# US 2014/0154234 A1 · Method for generating thrombin > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154234 A1 - Document ID: US20140154234A1 - Status: Published application - Kind: A1 - Application number: 14/063,648 - Filed: 2013-10-25 - Published: 2014-06-05 - Earliest claimed: 2010-07-30 - Assignees: Biomet Biologics, LLC - Applicants: Biomet Biologics, LLC - Inventors: Randel Dorian; Richard W. Storrs; Michael D. Leach - Web page: https://patentgrep.com/patent/US20140154234A1 ## CPC - C12N 9/6429: {Thrombin (3.4.21.5)} - A61K 38/4833: {Thrombin (3.4.21.5)} - A61P 7/00: Drugs for disorders of the blood or the extracellular fluid - C12Y 304/21005: Thrombin (3.4.21.5) - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12Y: ENZYMES ## Abstract Methods of generating thrombin and methods of applying a clotting tissue sealant to a site on a subject are provided. A blood component comprising platelets can be obtained from the subject. A hypotonic composition is contacted with a solid matrix to form a thrombin-containing liquid, where the hypotonic composition includes water, calcium, a blood component comprising platelets, and optionally a chelator. Calcium is present in the hypotonic composition in an amount greater than the amount of calcium that can be complexed by the chelator. Thrombin-containing liquid is then separated from the hypotonic composition and can be applied to the site on the subject to form a clot, for example, by combination with fibrinogen. ## Claims ### Claim 1 A method of generating thrombin comprising: contacting a hypotonic composition with a solid matrix to form a thrombin-containing liquid, the hypotonic composition comprising water, calcium, a blood component comprising platelets, and optionally a chelator, wherein the calcium is present in an amount greater than the amount of calcium that can be complexed by the chelator; separating the thrombin-containing liquid from the hypotonic composition. ### Claim 2 (depends on 1) The method of claim 1, wherein the water contains the calcium. ### Claim 3 (depends on 1) The method of claim 1, wherein the concentration of platelets in the hypotonic solution is less than about half of the concentration of platelets in whole blood. ### Claim 4 (depends on 1) The method of claim 1, wherein the concentration of platelets in the hypotonic solution is less than about one-third of the concentration of platelets in whole blood. ### Claim 5 (depends on 1) The method of claim 1, wherein the blood component comprises the chelator. ### Claim 6 (depends on 1) The method of claim 1, further comprising obtaining the blood component comprising platelets and adding a chelator to the blood component to prevent coagulation prior to the contacting step. ### Claim 7 (depends on 1) The method of claim 1, wherein the solid matrix comprises glass, titanium, or combinations thereof. ### Claim 8 (depends on 1) The method of claim 1, wherein at least about one minute passes between the contacting step and the separating step. ### Claim 9 (depends on 1) The method of claim 1, wherein the hypotonic composition and solid matrix are agitated prior to the separating step. ### Claim 10 (depends on 1) The method of claim 1, wherein the separating comprises removing the thrombin-containing liquid from the solid matrix using a syringe, by filtering, or by centrifuging. ### Claim 11 (depends on 1) The method of claim 1, further comprising at least one of: adding an alcohol to the thrombin-containing liquid; adding a chelator to complex calcium in the thrombin-containing liquid; adjusting the pH of the thrombin-containing liquid to below 7; and reducing the temperature of the thrombin-containing liquid. ### Claim 12 A method of applying a clotting tissue sealant to a site on a subject comprising: obtaining a blood component comprising platelets; contacting a hypotonic composition with a solid matrix to form a thrombin-containing liquid, the hypotonic composition comprising water, calcium, the blood component comprising platelets, and optionally a chelator, wherein the calcium is present in an amount greater than the amount of calcium that can be complexed by the chelator; separating the thrombin-containing liquid from the hypotonic composition; and applying the thrombin-containing liquid to the site on the subject to form a clot. ### Claim 13 (depends on 12) The method of claim 12, wherein the water contains the calcium. ### Claim 14 (depends on 12) The method of claim 12, wherein the concentration of platelets in the hypotonic solution is less than about half of the concentration of platelets in whole blood. ### Claim 15 (depends on 12) The method of claim 12, wherein the concentration of platelets in the hypotonic solution is less than about one-third of the concentration of platelets in whole blood. ### Claim 16 (depends on 12) The method of claim 12, wherein the blood component comprises the chelator. ### Claim 17 (depends on 12) The method of claim 12, further comprising adding a chelator to the blood component to prevent coagulation prior to the contacting step. ### Claim 18 (depends on 12) The method of claim 12, wherein the solid matrix comprises glass, titanium, or combinations thereof. ### Claim 19 (depends on 12) The method of claim 12, wherein at least about one minute passes between the contacting step and the separating step. ### Claim 20 (depends on 12) The method of claim 12, wherein the hypotonic composition and solid matrix are agitated prior to the separating step. ### Claim 21 (depends on 12) The method of claim 12, wherein the separating comprises removing the thrombin-containing liquid from the solid matrix using a syringe, by filtering, or by centrifuging. ### Claim 22 (depends on 12) The method of claim 12, further comprising at least one of: adding an alcohol to the thrombin-containing liquid; adding a chelator to complex calcium in the thrombin-containing liquid; adjusting the pH of the thrombin-containing liquid to below 7; and reducing the temperature of the thrombin-containing liquid. ### Claim 23 (depends on 22) The method of claim 22, wherein the alcohol is glycerol. ### Claim 24 (depends on 12) The method of claim 12, wherein the blood component comprising platelets is obtained from the subject. ### Claim 25 (depends on 12) The method of claim 12, wherein applying the thrombin-containing liquid to the site on the subject to form a clot comprises combining the thrombin-containing liquid with fibrinogen.