Acid soluble, pepsin resistant platelet aggregating material

Claim 1

A method of preparing a hemostatic agent comprising the steps of: (a) extracting cleansed, homogenized equine arterial tissue with dilute aqueous acid at from about 0° C. to 5° C., (b) dialyzing the extract against dilute acid, (c) subjecting the retentate to proteolysis with pepsin, and (d) precipitating the hemostatic agent by the addition of a water soluble salt to the resulting mixture.

Claim 2

A method of preparing a hemostatic agent comprising the steps of: (a) extracting cleansed, homogenized equine arterial tissue with dilute aqueous acid at from about 0° C. to 5° C., (b) dialyzing the extract against dilute acid, (c) subjecting the retentate to proteolysis with pepsin, and (d) precipitating the hemostatic agent by the addition of a water soluble salt to the resulting mixture.

Claim 14

A method of stimulating platelet aggregation in mammalian plasma or whole blood which comprises contacting said plasma or whole blood with an amount which is effective to stimulate platelet aggregation of an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000

Claim 15

A method of stimulating platelet aggregation in mammalian plasma or whole blood which comprises contacting said plasma or whole blood with an amount which is effective to stimulate platelet aggregation of an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000

Claim 17

A method of controlling external bleeding from a wound or incision comprising contacting said wound or incision of an amount which is effective to control bleeding of an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000

Claim 18

A method of controlling external bleeding from a wound or incision comprising contacting said wound or incision of an amount which is effective to control bleeding of an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000

Claim 19

A method of detecting abnormal platelet function comprising contacting platelets in plasma or whole blood under test with an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000 and comparing the aggregation response of said platelets to the response of normal platelets.

Claim 20

A method of detecting abnormal platelet function comprising contacting platelets in plasma or whole blood under test with an acid soluble, pepsin resistant, non-dialyzable, proteinaceous hemostatic agent isolated from equine arterial tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended periods of time at temperatures as low as -85° C., losing its platelet aggregating activity when exposed to collagenase at 37° C. or when heated at 100° C. for fifteen minutes; containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 23.8 Glycine 254.2 Histidine 10.4 Alanine 100.1 Hydroxylysine 5.5 Cysteine 10.7 Hydroxyproline 42.4 Valine 44.0 Aspartic Acid 75.1 Methionine 6.6 Threonine 36.0 Isoleucine 31.9 Serine 52.8 Leucine 52.1 Glutamic Acid 94.3 Tryosine 15.2 Proline 87.1 Phenylalanine 25.9 Arginine 33.9; ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: 322,000 300,000 250,000 220,000 163,000 132,000 116,000 100,000 61,500 51,500 45,000 36,000 and comparing the aggregation response of said platelets to the response of normal platelets.