Title:  Method for preparing thrombin for use in a biological glue

United States Patent:  6,472,162

Issued:  October 29, 2002

Inventors:  Coelho; Philip Henry (El Dorado Hills, CA); Kingsley; Phil (Sacramento, CA); Brausch; Jim (Sacramento, CA); Godsey; James H. (Folsom, CA); Rock; Gail (Ottawa, CA); Madsen; Trista K. (Elk Grove, CA); Frausto; Sona B. (Sacramento, CA)

Assignee:  ThermoGenesis Corp. (Rancho Cordovo, CA)

Appl. No.:  328350

Filed:  June 4, 1999

A sterile method for preparing stable thrombin component from a single donor's plasma in which the thrombin component and the clotting and adhesive proteins component are harvested simultaneously from the same donor plasma in less than one hour. The combined components provide an improved biological hemostatic agent and tissue sealant by virtue of its freedom from the risk of contaminating viruses or bacteria from allogenic human or bovine blood sources. The thrombin provides polymerization of the clotting and adhesive proteins in less than five seconds, and is sufficiently stable to provide that fast clotting over a six hour period. Further, the clotting times can be predictably lengthened by diluting the thrombin with saline.

SUMMARY OF THE INVENTION

The instant invention addresses the long felt need for a simple, practical, fast method of preparing stable human thrombin from a donor's blood, which will provide fast clots (<5 seconds) throughout a lengthy surgery (e.g. six hours) to combine with the clotting and adhesive proteins harvested and concentrated from the same unit of blood to form a biological sealant with no patient exposure to microbial or possible CJD or NVCJD contaminations. Previous works in the field (Hirsch, et al.) exemplified a thrombin with minimal stability in that the thrombin achieved rapid clotting of fibrinogen (i.e., less than 5 seconds) during only a very narrow four to five minute time period, or required so many steps and elapsed time it would not be suitable for perioperative preparation, both totally impractical for the broad range of surgeries.

The present invention provides a stable thrombin enzyme which can cause precise, repeatable fast or slow polymerization of clotting and adhesive proteins over a duration of up to six hours--throughout even a long surgery. Further, the use of clotting and adhesive proteins and thrombin all sourced from a single donor will eliminate various disease risks posed from the use of commercial fibrin glues where the fibrinogen is sourced from plasma pooled from thousands of donors and the thrombin is either sourced from a similar pool of human plasma or of bovine origin. The speed and simplicity of the production of stable thrombin by use of this invention allows it to be prepared just prior to or during operative procedures and it will provide fast clotting throughout even the longest surgeries. The thrombin produced by this invention can be diluted in saline, water and a dilute CaCl₂ solution (e.g. 125 mM CaCl₂) to provide precise, slower clotting times thereby allowing any preferred time from less than five seconds to longer than 2 minutes.

The procedure of the invention is preferably comprised of three steps, the first two of which should preferably occur at the same time:

1. Preparing a fraction enriched in prothrombin by use of Ethanol to substantially enhance the concentration of prothrombin and at the same time remove or denature naturally occurring ingredients within plasma, such as Fibrinogen and Antithrombin III which can bind to, block, interfere with or inhibit prothrombin or its subsequent activation to long-term functional thrombin.

2. Adding calcium ions to the enriched prothrombin solution and briefly agitating the solution to convert the pro-thrombin to stable, long term thrombin.

3. Expressing the thrombin solution through a filter to remove particulate matter which would prevent spraying the thrombin through a small orifice or expressing the thrombin through a thin tube onto a wound site.

Claim 1 of 119 Claims

We claim:

1. A method for extracting autologous thrombin from a patient, the steps consisting of:

obtaining a blood product from the patient;

sequestering plasma from the blood product;

adding ethanol to the plasma to prepare a solution containing prothrombin, wherein ethanol is present in the solution at a concentration between about 8% and about 20% volume per unit volume;

converting the prothrombin in the solution to thrombin;

filtering the thrombin to remove particulate matter; and

applying the thrombin to the patient.
 

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