IMPROVED EXTRACTION METHODS FOR PREPARING THROMBOPLASTIN REAGENTS

Abstract: The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using coumadin. Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the Prothrombin Time (PT) test. This invention describes the use of barium sulfate and chaotropic agents, and nonionic detergents, for the extraction of sensitive thromboplastin reagents from tissue. Extraction with sodium thiocyanate alone also greatly enhances thromboplastin sensitivity. This invention should be useful for all thromboplastins and will improve their sensitivity for all PT-based tests and specific assays.

Description: IMPROVED EXTRACTION METHODS FOR PREPARING THROMBOPLASTIN REAGENTS Field of the Invention Tnis invention relates to an improved method of extracting and preparing thromboplastin reagents. Background of the the Invention Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the prothrombin time (PT) test. The PT test is used to screen for blood coagulation factor deficiencies and for monitoring oral anticoagulant therapy (e.g. coumadin). Reagents for PT tests include tissue thromboplastin, also called tissue factor, and calcium ions as active ingredients diluted with appropriate buffers and stabilizers. Thromboplastin forms a complex with coagulation factor VII to greatly enhance its proteolytic activity. Thromboplastin may be derived from a variety of tissues of different animal sources. Each tissue has a characteristic activity and sensitivity to coagulation enzymes (i.e., factors); these properties are modulated by other constituents of the reagent. Thromboplastin sensitivity is defined as the prolongation of the clotting times of both coumadinized plasmas and plasmas deficient in clotting factors II, V, VII, and X. Sensitivity to coumadinized plasma is assessed by taking the ratio of an abnormal plasma sample to a normal plasma sample. SUBSTITUTESHEET Currently, the most sensitive thromboplastin reagents are derived from human brain and placenta. The limited availability of these materials, their cost and the potential for HIV virus contamination limit their universal acceptance. Thromboplast ns b derived from rabbit brain, the most common source, typically have relatively low sensitivity compared to thromboplastins derived from human tissues. The source of the thromboplastin, the method of extracting the thromboplastin and the reagent composition are all important parameters in determining reagent sensitivity, lϋ Variations in the composition of PT reagents can also be used to improve stability and adjust clotting times of plasma samples from normal individuals. Historically thromboplastin has been extracted from tissues lb by heating the tissue in water or saline solutions. Thromboplatin reagents made, by Baxter Healthcare Corporation, ϋade Division, contain thromboplastins extracted in saline-tartrate solutions (U.S. Patent No. 3,522,148 - July 25, 1970). These extracts are cent ifuged to remove large 20 particles. The supernate thromboplastin extract contains the active thromboplastin along with the sodium chloride and sodium tartrate from the extraction fluid. In Thromboplastin C the thromboplastin extract is added to a solution containing calcium lactate, sodium chloride, sodium tartrate, glycine and 2b carboxymethyl cellulose. The final concentration of extract is 25% of the final reconstituted volume. In Thromboplastin FS, the thromboplastin extract is added to a solution containing i idazole, calcium lactate, sodium chloride, 30 sodium tartrate, glycine and carboxymethyl cellulose. Because the final concentration of extract is 50% of the final reconstituted volume, Thromboplastin FS (TPES) made by Baxter Healthcare Corporation, Dade Division, has a relatively high concentration of rabbit brain extract. While more sensitive than other rabbit brain thromboplastins, the normal range PT values are longer than desired, the turbidity is high and the stability is less than optimal . b Boehringer Mannheim has developed a process to make rabbit brain thromboplastin more sensitive (DE 3150594A1). In their procedure, rabbit brain powder is mixed with equal parts of cellulose powder and washed with sodium acetate buffer at pH 1U 6.5-8 to remove contaminants such as hemoglobin. The brain residue is then extracted with surface active agents, such as sodium deoxycholate in the presence of calcium ions. The key constituent disclosed in the Boehringer Mannheim process is calcium ions, and, if needed, a surface active agent is used. lb The use of barium sulfate is discussed in the Boehringer Mannheim patent, but discounted: "according to our own experiences, the thromboplastin largely co-precipitates with the barium sulfate." In conventional procedures the relative insensitivity of thromboplastins is due, in part, to the presence of coagulation 0 factor VII, being tightly bound to the thromboplastin. Barium sulfate is commonly used as an adsorbing agent to remove vitamin -dependent coagulant proteins such as factors II, VII, IX, and X. Brief Summary of the Invention b The present invention uses various combinations of nonionic detergents, such as Triton* X-lϋϋ, chaotropic ions such as thiocyanate, iodide, guanidine and perchlorate, and barium sulfate to extract thromboplastin from a tissue source. Nonionic detergents greatly enhance the efficiency of removing the U coagulation factors bound to the thromboplastin. The approach described herein offers the advantage of enhanced sensitivity to factor deficiencies and coumadin therapy while maintaining a relatively short normal . range PT. A survey conducted by the CAP SUBSTITUTESHEET Hematology Resource Committee in 1978 revealed that the normal range PT was 9-15 seconds for 96% of the laboratories reporting (Triplett, D.A. , "How the Prothrombin Time Actually is Performed" in Standardization of Coagulation Assays: An Overview, ed. by 5 D.A. Triplett, College of American Pathologists, Skokie, 1982, pp. 113-119). The preferred PT range for normal donors is 10-14 seconds. Reagents prepared using the methods described here have enhanced reconstituted stability over Thromboplastin FS, use less brain extract and are less optically dense. The present 10 invention allows sensitivity to Factor VII deficient plasma using insensitive thromboplastin reagents to be set at any PT; the preferred PT time for Factor VII deficient plasma is greater than sixty seconds. The thromboplastin reagent of the present invention will have a ratio of the abnormal control (C0L2) to the lb normal control (COLl) greater than 1.8, however, preferably the ratio is in the range of 1.8 to 3.0. Alternatively, chaotropic ions, such as thiocyanate, guanidine, or iodide, used alone in the extraction fluid greatly 20 enhance sensitivity. Chaotropic ions are agents used in disrupting membranes and enzyme complexes by breaking noncovalent forces. (W. Hanstein, ϋestabilization of Membranes with Chaotropic Ions, Meth. Enzy. XXXI (1974)). Chaotropic ions typically have a low charge density with a large radius. 2b Commonly used ions include: tribromoacetate, trichloroacetate, guanidinium, thiocyanate, iodide, perchlorate, dichloroacetate, nitrate, bromide, trif1uoroacetate and chloroacetate. 30 Detailed Description and Best Mode In this invention tissue, such as acetone-dehydrated rabbit brain powder, is extracted with a combination of barium sulfate, nonionic detergents and chaotropic ions, such as thiocyanate, iodide, guanidine or perchlorate, to control reagent properties. BSTITUTESH This invention should be useful for all thromboplastins and will improve the sensitivity for all PT-based tests and specific assays. 5 PT values can be determined using automated coagulation analyzers, such as the MLA Electra 800, mechanical instruments, such as Fibro eters, or by manual techniques. The abnormal plasma for these studies has been either an abnormal control such as Baxter Healthcare Corporation, Dade Division, Ci-Trol ® Level 10 II (C0L2) or lyophilized coumadinized plasmas (LAC) for an anticoagulant control. Ci-Trol* Level I (COLl) or a pool of fresh normal citrated plasma (FNP) have been used to determine normal PT values. Sensitivity to factor deficiencies is assessed by measuring the PT of a factor Vll-deficient plasma (CF7). lb Rabbit brain thromboplastins available in the United States (Dade Thromboplastin C) yield ratios o 1.5 for C0L2/C0L1 and a 28-30 sec PT tor factor Vll-deficient plasma on the MLA Electra 800. Example I: General Process Used to Prepare Thromboplastins 2U To make the thromboplastin reagent, rabbit brain power (50gm) is extracted in an extraction fluid (lOOO L) containing sodium chloride (3ϋ-18UmM), a nonionic detergent, such as Triton® X-100 (0.01-0.25%) and a chaotropic ion, such as sodium thiocyanate (5-10UmM). Alternatively, the extraction fluid may contain only b sodium chloride (0-150mM) and the chaotropic ion (5-100mM). Barium sulfate powder is added to the extraction fluid at ϋ.l-l.ϋgm/gm brain powder. The extraction is performed at 43 to 47°C for fifteen (15) minutes; the extraction mixture is then centrifuged for ten (lϋ) minutes at 2500 RPM to remove the barium 0 sulfate and large particles. The extract is added to a base containing calcium ions (7-14mM), sodium chloride (7ϋ-15ϋmM), buffers and stabilizers. The product is then freeze-dried. SUBSTITUTE SHEET Example II: The Effect of Triton X-10U and Barium Sulfate in Extraction on Thromboplastin Sensitivity. Acetone-dehydrated rabbit brain powder (Igm) was extracted in b 2U mL of 0.63% sodium chloride (NaCl) containing 0.6 gm of barium sultate (BaS0 4 ) and 0 - 0.25% Triton X-100* (Rohm t. Haas, Philadelphia, PA) for fifteen (15) minutes at 45° C; the extraction mixture was then centrifuged at 2500 RPM for ten (10) minutes to remove the barium sulfate and large particles. The 1U extract was added to a base at a final concentration of 32% in 30mM (TAPSO) buffer, 5% glycine, 0.6% polyethylene glycol (PEG) with a molecular weight of 8000, 13.7mM calcium chloride (CaCl 2 ), lOOmM NaCl, pH 7.0. Prothrombin times (PT) in seconds were recorded using an MLA Electra 800. (Medical Laboratory lb Automation, Pleasantville, New York) Both the ratio of C0L2/C0L1 and PT of factor Vll-deficient plasma (CF7) increase substantially as the amount of detergent is increased (Table.I). 20 TABLE I Effect of Triton X-100 and Barium Sulfate in Extraction on Thromboplastin-bulk (MLA-800): Average of 2 replicates % Triton X-100 COLl CUL2 RATIO CF7 C0L2/C0L1 2b Base Formulation: 30mM TAPSU, 5% Glycine, 0.6% PEG, 13.7mM CaCl 2 , lOOmM NaCl, 32% 3U Extract pH 7.0 Brain extracted in 0.63% NaCl and detergent with barium sulfate at U.6 gm/gm brain in 20mL of extraction fluid Example III: The Effect of Sodium Thiocyanate (NaSCN) in Extraction on Thromboplastin Sensitivity. Acetone-dehydrated rabbit brain powder (lg ) was extracted in 5 20 mL of 0.63% sodium chloride (NaCl), except where noted, containing 0 (control), 10, 50, and lOOmM NaSCN for fifteen (15) minutes at 45°C; a sample containing lOOmM NaSCN, without NaCl was also evaluated. Barium sulfate was not used in this experiment. The extraction mixture was centrifuged as described 1U in Example I. Prothrombin times (PT) in seconds were recorded using an MLA Electra 800. Both the ratio of C0L2/C0L1 and PT of factor Vll-deficient plasma (CF7) increase substantially as the amount of NaSCN is lb increased (Table II). Sodium thiocyanate alone increased thromboplastin sensitivity. TABLE II Effect of Sodium Thiocyanate (NaSCN) in Extraction on Thromboplastin - bul (MLA-δOO) * 20 25 Base Formulation: 3UmM TAPSO, b% Glycine, 0.6% PEG, 13.7mM CaCl 2 , lOOmM " NaCl , 32% Extract, pH 7.0 3U SUBSTITUTE SHEET Example IV: Comparison of Various Compositions of Extraction Fluids and Percentage of Extract on Thromboplastin Sensitivity. Rabbit brain powder was extracted in extraction fluids 5 containing two (2) different compositions of NaCl, Triton X-100, NaSCN and barium sulfate. In Table III, Extract E contained 130mM NaCl, 5ϋmM NaSCN, 0.05% Triton X-100 and 0.3gm barium sulfate/gm brain powder; Extract N contained 50mM NaCl, lOmM NaSCN, 0.02% Triton X-100 and ϋ.4gm barium sulfate/gm brain 10 powder. The brain powder was extracted, centrifuged and the extracts were added to a base to the final concentrations of 32% or 50% as described in Example I. Prothrombin Times (PT) in seconds were recorded using an MLA Electra 800. lb Table III shows that the components of the extraction fluids and the amount of. extract can vary considerably to yield thromboplastins with enhanced sensitivity over rabbit brain thromboplastins, such as Dade Thromboplastin C (ratio C0L2/C0L1 - l.b). 2ϋ 25 30 SUBSTITUTE SHEET TABLE III Comparison of Various Compositions of Extraction Fluids and Percentage of Extract on Thromboplastin - Bulk (MLA-800) 10 Thromboplastin FS 13.3 24.5 1.84 13.0 31.4 2.42 64.6 Base Formulation: 30mM TAPSO, b% Glycine, 0.6% PEG, 13.7mM CaCl 2 , lOOmM NaCl, pH 7.0 lb Extract E: Brain Powder extracted in 130mM NaCl, 50mM NaSCN, 0.05% Triton X-100, 0.3gm barium sulfate/gm brain powder Extract N: Brain Powder extracted in 50mM NaCl, lO M NaSCN, 0.02% Triton X-100, ϋ.4gm barium sulfate/gm brain powder 20 b U SUBSTITUTE SHEET Example V: Comparison of Various Formulations of Sensitive Thromboplastins. Acetone-dehydrated rabbit brain powder was extracted in a b solution containing bOmM NaCl, lOmM NaSCN, 0.02% Triton X-100 and ϋ.4gm barium sulfate/gm brain powder. The brain powder was extracted as described in Example I. The extracts were added to bases for two (2) different formulations: Formulation D contained 35% extract in 4ϋmM bicine buffer, 5.25% glycine, 0.6% 10 PEG, lOmM CaCl 2 , 134mM NaCl, pH 7.1: Formulation T contained 36% extract in 80mM TAPSO, 5.25% glycine, 0.6% PEG, lOmM CaCl 2 , 118mM NaCl, pH 7.4. Both formulations were lyophilized. After reconstitution, PT in seconds was recorded using an MLA Electra 80U. lb Table IV shows that the composition of the formulation can vary considerably to yield thromboplastins with enhanced sensitivity and other properties superior to Thromboplastin FS, a relatively sensitive rabbit brain thromboplastin. 20 2b 3U TABLE IV Comparison of Various Formulations of Sensitive Thromboplastins - Bulk (MLA-80U) 10 Thromboplastin FS 14.2 26.7 1.88 13.4 30.5 2.28 58.9 Extract Mixture: Brain Powder extracted in 5ϋmM NaCl, lOmM NaSCN, 0.02% Triton X-100, U.4gm barium sulfate/gm brain powder Formulation D: 15 40mM Bicine, 5.25% Glycine, 0.6% PEG, lOmM CaCl 2 , 134mM NaCl, pH 7.1 Formulation T: 80mM TAPSϋ, 5.25% Glycine, 0.6% PEG, lOmM CaCl 2 , llδ M NaCl, pH 7.4 -20 25 3U SUBSTITUTE SHEET Example VI: Preparation of Thromboplastins with sensitivity typical of those sold in the U.S. Acetone-dehydrated rabbit brain powder was extracted in b solution containing 5UmM NaCl, lϋmM NaSCN, 0.02% Triton X-100 and U.4gm barium sulfate/gm brain powder. The brain powder was extracted as described in Example 1. The extracts were added to bases for three different formulations: Formulation E contained 10% extract in 53mM TAPSϋ buffer, 4.00% glycine, 0.3% PEG, llmM 0 CaCl 2 , 50mM NaCl, pH 7.4; Formulation F contained 12% extract in 50mM TAPSϋ buffer, 4.00% glycine, 0.3% PEG, llmM CaC , 50mM NaCl, pH 7.4; Formulation G contained 10% extract in 53mM TAPSO buffer, 4.00% glycine, 0.3% PEG, llmM CaCl 2 , 65mM NaCl, pH 7.4. The formulations were lyophilized. After reconstitution, 5 PT in seconds were records using an MLA Electra 800. Table V shows that the extraction fluids described herein can be used to make conventional rabbit brain thromboplastins available in the U.S. similar to Thromboplastin C. Relatively small amounts of extract (10-12% versus 25% for Thromboplastin C) U are required to make a thromboplastin with lower COL/COLl ratio and LAC/FNP ratio. However, the sensitivity to factor VII is enhanced. b 0 TABLE V Comparison of Various Formulations of Thromboplastins with Sensitivity Typical of those sold in the U.S. (MLA-800) 5 Formulation COLl C0L2 RATIO FNP LAC RATIO CF7 C0L2/C0L1 LAC/FNP E 11.8 20.4 1.73 11.6 21.7 1.87 42.1 F 11.7 20.4 1.74 11.3 21.3 1.88 40.6 G 11.4 19.7 1.73 11.1 21.0 1.89 40.4 0 Tnromboplastin C 11.9 20.2 1.70 11.5 20.2 1.76 27.7 Extract Mixture: Brain Powder extracted in 50mM NaCl, lOmM NaSCN, 0.02% Triton X-100, Q.4gm barium sulfate/gm brain powder Formulation E: 10% Extract, 53mM TAPSO, 4.00% Glycine, 0.3% PEG, UmM CaCl 2 , 50mM 5 NaCl, pH 7.4 Formulation F: 12% ' Extract, 50mM TAPSϋ, 4.00% Glycine, 0.3% PEG, llmM CaCl 2 , 50mM NaCl, pH 7.4 Formulation G: 0 12% Extract, 50mM TAPSO, 4.00% Glycine, 0.3% PEG, llmM CaCl 2 , 65mM NaCl, pH 7.4 b U SUBSTITUTE SHEET Variants or Equivalents of the Invention The performance and sensitivity of a PT reagent is the result of interactions between all of the constituents of the reagent, b Generally, changes in formulation which increase the sensitivity also increase the value of the normal PT. In the present invention, the components of the extraction fluid are carefully balanced for best normal PT and sensitivity. In addition, the individual constituents of the extraction mixture influence 10 reagent performance in specific ways. Varying the concentrations of the various components also alters the properties of the prepared extract; therefore, extracts with different properties can be prepared depending on the extraction fluid composition. lb Barium sulfate and any nonionic detergent, such as Triton® X-lϋϋ, Bπ ' j-3b, Nonidet* P4U, FSN* and Tergitol* greatly enhance sensitivity, especially to specific coagulation factor deficiencies such as factor VII. Chaotropic ions (thiocyanate, guanidine, iodide, perchlorate) alone in the extraction enhances 20 sensitivity to both coumadinized patient samples and specific factor deficiencies such as factor VII. To make the thrombopl stin reagent, the tissue is extracted in an extraction fluid containing sodium chloride (30-180mM), the nonionic detergent (0.01-0.25%) and the chaotropic ion such as 25 thiocyanate, guanidine, iodide and perchlorate (5-lOOmM). Barium sulfate powder is added to the extraction fluid at 0.1-1.0gm/gm brain powder. Alternatively, the extraction fluid may contain only sodium chloride (0-150mM) and sodium thiocyanate (5-100mM). The extraction is performed at 43 to 47°C for fifteen (15) 30 minutes; the extraction mixture is then centrifuged for ten (10) minutes at 2500 RPM to remove the barium sulfate and large particles. The extract is added to a base containing calcium ions (7-14mM), sodium chloride (70-150mM), buffers and stabilizers. The buffers and stabilizers can be varied to improve the stability of the product as well. The extraction method and components can be used with any source of tissue containing thromboplastin, such as rabbit brain and lung, bovine brain and lung, ovine brain and lung, and human brain, lung and placenta. lϋ 15 U b 0 SUBSTITUTESHEET

Claims: WHAT IS CLAIMED IS: 1. A method for extracting thromboplastins comprising: (a) contacting tissue containing thromboplastin with an b effective amount of extraction fluid comprising barium sulfate, nonionic detergent, chaotropic ion, and a salt under conditions which result in the extraction of thromboplastin, and iϋ (b) separating said extracted thromboplastin from said depleted tissue and barium sulfate. 2. The method of Claim 1 wherein the effective amount of said extraction fluid is about lOOmL for every 5 grams of tissue. 15 3. The method of Cl aim 1 wherei n the said noni oni c detergent i s at about 0.01-0.25%, said chaotropic ion is at 5-100mM, and the salt is at about 30-180mM. 20 4. The method of Cl aim 1 wherein the effecti ve amount of barium sulfate is about 0.1-1.0 gm/gm tissue containing thrombopl astin. 5. The method of Cl aim 1 wherein the extraction is conducted at 2b between 43 to 47°C for approximately fifteen (15) minutes . 6. Tne method of Cl aim 1 wherein said tissue is rabbit brai n powder. 3ϋ 7. A method for extracting thrombopl astins comprising: (a) contacting tissue containing thromboplastin with an effective amount of extraction fluid comprising chaotropic ions, and under conditions which result in the extraction of thromboplastin; and b (b) separating said extracted thromboplastin from said depleted tissue. 8. The method of Claim 7 wherein the effective amount of said 10 extraction fluid is about 100 mL for every 5 grams of tissue. 9. The method of Claim 7 wherein the concentration of the chaotropic ion is about 5-100 mM. lb . lϋ. The method of Claim 7 wherein the extraction is conducted at between 43 to 47°C for approximately fifteen (15) minutes. 11. The method of Claim 7 wherein said tissue is rabbit brain powder. • 12. A method for extracting thromboplastins comprising: (a) contacting tissue containing thromboplastin with an effective amount of extraction fluid comprising 25 chaotropic ions and a salt, under conditions which result in the extraction of thromboplastin; and (b) separating said extracted thromboplastin from said depleted tissue. 30 SUBSTITUTE SHEET 13. The method of Claim 12 wherein tne effective amount of said extration fluid is about 100 mL for every 5 grams of tissue. 14. The method of Claim 12 wherein the concentration of the b chaotropic ion is about 5-100 M and the concentration of the salt ranges from about 30-180 mM. lb. The method of Claim 12 wherein the concentration of the chaotropic ion is about 10-100 mM. lϋ 16. The method of Claim 12 wherein the extraction is conducted at between 43 to 47°C for approximately fifteen (15) minutes. 17. The method of Claim 12 wherein said tissue is rabbit brain 15 powder. 18. A method for extracting thromboplastin reagents comprising: (a) contacting tissue containing thromboplastin with an 20 effective amount of extraction fluid comprising barium sulfate, nonionic detergent and salt, under conditions which result in the extraction of thromboplastin, and (b) separating said extracted thromboplastin from said 2b depleted tissue. ly. The method of Claim 18 wherein the effective amount of said extraction fluid is about 100 mL for every 5 grams of tissue. 3U UBSTITUTESHEE 20. The method of Claim 18 wherein the effective amount of barium sulfate is about 0.1-1.0 gm/gm tissue containing thromboplastin. The effective amount of detergent is 0.01-0.26%. 5 21. The method of Claim 18 wherein the extraction is conducted at between 43 to 47°C for approximately fifteen (15) minutes. 22. The method of Claim 18 wherein said tissue is rabbit brain 10 powder. 23. A method for extracting a thromboplastin reagent comprising: (a) contacting tissue containing thromboplastin with an lb effective amount of extraction fluid comprising a salt, a chaotropic ion, and barium sulfate, under conditions which result in the extraction of thromboplastin, and (b) separating said extracted thromboplastin from said U depleted tissue. 24. The method of Claim 23 wherein the effective amount of said extraction fluid is about 100 mL for every 5 grams of tissue. b 25. The method of Claim 23 wherein said chaotropic ion is at about 5-100 mM, and the salt is at about 30-180 mM. 26. The method of Claim 23 wherein the effective amount of barium sulfate is about 0.1-1.0 gm/gm tissue containing 0 thromboplastin. SUBSTITUTE SHEET 27. The method of Claim 23 wherein the extraction is conducted at between 43 to 47°C for approximately fifteen (15) minutes. 28. The method of Claim 23 wherein said tissue is rabbit brain b powder. 29. An extracted thromboplastin composition having approximately normal prothrombin times and enhanced sensitivity to factor deficiencies and coumadin therapy made the process of Claim 1. 0 30. The composition of Claim 29 wherein the normal prothrombin time ranges from about 9 to 15 seconds. 31. The composition of Claim 29 wherein said enhanced sensitivity 5 to coumadin therapy shown by C0L2/C0L1 ratio ranges from about 1.8 to 3.0. 32. An extracted thromboplastin composition having approximately normal prothrombin times and enhanced sensitivity to factor 0 deficiencies and coumadin therapy made using the process of Claim 7. 33. The composition of Claim 32 wherein the normal prothrombin time ranges from about 9 to 15 seconds. b 34. The compositi on of Cl aim 32 wherei n said enhanced sensitivity to coumadi n therapy shown by C0L2/C0L1 rati o ranges from about 1.8 to 3.0. ϋ 35. An extracted thromboplastin composition having approximately normal prothrombi n times and enhanced sensitivity to factor defi cienci es and coumadin therapy made usi ng the process of Cl aim 12. 36. The composition of Claim 35 wherein the normal prothrombin time ranges from about 9 to 15 seconds. 37. The composition of Claim 35 wherein said enhanced sensitivity b to coumadin therapy shown by C0L2/C0L1 ratio ranges from about 1.8 to 3.0. 38. An extracted thromboplastin composition having approximately normal prothrombin times and enhanced sensitivity to factor 1U deficiencies and coumadin therapy made using the process of Claim 19. 39. The composition of Claim 38 wherein the normal prothrombin time ranges from about 9 to 15 seconds. lb 4U. The composition of Claim 38 wherein said enhanced sensitivity, to coumadin therapy shown by C0L2/C0L1 ratio ranges from about 1.8 to 3.0. 20 b ϋ SUBSTITUTESHEET

Inventors: HAWKINS PAMELA L H, MAYNARD JAMES R, HAWKINS, PAMELA, L., H., MAYNARD, JAMES, R.
Applicants: BAXTER INT, BAXTER INTERNATIONAL INC.
Application year: 1989
Main classification: C07K3
IPC codes: A61K35, A61K38, A61P7, C07K1, C07K14, C12P21