WO1989001512A1

WO1989001512A1 - A sponge enzyme having transglutaminase-like activity

Info

Publication number: WO1989001512A1
Application number: PCT/US1988/002857
Authority: WO
Inventors: Laszlo Lorand; Gerald Weissmann
Original assignee: The Liposome Company, Inc.; New York University; Northwestern University
Priority date: 1987-08-17
Filing date: 1988-08-17
Publication date: 1989-02-23
Also published as: AU2423988A

Abstract

The present invention relates to the isolation of a marine sponge enzyme having properties similar to transglutaminase, from the tissues of the marine sponge, specifically Microciona prolifera. Transglutaminases and transglutaminase-like substances such as this may be used in the treatment of bleeding disorders, such as hemophilia, or in the treatment of excess bleeding as may arise in a surgical or dental procedure. The transglutaminase may be administered alone or in the form of a sustained delivery vehicle, such as in liposomes, and alternatively, with or without fibronectin.

Description

A SPONGE ENZYME HAVING TRANSGLUTAMINASE-LIKE ACTIVITY

The invention described herein was made in the course of work under a Public Health Service Research Career Award, and a grant from the National Institutes of Health.

CORRESPONDING U.S. PATENT APPLICATION DATA

This application is a continuation-in-part of copending U.S. Patent Application Serial No. 085,733, filed August 17, 1987.

BACKGROUND OF THE INVENTION

The present invention is directed to the isolation and use of an enzyme having transglutaminase-like activity, isolated from marine sponges, and the use of transglutaminases from other sources, for the treatment of bleeding conditions. Such conditions may result from surgical or dental procedures, or bleeding disorders such as hemophilia or hemophilia-like disorders. Marine sponge enzyme (MSE) isolated by the methods of the present invention may be used in the treatment of such disorders. For the purposes of this application, the term "transglutaminase" shall be taken to include transglutaminase-like substances, such as MSE.

The process of clot formation is initiated when either intrinsic or extrinsic pathways are activated. The intrinsic pathway is initiated by the blood itself, such as when blood comes into contact with a substance such as glass, or in vivo, when antigen-antibody reactions or other trauma to the blood occur. The extrinsic pathway is initiated by the contact of blood with traumatized vascular wall or extravascular tissues, e.g., in injury. In either case, the enzyme prothrombin is converted to thro bin by a complex of substances known as prothrombin activator. Thrombin, a proteolytic enzyme, then catalyzes the reaction of fibrinogen, a soluble circulating protein, into insoluble fibrin. The mechanism by which this occurs is the removal of four low molecular weight peptides from each fibrinogen molecule, forming a fibrin monomer, which then polymerizes with other fibrin monomers to form long fibrin threads. Such threads form the substance (the reticulum) of the clot. In the early stages of the clot, weak non-covalent bonds hold the threads together and they thus may be easily broken apart. However, in the ensuing minutes of clot formation, thrombin activates fibrin-stabilizing factor (Factor XIII) to Factor XIII (activated form), which causes formation of covalent bonds as well as multiple cross-linkings between the fibrin threads, increasing the clot strength.

There are many unusual situations and disease conditions that make clot formation desirable; surgical and dental techniques that damage tissues of patients whose clotting is abnormal, due to drug-induced or other reasons, can cause great loss of blood. Such patients may be treated with anticlotting drugs, such as coumadin or heparin. Hemophiliacs or persons having hemophilia-like disorders have a bleeding tendency that necessitates their use of clotting factors. Such factors (especially Factor XIII , and transglutaminases) have heretofore been purified from pooled blood from human donors, and administered to human subjects in need of such therapy.

We have identified a non-blood source of a compound with activities similar to that of blood-derived transglutaminase, located in the tissues of the marine sponge Microciona prolifera. We disclose methods for isolation of this transglutaminase-like substance from the sponge, discuss the criteria by which this transglutaminase-like enzyme was identified, and disclose a mode of administration of MSE either in its free form or using a drug delivery system, such as liposomes. The present invention shows that transglutaminase activity is present in species without blood, hemolymph, or closed coelom.

The lack of blood products employed by this invention makes it an improved starting material for a transglutaminase-like substance, as such blood products have of late been the source of serious contaminants such as virus particles and byproducts (e.g., hepatitis and HTLV-III virus) that are found in the final products.

The delivery of pro-clotting enzymes to the patient has been via intravenous injection (in the case of hemophiliacs), or topical administration in the case of surgical or dental patients. In the latter case, the substance is administered to the bleeding site via application on gauze or sponges. Such administration promotes the formation of a clot at the bleeding site. Transglutaminase is such a pro-clotting enzyme but has not been previously so employed. This application is continued as needed to the site until bleeding is arrested. In the present invention, any supporting matrix that supports enzymatic activity, for example, a balloon, a heteropolymer, a polymer matrix, a gauze applicator, a sponge, or a bead, to name a few, may be used. In such a case, the MSE may be administered topically. The use of a delivery system that sustains the release of the substance at the site is a possible enhanced mechanism for its application. Such a delivery system, for example, is liposomes.

Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unila ellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer) . The bilayer is composed of two lipid monolayers having a hydrophobia "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.

The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 12:238-252 involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles.

Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Publication No. 87/00238, January 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 n filter; such vesicles are known as VET...S.

Another class of liposomes that may be used are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Patent No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Patent No. 4558,579 to Fountain, et al., and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, January 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies". Other techniques that are used to prepare vesicles include those that form reverse-phase evaporation vesicles (REVs), Papahadjopoulos et al., U.S. Patent No. 4,235,871, issued November 25, 1980.

A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., PCT Publication No. 85/04578, October 24, 1985, entitled "Steroidal Liposomes." Mayhew et al., PCT Publication No. 85/00968, March* 14, 1985, described a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivatives thereof. Also, a variety of tocopherols and their water soluble derivatives have been used to form liposomes, see Janoff et al., PCT Publication No. 87/02219, April 23, 1987, entitled "Alpha Tocopherol-Based Vesicles."

In a liposome-drug delivery system, a bioactive agent such as a drug is entrapped in or associated with the liposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871; Schnieder, U.S. Patent No. 4,114,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountain et al., U.S. Patent No. 4,588,578.

In the present invention, the transglutaminase-like substance, once purified from the sponge, may be entrapped in the aqueous space of the liposomes by any of the known procedures for forming the liposomes, and administered to the subject.

In another aspect of the invention, we disclose the topical application of transglutaminases and transglutaminase-like substances to promote clotting of the blood at epithelial sites. Further, transglutaminases are shown in the present invention to form complexes with fibronectin in blood plasma. Such complex formation was detected by a shift in transglutaminase electrophoretic (anodic) mobility, when mixtures of plasma (containing fibronectin) and transglutaminase were applied to electrophoretic gels. This binary system of transglutaminase and fibronectin can enhance the activity of topically-acting clot formation of the transglutaminase. Alternatively, (1) tertiary and (2) quarternary complexes, respectively, can be formed with (1) transglutaminase, fibronectin, and liposomes, and (2) transglutaminase, fibronectin, liposomes and a gelatin, or any combination of these components.

SUMMARY OF THE INVENTION

The present invention relates to the isolation of a marine sponge enzyme having properties similar to transglutaminase, from the tissues of the marine sponge, specifically Microciona prolifera. The transglutaminase-like substance can be used in the treatment of bleeding disorders, such as hemophilia, or in the treatment of excess bleeding as may arise in a surgical or dental procedure. The transglutaminase can be administered alone or in the form of a sustained delivery vehicle, such as in liposomes. Alternatively, a binary complex of transglutaminase and fibronectin can be prepared, or a tertiary complex of transglutaminase, fibronectin, and a liposome, or a tertiary complex of transglutaminase, fibronectin, and gelatin. Further, a quarternary complex of transglutaminase, fibronectin, a liposome, and gelatin can be administered. Such compositions can be administered topically to a subject in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWING

FIGURE 1 depicts two reactions that are catalyzed by transglutaminase. (A) Protein crosslinking via gamma-glutamyl:epsilon-lysine bonds. (B) A ine incorporation into proteins (e.g. dimethylcasein)

FIGURE 2 is a graph demonstrating the incorporation of putrescine into dimethylcasein as it varies with time and transglutaminase enzyme concentration. FIGURE 3 is a bar graph demonstrating the effect of primary amines on the inhibition of transglutaminase. A - Control, B&C - dansylcadaverine, D&E - histamine, F&G - [Di-allyl] [amino-propionyl] [benzothiophene] , H&I - dimethyldansylcadaverine.

FIGURE 4 is a graph demonstrating that the effect of transglutaminase on clotting of (A) lobster plasma and (B)

14

C-putrescine uptake is identical.

FIGURE 5 is a graph demonstrating that uptake of putrescine is possible only when both transglutaminase and substrate are present together. (A) 1:1 Mix 8-7 dialysate, 8-5 Fraction 16, (B) 8-7 dialysate, (C) 8-5 Fraction 16.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the isolation and purification of MSE from the marine sponge, and the method of use of this substance for the medical use of clotting of blood. The invention also involves the method of treating abnormal clotting disorders such as hemophilia or hemophilia-like disorders, and treatment of bleeding during trauma or surgical procedures using this substance. It also involves the administration of the substance in a sustained delivery system, for example, in liposomes. A further embodiment is the use of a tra sglutaminase-fibronectin (binary) complex at a topical site to promote and enhance clot formation. In addition, tertiary and quarternary complexes including transglutaminase, fibronectin and liposomes, and transglutaminase, fibronectin, liposomes, and gelatin, respectively can be formed, and delivered to a subject requiring treatment with clot-promoting agents. The use of liposomes in combination with gelatin matrices for delivery of drug and other substances contained therein is disclosed in U.S. Patent No. 4,708,861, to Popescu et al., issued Nov. 24, 1987, relevant portions of which are hereby incorporated by reference. The transgluta inase-fibronectin binary complex, and MSE-fibronectin complexes, disclosed in this invention, enable enhancement of the clotting capabilities of transglutaminase by maintaining a clotting-effective amount of the transglutaminase at the bleeding site by use of fibronectin, a substance normally present at clotting sites, or by the use of novel drug delivery systems such as liposomes and gelatins, alone or in combination. For example, another binary complex such as transglutaminase and liposomes can also be used. In this complex, the transglutaminase can be entrapped in or associated with the liposomes. A tertiary complex such as transglutaminase, fibronectin and liposomes can be used to both enhance delivery, both topically and parenterally, to the desired site, and to enhance the clotting capabilities by the presence of fibronectin. Finally, a quarternary complex, such as for example, transglutaminase, fibronectin, liposomes, and a gelatin, within which the other components can be contained, is also embodied by the present invention. For these binary, tertiary and quarternary complexes, any compatible transglutaminase or transglutaminase-like substance can be employed, the MSE of the present invention being among those transglutaminases.

These complexes may be formed by simple admixing of the components, for example, combination of the transglutaminase and the fibronectin. When liposomes are used, the transglutaminase and alternatively or additionally, the fibronectin, can be entrapped in or associated with the liposomes. When gelatin is used, any or all of the components, such as the transglutaminase alone or in combination with the fibronectin or the liposomes can be admixed with the gelatin and administered to the site. The formation of these binary, tertiary, or quarternary complexes requires the use of compatible solvents and ingredients to achieve the complex.

We have observed that clumped marine sponge cells resemble a blood clot, evidenced by a clump of cells surrounded by a fibrous matrix. Such matrix suggests the presence of a stabilizing factor as is present in blood clots, such substance being the transglutaminases. There are two reactions that are known to be catalyzed by transglutaminase; the first is protein crosslinking via gamma glutamyl-epsilon lysine bridges, and the second amine (primary amine) incorporation into proteins (Figure 1). We have determined that the marine sponge cells contain transglutaminase-like activity (according to procedures in Lorand et al., 1984, Molec. Cell. Biochem., 58:9-35). Unbroken cells show no such activity, but supematants of centrifuged, sonicated cells show transglutaminase activity as demonstrated by standard qualitative test criteria. In such cases, the enzyme is substantially free of sponge cells. The enzyme-like activity is also present in preparations containing the enzyme on the surface of the cells. In such case, the enzyme is exogenous to the sponge cells. Several tests were carried out on the supematants of sonicated sponge cells; such tests are standard assays for the presence of transglutaminase in cells.

One method for testing transglutaminase activity is to assay 14 equation B (Fig. 1); the incorporation of radiolabelled ( C) putrescine into dimethylcasein. Putrescine concentration was both time an enzyme concentration dependent, and it also was

+ +++ CCaa -- aanndd ssuubbssttrraattee ddee]pendent (requiring dimethylcasein as substrate) (Figure 2).

Another test is that of inhibition by low molecular weight inhibitors, primary amines (e.g., histamine, dansylcadaverine) but not tertiary amines (dimethyldansylcadaverine). As are other transglutaminases, the MSE was inhibited by primary amines as named above, as well as histamine and [Di-allyl] [amino-propionyl] [benzothiophene] , (DAPBT). It was not inhibited by the tertiary amines dimethyldansylcadaverine (DMDC) (Figure 3). As may be seen in Figure 3, on the left, is the control (no amine) followed by three pairs of inhibitors, dansylcadaverine, histamine, and DAPBT, at two concentrations each. These were effective inhibitors at concentrations previously shown effective with transglutaminase from other sources. To the right are two concentrations of DMDC which did not inhibit the enzyme.

Yet another test was the clotting of citrated lobster plasma by gamma glutamyl-epsilon amino lysine crosslinking of fibrinogen.

The marine sponge enzyme was first isolated by the following technique as outlined in the purification table (Table 1): The sponge cell extract containing the transglutaminase was isolate*! by a process involving several steps, one of which is the disruption of the sponge cells. About 100 ml of sponge are dissociated in 50 ml calcium/magnesium free sea water (CMFSW) (460mM NaCl, 7mM a₂S0₄, lOmM KC1, lO M Hepes, 2.5mM EDTA) to make 150 ml total of thick cell suspension. The cells are then separated into 12 centrifuge tubes containing 12.0 ml each of cell suspension, and are then centrifuged at approximately 2000 rpm for 5 minutes in a clinical centrifuge, and the supernatant decanted. About 10.0 ml of the CMFSW is added to the pellet, and the pellet resuspended, centrifuged as before, and the supernatant again decanted. A solution of 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5 was added to each tube, and the tubes resuspended. The tubes were spun as above and the supernatant decanted. The cells are then centrifuged a third time in 10 mM EDTA/10 mM Benzamidine/50mM Tris, and sonicated at moderate power for 15 one-second bursts separated by four-second periods of rest. The samples were then ultracentrifuged at 150,000g for 60 minutes at 4°C and the supernatant dialyzed for 18 hours against four separate washes of 2.0 liters of 0.8 M sucrose, 1 mM EDTA, 0.5mM Benza idine, 15mM Tris-HCl, at pH 7.5. The dialysate was then further purified on a Sephadex G100 column to obtain an enzyme product that had peak activity when concentrated purified greater than 25 times over the starting (cell homogenate) material. The resulting material is MSE, substantially free of marine sponge cells.

When lobster plasma was combined with purified enzyme as prepared above, the dose response curve for the velocity of 14 lobster clotting time (Figure 4A) and C-putrescine uptake

(Figure 4B) were identical. Figure 4a also shows that the purified enzyme reacted with dimethylcasein, but not DMDC, as before.

Another test was run to determine whether exogenous substrate for the transglutaminase enzyme was present in marine sponges. For this test, various Sephadex G-100 column fractions of enzyme were combined with a crude extract found in the dialysate of thte sample, containing the putative substrate. These combined

14 samples were added to C-putrescine, and uptake of putrescine was measured over time. As seen in Figure 5, in the absence of exogenous substrate, there is increasing incorporation of putrescine over two hours. With only one or the other of enzyme or substrate, there is no incorporation of putrescine.

The MSE as disclosed in the present invention may be used in any situation where a clotting factor is indicated; for example, it may be used topically to arrest bleeding during surgical or dental procedures. It may also be used in vivo in the intravenous treatment of bleeding disorders such as hemoplllia or hemophilia-like disorders. Since persons with hemophilia or hemophilia-like disorders often experience arthritic symptoms due to bleeding into the joints, the MSE of the invention may be injected intrasynovially (injected directly into the joint), thereby arresting the bleeding into the joint. The MSE likewise may be injected into any other similarly closed space in the body. The MSE may be used in its purified form alone, or in combination with other pharmaceutical carriers or solutions. It may also be incorporated into a delivery system, such as liposomes, and delivered by any of the methods known for administering liposomes.

The MSE resulting from the processes of the present invention whether used in their free-, liposome-encapsulated, fibronectin-complexed, gelatin-associated or other binary, tertiary, or quarternary forms can be used therapeutically in mammals, including man, in the treatment of conditions which favor or require use of a clotting agent. In the liposome delivery aspect of the invention, it may be used for the sustained delivery of the transglutaminase in its oactive form. Such conditions include but are not limited to disease states such as those that can be treated with clotting agents, such as thrombin.

In the complex formation embodiments of the invention, the transglutaminase may be used in combination with fibronectin, as a transglutaminase-fibronectin complex has been shown to form in mixtures of purified and transglutaminase-containing lysate of membrane-depleted red blood cells with fibronectin-containing plasma. These complexes were detected by nondenaturing electrophoresis, by observation of a shift in the mobility of transglutaminase in three electrophoretic systems as a function of transglutaminase admixture with fibronectin-containing human blood plasma, according to the methods of Lorand et al., 1988, Proc. Natl. Acad. Sci., USA, 85:1057-1059, the preparations and methods contained therein, including those relating to the complexes, are incorporated herein by reference. In these methods, the^' anodic mobility of the erythrocyte protein (transglutaminase) was reduced to approximately half of its value in the presence of such plasma. For example, when the relative ratio of plasma to erythrocyte lysate was increased, the free transglutaminase species was gradually replaced by a slower migrating transglutaminase species (Lorand et al., supra.). Furthermore,- absence of transglutaminase in the electrophoretic gel area between the two discrete electrophoretic zones (fast and slow moving transglutaminase) can be taken as a sign of tight complex formation.

The mode of administration of the preparation may determine the sites and cells in the organism to which the compound will be delivered. When the transglutaminase is administered in liposomes, or in other binary, tertiary or quarternary complexes, the liposomes of the present invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intra-arterially or intravenously. The preparations may also be administered via oral, subcutaneous, or intrasynovial, or intramuscular routes. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic. Other uses, depending * upon the particular properties of the preparation, may be envisioned by those skilled in the art.

For the topical mode of administration, the liposomes of the present invention may be incorporated into dosage forms such as gels, oils, emulsions, and the like. Such preparations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.

For the administeration of the transglutaminase or transglutaminase-like compounds of the present invention, a number of sustained drug delivery systems can be employed, for example, liposomes, transdermal systems, nasal delivery, carrier molecules, monoclonal antibodies, osmotic pumps, and the like. In the present invention, the use of liposomes as the drug delivery system is preferred.

For administration to humans in the curative treatment of disease states responding to blood clotting therapy, the prescribing physician will ultimately determine the appropriate dosage for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease. The dosage of the transglutaminase or transglutaminase-like substance of the present invention will be that necessary to cause clotting of the blood. The dosage of the transglutaminase or transglutaminase-like substance in liposomal form will be about that employed for the free drug. In some cases, however, it may be necessary to administer dosages outside these limits.

The following example is given for purposes of illustration only and not by way of limitation on the scope of the invention.

EXAHEEJL

100 ml of sponge were mechanically dissociated in 50 ml calcium/magnesium free sea water (CMFSW) (460mM NaCl, 7mM Na.SO., lOmM KC1, lOmM Hepes, 2.5mM EDTA) to make 150 ml total of thick cell suspension. The cells were then separated into 12 centrifuge tubes containing 12.0 ml each of cell suspension, and then centrifuged at 2000 rp for 5 minutes in a clinical centrifuge, and the supernatant was decanted. About 10.0 ml of the CMFSW was added to the pellet, and the pellet resuspended, centrifuged as before, and the supernatant again decanted. A solution of 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5 was added to each tube, and the tubes resuspended. The tubes were spun as above and the supernatant decanted. The cells were then centrifuged a third time in 10 mM EDTA/10 mM Benzamidine/50mM Tris, and sonicated at moderate power for 15 one-second bursts separated by four-second periods of rest. The samples were then ultracentrifuged at 150,000g for 60 minutes at 4^CC and the supernatant dialyzed for 18 hours against four separate washes of 2.0 liters of 0.8 M sucrose, 1 mM EDTA, 0.5mM Benzamidine, 15mM Tris-HCl, at pH 7.5. The dialysate was then further purified on a Sephadex G100 column to obtain an enzyme product that had peak activity when concentrated purified greater than 25 times over the starting (cell homogenate) material.

Claims

We claim:

1. Marine sponge enzyme having transglutaminase-like activity wherein the enzyme is exogenous to the sponge cells.

2. The marine sponge enzyme of claim 1 wherein the marine sponge is MJcrocjona pro fera.

3. Marine sponge enzyme having transglutaminase-like activity,* which is substantially free of sponge cells.

4. The marine sponge enzyme of claim 3 wherein the marine sponge is Microciona prolifera.

5. A method of treating bleeding disorders with the Marine Sponge Enzyme of claim 2 or 4 comprising administering the Marine Sponge Enzyme to a subject in need of such treatment.

6. The method of claim 5 wherein the bleeding disorder is hemophilia or a hemophilia-like disorder.

7. The method of claim 5 wherein the bleeding disorder results from a surgical or dental technique.

8. A method of administering marine sponge enzyme to a subject comprising associating marine sponge enzyme with a drug delivery vehicle.

9. The method of claim 8 wherein the drug delivery vehicle is a liposome.

10. The method of claim 8 wherein the drug delivery vehicle is a supporting matrix that supports enzymatic activity.

11. A method of obtaining marine sponge enzyme having transglutaminase- like activity from marine sponge cells comprising the step of disrupting the sponge cells so that the marine sponge enzyme is exogenous to the sponge cells.

12. A binary complex of transglutaminase and fibronectin, effective for clotting blood in an animal.

13. A tertiary complex of transglutaminase, fibronectin, and a liposome.

«

14. A tertiary complex of transglutaminase, fibronectin, and gelatin.

15. A quarternary complex of transglutaminase, fibronectin, a liposome, and gelatin.

16. A method of administering a composition comprising a clotting-effective amount of transglutaminase wherein the transglutaminase is administered topically to a subject in need of such treatment.

17. The method of claim 16 wherein the composition additionally comprises fibronectin.

18. The method of claim 17 wherein the composition additionally comprises liposomes.

19. The method of claim 17 wherein the composition additionally comprises gelatin.

20. The method of claims 17 wherein the composition additionally comprises liposomes and gelatin.