HUT67693A - Hemostatic composition for arresting local bleedings - Google Patents

Abstract

Method for arresting local bleedings by topical use of FVIIa and a hemostatic composition containing FVIIa together with a biologically compatible carrier which permits said FVIIa to remain in contact with said bleeding wound.

Description

The present invention relates to a method for stopping local bleeding by the use of factor VIIa and hemostatic compositions containing this factor.

When blood vessels are damaged by physical trauma, including surgery, bleeding occurs. Normal physiological pathways as vazokontrakció, platelet adhesion and aggregation to the injured vessel wall and the coagulation factors of the plasma activation due to blood vessel damage to the shut-off plug ¹ 'is formed. This process is called physiological hemostasis, which is described in detail below.

Coagulation is facilitated by two separate processes, intrinsic and extrinsic.

Only coagulation factors present in plasma are involved in the intrinsic process. In an intermediate step in the intrinsic pathway, inactive factor IX is activated to factor IXa; this reaction is catalyzed by factor Xla and calcium ions. Factor IXa then participates in the activation of Factor X to Factor Xa in the presence of Factor VIIIa, phospholipid and calcium ions.

Plasma factors and components of tissue extracts play a role in the extrinsic pathway. Factor VII, a proenzyme in plasma, is also involved in the extrinsic process of coagulation, when it is activated to factor VIIa and converts factor X into factor Xa in the presence of tissue factor and calcium ions.

Factor Xa then promotes the conversion of prothrombin-thrombin in the presence of calcium ions and phospholipid. Finally, thrombin converts plasma fibrinogen to fibrin, which in the presence of Factor XIIIa and calcium ions, polyserizes into a fibrous network and thereby forms a blood clot.

Plasma factors obtained from natural sources or purified by recombinant techniques, such as factor VIII: C / see 4,831,119; 4,868,112; 4,886,876; 4,657,894; Re. U.S. Patent Nos. 32,011 and 4,649,132; and Factor VIIa, see U.S. Patent 4,784,950. 4,382,083; U.S. Patent Nos. 4,479,938 and 4,357,321 are used to treat patients with haemophilia or inhibitory body hemophilia due to lack or insufficient synthesis of coagulation factors. These coagulation factors are administered to the patient in need of treatment in the form of an aqueous solution by infusion or bolus injection, depending on the plasma factors to be used and the patient's condition. Typically, after the administration of the coagulation factor, the bleeding ceases within 14 minutes to 3 hours or later.

However, faster stopping of bleeding is required in the case of severe bleeding from extensive lesions of larger arteries, or bleeding from larger mucosal surfaces, or bleeding from unexcavated cavities.

Even if the bleeding is shorter, it can cause excessive blood loss, which can adversely affect the normal functioning of the body. Even in the case of bleeding in bony, non-expanding cavities, accumulation of leaking blood can cause localized soft tissue damage due to increased pressure. Conventional treatment of such conditions involves surgical and / or medicated hemorrhage.

Surgical sedation consists of ligation or suture of ruptured blood vessels, swabing of cavities, coagulation of tissue surfaces, including contacts of ruptured blood vessels, or use of cautery agents or heated air.

Surgical hemostasis can also be facilitated by the use of appropriately sized blocks, plates, or bio-absorbable haemostatic sponges.

In some places, pharmaceutical preparations containing oxythrombin or other coagulant activating factors such as factor VIII, XIII or calcium ions are used as surgical hemostatic excipients. These excipients are, for example, sprayed into solution at the bleeding site (U.S. Patent No. 4,298,598). Textiles soaked in one or more hemostatic compounds, such as gauze or cotton wool or bio-absorbent sponges, are also used prior to use (U.S. Patent No. 4,363,319).

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U.S. Pat. No. 2,558,395 discloses ready-to-use sponges consisting of undenatured gelatin containing thrombin. United States Patent 4,265,233 describes a wound healing agent consisting of gelatin, collagen, polyglycolic acid and polyacetic acid, to which FXIII is covalently bonded. EP 277096 A discloses a wound dressing comprising a porous sponge containing a stable thrombin composition and a substrate such as collagen or denatured gelatin. WO 90/13320 describes an effective antihypertensive amount of a porous sponge containing thrombin and one or more coagulation factors other than thrombin. U.S. Patent Nos. 4,563,387 and 4,642,111, respectively, relate to a method and device for treating tumors and disclose an anticancer agent attached to a structure such as a polymer and a blood coagulation factor that can be administered by injection at the site of bleeding directly induced by tumor treatment.

Adhesive containing an aerosol containing FXIII and thrombin in Japanese Patent Application No. 59-116213, and adhesive for living tissues in Japanese Patent Application 02-167234, containing fibrinogen, prothrombin, FVII, FIX, FX, FXIII, antithrombin, protease benzoate and calcium ions. are described.

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- 6 In recent years, there has been increasing concern about the safe use of ox products, such as thrombin or prothrombin, in medicinal products for human use. Describe the potential risk of transmitting infectious material to cattle and ox spongiform encephalopathy (Bovine spongiform encephalopathy), which is one or more well-known diseases associated with degenerative encephalopathy, such as Creutzfeldt's disease. cause. In addition, clinical researchers have observed that the use of oxytrombin in humans leads to the production of antibodies that cross-react with human thrombin and may cause bleeding problems.

Therefore, it is an object of the present invention to provide a safe and effective means of locally producing bleeding at the site of injury.

The present invention is based on the surprising discovery that, when applied at the site of injury, FVIIa is capable of instantly stopping bleeding in the absence of thrombin and other coagulation factors, and when incorporated or incorporated into a biocompatible carrier (s) or substance (s). wash away from the site of injury.

According to the present invention, FVIIa is incorporated into a carrier material which is free of thrombin and is not accompanied or contaminated by other coagulation factors.

Thus, the present invention relates to a hemostatic composition which, when incorporated into a biocompatible carrier, contains an effective amount of FVIIa and does not contain thrombin.

More particularly, the present invention relates to a haemostatic haemostatic hemostatic composition which is not contaminated or coagulated by other coagulation factors and contains FVIIa sufficiently active to produce a haemorrhagic effect in combination with a biocompatible carrier that allows attached to, attached to, or in contact with.

Before describing the invention, it may be necessary to interpret certain of the definitions used below in order to better understand it.

Haemostatic or haemostatic agent: a haemostatic agent.

Haemostatic or haemostatic preparation: A preparation containing a haemostatic or haemostatic agent.

Blood clot: the end result of the cascade system of the blood coagulation process, which consists in the conversion of soluble plasma fibrinogen to insoluble fibrin. Fibrin Physically *

It stops bleeding. The blood clot covers the surface, holds the edges of the wound together and forms a matrix for subsequent cell proliferation and wound healing.

Coagulation factors: plasma proteins involved in the coagulation cascade.

Activated Coagulation Factors: Coagulation factors that are converted to active enzymes by an activator, where the activator is often an activated coagulation factor itself. The active form of the factor is usually given the index (e.g. factor VIIa).

Proenzymes: Inactive preforms of enzymes that are converted to active enzymes after peptide bond (s) / cleavage.

Biodegradable material: A substance that is degraded to smaller molecules in the body, where the size of the molecules allows for transport to the bloodstream and gradual removal from the application site.

Sponge: A porous, networked structure with a significantly larger internal surface than the outer surface. The porous structure contains hollow spaces within the mesh-like structure and is capable of absorbing a volume of fluid that is many times its own weight.

Covalent bond: Two atoms combine to form a common pair of electrons so that the two electrons that make up the bond belong to both atoms that make up the molecule.

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Bandage: A wound-applied substance that, when fixed in place, provides protection and promotes healing.

Local: Local.

Biocompatible: a substance absorbed by the body that remains functional for a period without rejection.

Gel: A colloidal system consisting of a solid and a liquid phase in the form of a solid or semi-solid mass.

Paste: An ointment consisting of one or more hydrogel or grease based substances. It is less greasy than ointment and absorbs better.

Granule: a small particle or mass.

Film: Thin coating or layer.

Patch: A substance for external use that adheres to the skin by its material and texture.

Bandage: a gauze, muslin, flannel or other material used to fix the bandages or stop bleeding.

FVIIa is administered in an effective hemostatic amount. An effective hemostatic amount preferably stops bleeding when left in contact with the site of injury for a sufficient period of time; this time is at least about 60 seconds for a normal coagulation mechanism, and if the coagulation process is * <* *.

-10 does not run flawlessly, less than about 10 minutes. FVIIa is to be administered in an amount of about 0.2 to 2.0 mg, preferably about 0.5 to 1.5 mg, most preferably about 0.9 to 1.1 mg per application.

PVIIa can be obtained from plasma (EP 0082182B) or by recombinant DNA technology (EP 0200421A). Purified human factor VIIa is preferably by the methods of Broze and Majerus / J. Biol. Chem. 255, 1242-1247 (1980) and according to Hedner and Kisiel, J. Clin. Invest. 71, 1836-1841 (1983). By these methods, factor VII can be produced without detectable levels of other coagulation factors.

Further purified Factor VII formulations may be obtained by a final purification involving gel filtration. Factor VII can then be converted to activated Factor VIIa by known methods, for example, various plasma proteins such as XIIa, IXa or Xa. Bjoern et al., Activation of Coagulation Pactor VII to VIIa, Research Disclosure 269, 564-565 / 1986, can be activated by ion exchange chromatography such as MonoQ column (Pharmacia Pine Chemicals, Uppsala, Sweden) and others.

One skilled in the art will recognize that the factor VIIa useful in the present invention may also be produced by recombinant DNA technology, e.g., the gene encoding cDNA or factor VII / Hagen et al., Proc. Natl. Acad. Sci. USA 83, 2412-2410 * · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ics · what →

- 11/1986 // by inserting into a suitable vector, transforming suitable cell lines with a vector and culturing the transformed cells in a suitable medium, then isolating the expressed product and activating it into factor VIIa. The preparation of FVII by recombinant DNA technology is also disclosed in U.S. Patent No. 4,784,950, which is incorporated herein by reference. Factor Vlla obtained by recombinant DNA technology may be authentic Factor VIIa or more or less modified Factor VIIa, provided that the modified Factor VIIa has the same biological activity as authentic Factor VIIa. The preparation of such modified factor VIIa occur encoding Factor VII DNA sequence is modified by altering the amino acid codons or remove existing amino acid codons in the natural gene by means of some, _r using known means, such as site-directed mutagenesis.

It will be appreciated that the above methods are independent of the method of recovering factor VIIa and therefore the invention encompasses any factor VIIa suitable for use in the present invention.

The carrier may be a gel, paste, solid or other suitable biocompatible / acceptable carrier for topical administration of drugs or other biologically active compositions.

Preferably, the gel or paste has a viscosity of from 0.2 to 30 ···· ···········································iting date (s)

- 12 Pa · s.

Typically, the biocompatible carrier will comprise natural macromolecules, raint gelatin, collagen, alginic acid, cellulose, chitin, fibrinogen, fibrin cleavage products, fibronectin fragments, globulin, myoglobulin, casein, keratin, albumin, polysaccharides, dextrans, glycosarines, or chemically modified natural molecules such as denatured gelatin, alginic acid alginates such as calcium alginate, oxidized cellulose, substituted cellulose ethers such as glycol cellulose, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, substituted cellulose esters such as acetylated cellulose, substituted cellulose ether esters such as acetylated ethyl cellulose, chitosan, or synthetic polymers such as vinyl polymers such as polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, polyglycolic acid, polyacetic acid, polidex ropes or copolymers such as polyoxyethylene-polyoxyioropylene copolymers or natural fibers, synthetic fibers or mixtures of the above substances / compounds.

Solid, biocompatible carriers are preferably granules, powders, foams, films, patches, surgical dressings or bandages.

Solid biocompatible carriers are generally selected from those used as haemostats.

♦ «<··· ···« ····· 4 ··· · · · · · · · · · · · ·

These include modified polymers of cellulose, collagen, gelatin, alginate or synthetic.

The biocompatible carrier may further comprise a fibrinolysis inhibitor, such as aprotinin, ε-capric acid or tranexamic acid. They may also contain a stabilizer such as naturally occurring amino acids, mono- or disaccharides, polyglycols, glycerol, proteins or metal salts as calcium salts, and mixtures of the above. Buffering salts may also be added, such as alkali metal acetates, alkali metal carbonates or bicarbonates, alkali metal citrates, alkaline metal phosphates or hydrogen phosphates, alkali metal succinates, imidazole / methane hydroxy, buffering systems and mixtures thereof. In addition, antimicrobial or bactericidal agents such as antibiotics, sulfonamides, antifungal agents, antiviral compounds and preservatives may be used.

The method and haemostatic composition of the present invention are surgical interventions or other injuries such as accidental injuries to the skin and / or surrounding tissues or major abdominal organs / liver, spleen or intestines that may be used to promote blood coagulation; Lung Surgery; neurosurgery to prevent pressure damage to brain and nerve tissue; in the case of orthopedic surgery, which may be otherwise difficult to prevent, often extensive bleeding may occur; and to stop leakage from the suture site in vascular surgery; in oral and dental surgery as in tooth extraction; and epistaxis.

In ready-to-use formulations, FVIIa can be incorporated into the carrier in a variety of known ways, such as co-precipitation, swelling, dispersing, mixing, impregnating, spraying, embedding, injection, or a combination thereof.

If the carrier is a gel or a paste, the factor VIIa is preferably introduced into the carrier under non-infectious conditions. This may be accomplished by adding a suitable solution of PVIIa to the carrier and then gently agitating with a suitable device so that PVIIa is uniformly distributed in the gel or paste. The PVIIa-containing carrier is then transferred to a suitable formulation, for example, in a tube, plastic container or syringe. The final sterilization may be carried out, for example, by heat or ionizing radiation.

When the carrier is solid, factor VIIa is delivered by placing the carrier in a suitable solution of PVIIa for a period of time sufficient to impregnate with factor VIIa solution. FVIIa can also be introduced into the solid support by spraying, embedding or multiple injections. After the excess water has been removed from the FVII impregnated substrate by vacuum drying or by rapid-drying, it is suitably packaged in, for example, a paper bag or so-called. in a blister pack and finally sterilized by, for example, heat, ethylene oxide or ionizing radiation.

FVIIa can also be attached to the support by electrostatic interaction between FVIIa and the support.

FVIIa may also be covalently linked to the support by chemical crosslinking reagents such as bifunctional N-hydroxysuccinimide esters or other bifunctional chemical crosslinking reagents.

Finally, FVIIa can be attached to the carrier by physical post-absorption, dispersion or adsorption.

Factor VIIa may be added to the carrier immediately prior to use. For example, a suitable solution of FVIIa is sprayed onto the carrier or embedded in the FVIIa solution. The FVIIa solution may also be injected into the vehicle.

A preferred carrier is a sponge material known as a biodegradable haemostatic sponge. Biodegradable or absorbable compounds such as collagen, gelatin, chitin, cellulose, polyglycolic acid and polyacetic acid are used to produce hemostatic sponges. Such absorbable hemostatic agents may be left at the site of bleeding after suturing internal injuries, and may act over a period of time depending upon their water solubility, degradability, and size.

The properties of the above materials can be controlled by various chemical or physical treatments, for example leading to improved mechanical strength of the structure or reducing the water solubility of the material, thereby delaying the rate of absorption, which prolongs the duration of the hemostatic effect.

For example, gelatin can be denatured at 100-160 ° C for several hours. Ξ After treatment, the initially water-soluble gelatin becomes essentially insoluble in water, but the body's protein-degrading enzymes can still break it down into absorbable molecules.

In contrast, hemostatic sponges made from non-denatured gelatin dissolve quite rapidly and, upon contact with aqueous solutions or bleeding wounds, convert to a soft gel.

The hemostatic foam containing FVIIa is prepared by foaming the non-denatured gelatin and FVIIa and freeze-drying the foam, or saturating the preformed dried foam with FVIIa solution and then freeze-drying the foam.

The latter technique allows the use of a water-insoluble sponge material, which is advantageous because such sponges, when applied at the site of bleeding, retain a much longer physical structure than non-denatured sponges.

In a preferred embodiment of the invention, the carrier is a ready-to-use haemostatic sponge to which VHa factor is added prior to formulation and final sterilization.

Example 1

Four 5 mm cores were cut from a gelatin sponge / Spongostan using Novo Nordisk A / S7 punch. Two of these were soaked in sterile water and the other two in 2 ml of sterile water containing 1.13 mg factor VIIa. Before application to the bleeding site as described below, the soaking time is approximately 5 minutes.

Sprague-Dawley rats (450 g) were anesthetized with halothane and administered 0.2 ml / kg of anesthetic stock solution intraperitoneally.

The rat is placed on a heating pad and the liver is exposed by opening the abdomen with a longitudinal centerline cut. The intestinal contents were removed with a swab moistened with warm saline.

Using a piece of steel placed behind the liver, a solid bed is formed. Four 5 mm tissue cores were cut through the entire thickness of the liver and the four prepared gelatin sponges were inserted into the wells.

Ξ Observe four sites for 20 minutes, then cut the liver and attempt to remove the gel plugs with a fine-toothed tweezer and gently pull.

At two sites filled with gelatin sponge impregnated with factor VIIa, bleeding stopped and the other two sites continued to leak. None of the plugs in the liver biopsy sites were difficult to remove, but removal of the factor VIIa-plated plug appeared to be more difficult.

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- 18 2, Example

Prior to surgery, small pieces (45 x 20 x 10 mm) were cut from the dry gelatin sponge (Spongostan standard). The above dimension was chosen to ensure that the sponge accurately covers the 25mm long incision with 10mm overlap.

Freeze-dried Factor VIIa was prepared in an aqueous solution (1.0 mg per ml) containing calcium ions (10.0 mmol) and stored at room temperature prior to surgery.

The abdominal cavity of the anesthetized guinea pig is opened by a midline incision and the spleen is visualized in the wound. A 3.0 mm deep and 25 mm hossu incision was made using a special tool made of scalpels assembled with locking blocks and longitudinal grooves. The first incision serves as a control incision, which is left to bleed for 12 minutes to exclude the possibility of spontaneous coagulation.

A second incision is then made 30 mm from the first cut and left to bleed freely for 60 seconds. Then, a piece of gelatin sponge is carefully placed on the incision, 1.0 ml of factor VIIa solution is added dropwise and gently massaged into the sponge for 30 seconds with gentle finger pressure. Full blood coagulation occurs immediately.

Four different commercially available haemostatic sponges are used in the test series.

isotonic with sodium chloride solution. The specific time of hemostasis ranged from 1.8 to 7.5 minutes.

In a similar test procedure, oxygen thrombin / 50 NIH E / ml_7 in aqueous solution also causes sudden hemostasis.

Example 3

Without incorporation into the matrix, an aqueous solution of FVIIa is sprayed locally to stop venous bleeding from the gallbladder bed and abdominal surgery. The study is conducted in a total of 8 patients divided into two groups, randomized to a double-blind study (the clinician does not know whether it is effective or gives the patient a placebo).

The contents of vials containing 562.5 µg of lyophilized Factor VIIa or placebo formulations similar to Factor VIIa were reconstituted with 3.7 ml sterile water immediately before use and transferred to syringes with spray needles. For each application, a total volume of 3.7 ml is injected.

Four patients undergoing surgical removal of the gallbladder were examined, two of whom had Factor VIIa. receive a placebo. After removal of the gallbladder, factor VIIa or placebo is injected onto the gallbladder bed. Efficacy was evaluated after 2 minutes. In each case, the efficacy of the formulation is determined by comparing the leak before and after application.

Four other patients undergoing general elective abdominal surgery are being examined. The incisions are extended down to the peritoneum without perforation of the peritoneum, and arterial spattering is prevented by conventional surgical techniques. Immediately after the surgical incision, the middle of the wound is covered with a thick swab and Factor VIIa is injected at one end of the wound and the control placebo at the other end. Effectiveness is evaluated 3 minutes after wound injection. The Surgeon Blindly ”Judges which end of the wound is bleeding less.

In these experiments, factor VIIa did not affect venous bleeding. This is probably because Factor VIIa is washed away from the wound when applied only in aqueous solution and not incorporated into a matrix or biocompatible carrier that would allow FVIIa to remain in contact with the bleeding wound.

The scope of the invention is not limited by the above examples, which are intended to illustrate the invention only. From the foregoing description, it will be apparent to those skilled in the art that various modifications may be made to the invention other than those disclosed and described. Such modifications are intended to be within the scope of the claims.

Claims (31)

Claims A hemostatic composition for inducing haemostasis of a bleeding wound, characterized in that an effective amount of factor VIIa alone, sufficiently free of other coagulation factors and sufficient to exert a hemostatic effect, is used together with a biocompatible carrier capable of maintaining factor VIIa in contact with the bleeding wound. The hemostatic composition of claim 1, wherein the biocompatible carrier is a gel or paste. The hemostatic composition of claim 2, wherein the gel or paste has a viscosity in the range of about 0.2 to about 30 Pa. 4. The hemostatic composition of claim 1, wherein the biocompatible carrier is in a solid state. 5. The hemostatic composition of claim 1, wherein the biocompatible carrier is a natural macromolecule, a chemically modified natural molecule, a synthetic polymer, a natural or synthetic fiber, or mixtures thereof. 6. The haemostatic composition of claim 5, wherein said biologically active agent is a biologically active agent. 22 compatible carriers are polysaccharides or proteins or mixtures thereof. 7. The haemostatic composition of claim 4, wherein the biocompatible carrier is a granule, powder, sponge, film, patch, surgical dressing or bandage. 8. The hemostatic delay according to claim 4, characterized in that the biocompatible carrier is composed of modified cellulose, collagen, gelatin or natural or synthetic fibers. The hemostatic composition of claim 1, wherein the factor Vlla is fixed to the biocompatible carrier by electrostatic interaction between FVIIa and the biocompatible carrier, or covalently linked to the biocompatible carrier. 10. The haemostatic composition of claim 9, wherein factor VIIIa is covalently bound to a biocompatible carrier by chemical crosslinking agents such as bifunctional N-hydroxysuccinimide esters or other bifunctional chemical crosslinking agents. 11. The haemostatic composition of claim 1, wherein the FVIIa binds to the biocompatible carrier by physical means such as absorption, dispersion or adsorption. 12. The hemostatic composition of claim 1, wherein the amount of FVIIa is from about 0.2 mg to about 2.0 mg. 13. The hemostatic composition of claim 12, wherein the amount of FVIIa is from about 0.9 to about 1.1 mg. 14. The antihypertensive composition of claim 1, wherein the fibrinolysis inhibitor is selected from the group consisting of aprotinin, E-aminocaproic acid and tranexamic acid. 15. The hemostatic composition of claim 1, further comprising a stabilizer. The hemostatic composition according to claim 15, wherein the stabilizer is naturally occurring amino acids, mono- or disaccharides, polyglycols, glycerol, proteins, or divalent metal ions or mixtures thereof. The hemostatic composition according to claim 1, wherein it contains one or more buffering agents such as alkali metal acetates, alkali metal carbonates or bicarbonates, alkali metal succinates, imidazole, tris buffer or a twin anionic buffer system, or mixtures of these. The hemostatic composition according to claim 1, characterized in that it contains one or more antimicrobial or bactericidal agents such as antibiotics, sulfonamides, antifungal agents, antiviral compounds and preservatives. 19. A method of generating haemostasis at the site of a bleeding wound, wherein, at the site of the bleeding wound, a factor VIIa sufficiently active alone is used to exert a haemostatic effect and is compatible with a biocompatible carrier that allows FVIIa to stay in contact with bleeding wounds. 20. The method of claim 19, wherein the biocompatible carrier is a gel or paste. 21. The method of claim 20, wherein the gel or paste has a viscosity in the range of about 0.2 to about 30 Pa. 22. The method of claim 19, wherein the biocompatible carrier is a solid. 23. The method of claim 19, wherein the biocompatible carrier comprises natural macromolecules, chemically modified natural molecules, synthetic polymers, natural or synthetic fibers, or mixtures thereof. • · · 24. The method of claim 19, wherein It is understood that the biocompatible carrier consists of polysaccharides or proteins or mixtures thereof. 25. The method of claim 19, wherein the biocompatible carrier is a granule, powder, sponge, film, patch, surgical dressing, or bandage. 26. The method of claim 25, wherein It is understood that the biologically acceptable carrier is modified cellulose, collagen, gelatin or natural or synthetic fibers. 27. The method of claim 19, wherein the factor VIIa is attached to the biocompatible carrier by electrostatic interaction between FVIIa and the biocompatible carrier, or covalently linked to the biocompatible carrier. 28. The method of claim 27, wherein factor VIIa is covalently bound to a biocompatible carrier by chemical crosslinking agents such as bifunctional N-hydroxysuccinimide esters or other bifunctional chemical crosslinking reagents. 29. The method of claim 19, wherein the FVIIa binds to the biocompatible carrier by physical means such as absorption, dispersion, or adsorption. • · · · · · 9 * * · 4 ^Λ ··· • 9 9 9 9 · «· * ·« «« ·· · 30. The method of claim 19, wherein the amount of FVIIa is from about 0.2 mg to about 2.0 mg. 31. The method of claim 30, wherein the amount of FVIIa is from about 0.9 to about 1.1 mg.