS, although there are a few endotoxins that are not related to LPS, such as the so-called delta endotoxin proteins produced by Bacillus thuringiensis.

<span class="mw-page-title-main">Factor XIII</span> Class of enzymes

Factor XIII or fibrin stabilizing factor is a zymogen found in blood of humans and some other animals. It is activated by thrombin to factor XIIIa. Factor XIIIa is an enzyme of the blood coagulation system that crosslinks fibrin. Deficiency of XIII worsens clot stability and increases bleeding tendency.

<span class="mw-page-title-main">Factor X</span> Mammalian protein found in Homo sapiens

Factor X, also known by the eponym Stuart–Prower factor, is an enzyme of the coagulation cascade. It is a serine endopeptidase. Factor X is synthesized in the liver and requires vitamin K for its synthesis.

<span class="mw-page-title-main">Innate immune system</span> Immunity strategy in living beings

The innate immune system or nonspecific immune system is one of the two main immunity strategies in vertebrates. The innate immune system is an alternate defense strategy and is the dominant immune system response found in plants, fungi, prokaryotes, and invertebrates.

The prothrombinase enzyme complex consists of factor Xa (a serine protease) and factor Va (a protein cofactor). The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin (factor II), an inactive zymogen, to thrombin (factor IIa), an active serine protease. The activation of thrombin is a critical reaction in the coagulation cascade, which functions to regulate hemostasis in the body. To produce thrombin, the prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320. Although it has been shown that factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances. The prothrombinase complex can catalyze the activation of prothrombin at a rate 3 x 105-fold faster than can factor Xa alone. Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.

<span class="mw-page-title-main">Limulus amebocyte lysate</span> Chemical used for the detection and quantification of bacterial endotoxins

Limulus amebocyte lysate (LAL) is an aqueous extract of motile blood cells (amebocytes) from the Atlantic horseshoe crab Limulus polyphemus. LAL reacts with bacterial endotoxins such as lipopolysaccharides (LPS), which are components of the bacterial capsule, the outermost membrane of cell envelope of gram-negative bacteria. This reaction is the basis of the LAL test, which is widely used for the detection and quantification of bacterial endotoxins.


Depyrogenation refers to the removal of pyrogens from solutions, most commonly from injectable pharmaceuticals.

<span class="mw-page-title-main">Amebocyte</span>

An amebocyte or amoebocyte is a motile cell in the bodies of invertebrates including cnidaria, echinoderms, molluscs, tunicates, sponges, and some chelicerates.

Limulus clotting factor overbar C is an enzyme. This enzyme catalyses the following chemical reaction

Limulus clotting factor B is an enzyme. This enzyme catalyses the following chemical reaction

Limulus clotting enzyme is an enzyme. This enzyme catalyses the following chemical reaction:

<span class="mw-page-title-main">Contact activation system</span>

In the contact activation system or CAS, three proteins in the blood, factor XII (FXII), prekallikrein (PK) and high molecular weight kininogen (HK), bind to a surface and cause blood coagulation and inflammation. FXII and PK are proteases and HK is a non-enzymatic co-factor. The CAS can activate the kinin–kallikrein system and blood coagulation through its ability to activate multiple downstream proteins. The CAS is initiated when FXII binds to a surface and reciprocal activation of FXII and PK occurs, forming FXIIa and PKa. FXIIa can initiate the coagulation cascade by cleaving and activating factor XI (FXI), which leads to formation of a blood clot. Additionally, the CAS can activate the kinin–kallikrein system when PKa cleaves HK to form cHK, releasing a peptide known as bradykinin (BK). BK and its derivatives bind to bradykinin receptors B1 and B2 to mediate inflammation.

<span class="mw-page-title-main">Fred Bang</span> Medical researcher

Frederik Barry Bang (1916–1981) was an American medical researcher who developed the Limulus amebocyte lysate (LAL) test for bacterial endotoxins. He was influential in applying marine biology to medical research, especially immunology.

Jack Levin is an American physician-scientist and hematologist who, with Fred Bang, developed the Limulus amebocyte lysate (LAL) test for bacterial endotoxins.

References

  1. 1 2 3 Srimal S, Miyata T, Kawabata S, Miyata T, Iwanaga S (August 1985). "The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda". Journal of Biochemistry. 98 (2): 305–318. doi:10.1093/oxfordjournals.jbchem.a135283. PMID   3905780.
  2. 1 2 Miyata T, Usui K, Iwanaga S (June 1984). "The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas". Journal of Biochemistry. 95 (6): 1793–1801. doi:10.1093/oxfordjournals.jbchem.a134792. PMID   6469947.
  3. ↑ Loof TG, Mörgelin M, Johansson L, Oehmcke S, Olin AI, Dickneite G, et al. (September 2011). "Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense". Blood. 118 (9): 2589–2598. doi: 10.1182/blood-2011-02-337568 . PMID   21613262. S2CID   16041445.
  4. 1 2 Hyronimus B, Le Marrec C, Urdaci MC (July 1998). "Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4". Journal of Applied Microbiology. 85 (1): 42–50. doi: 10.1046/j.1365-2672.1998.00466.x . PMID   9721655. S2CID   27449682.
  5. 1 2 3 4 5 6 7 Iwanaga S (May 2007). "Biochemical principle of Limulus test for detecting bacterial endotoxins". Proceedings of the Japan Academy. Series B, Physical and Biological Sciences. 83 (4): 110–119. Bibcode:2007PJAB...83..110I. doi:10.2183/pjab.83.110. PMC   3756735 . PMID   24019589.
  6. 1 2 Bergner A, Oganessyan V, Muta T, Iwanaga S, Typke D, Huber R, Bode W (December 1996). "Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor". The EMBO Journal. 15 (24): 6789–6797. doi:10.1002/j.1460-2075.1996.tb01070.x. PMC   452504 . PMID   9003754.
  7. 1 2 3 4 5 6 7 8 Iwanaga S, Kawabata S (September 1998). "Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab". Frontiers in Bioscience. 3 (4): D973–D984. doi: 10.2741/A337 . PMID   9727083.
  8. ↑ "Hemolymph coagulation". QuickGO. European Bioinformatics Institute. GO:0042381.
  9. 1 2 Osaki T, Kawabata S (June 2004). "Structure and function of coagulogen, a clottable protein in horseshoe crabs". Cellular and Molecular Life Sciences. 61 (11): 1257–1265. doi:10.1007/s00018-004-3396-5. PMID   15170505. S2CID   24537601.
  10. 1 2 Tinker-Kulberg R, Dellinger K, Brady TE, Robertson L, Levy JH, Abood SK, et al. (2020-04-01). "Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source". Frontiers in Marine Science. 7. doi: 10.3389/fmars.2020.00153 . ISSN   2296-7745.
  11. ↑ Smith SA, Travers RJ, Morrissey JH (2015-07-04). "How it all starts: Initiation of the clotting cascade". Critical Reviews in Biochemistry and Molecular Biology. 50 (4): 326–336. doi:10.3109/10409238.2015.1050550. PMC   4826570 . PMID   26018600.
  12. 1 2 "Facts About Horseshoe Crabs and FAQ". Florida Fish And Wildlife Conservation Commission. Retrieved 2023-11-27.
  13. ↑ Novitsky TJ (December 1994). "Limulus amebocyte lysate (LAL) detection of endotoxin in human blood". Journal of Endotoxin Research. 1 (4): 253–263. doi: 10.1177/096805199400100407 . ISSN   0968-0519. S2CID   85838507.
  14. ↑ "Bacterial Endotoxin test". Wako LAL System. Retrieved 2023-11-27.
  15. 1 2 Sandle T (2016). "Endotoxin and pyrogen testing". Pharmaceutical Microbiology. Elsevier. pp. 131–145. doi:10.1016/b978-0-08-100022-9.00011-6. ISBN   9780081000229.
  16. ↑ Coban A, Bornberg-Bauer E, Kemena C (December 2022). "Domain Evolution of Vertebrate Blood Coagulation Cascade Proteins". Journal of Molecular Evolution. 90 (6): 418–428. Bibcode:2022JMolE..90..418C. doi:10.1007/s00239-022-10071-3. PMC   9643190 . PMID   36181519.
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