Acid soluble platelet aggregating material isolated from human umbilical cord

Claim 1

An acid soluble, pepsin sensitive non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord tissue characterized by the ability to enhance platelet aggregating activity in mammalian blood, stable for extended period of time at temperatures as low as -85° C., containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 30.1 Glycine 287.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________

Claim 7

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 acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 30.1 Glycine 287.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________

Claim 8

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 acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 30.1 Glycine 287.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________

Claim 10

A method of controlling external bleeding from a wound or incision comprising contacting said wound or incision with an amount which is effective to control bleeding of an acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 30.1 Glycine 257.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________

Claim 11

A method of controlling external bleeding from a wound or incision comprising contacting said wound or incision with an amount which is effective to control bleeding of an acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino acid residues: ______________________________________ Lysine 30.1 Glycine 257.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________

Claim 12

A method of detecting abnormal platelet function comprising contacting platelets in plasma or whole blood under test with an acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino residues: ______________________________________ Lysine 30.1 Glycine 287.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________ and comparing the aggregation response of said platelets to the response of normal platelets.

Claim 13

A method of detecting abnormal platelet function comprising contacting platelets in plasma or whole blood under test with an acid soluble, pepsin sensitive, non-dialyzable, proteinaceous hemostatic agent isolated from human umbilical cord 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., containing the following average number of amino acid residues per 1,000 total amino residues: ______________________________________ Lysine 30.1 Glycine 287.8 Histidine 14.4 Alanine 111.9 Hydroxylysine 6.4 Cysteine 6.3 Hydroxyproline 36.1 Valine 41.2 Aspartic Acid 51.3 Methionine 8.1 Threonine 19.6 Isoleucine 18.9 Serine 38.6 Leucine 41.1 Glutamic Acid 90.3 Tryosine 7.8 Proline 100.8 Phenylalanine 20.1 Arginine 69.2 ______________________________________ which on SDS-PAGE electrophoresis produces bands characteristic of the following molecular weights: ______________________________________ 300,000 200,000 250,000 132,000 220,000 116,000 ______________________________________ and comparing the aggregation response of said platelets to the response of normal platelets.

Claim 14

A method of preparing a hemostatic agent comprising the steps of: (a) extracting cleansed, homogenized, human umbilical cord tissue with dilute aqueous acid at from 0° to 5° C., (b) separate and recover a solid precipitate by centrifugation, homogenize and again extract with dilute aqueous acid at from 0° to 5° C., (c) separate solid precipitate by centrifugation and recover aqueous phase, (d) filter aqueous phase and dialyze against dilute aqueous acid at from 0° to 5° C. to produce a concentrated retentate containing the hemostatic agent; said dilute aqueous acid being at a concentration of from 0.05 to 0.1 molar.

Claim 15

A method of preparing a hemostatic agent comprising the steps of: (a) extracting cleansed, homogenized, human umbilical cord tissue with dilute aqueous acid at from 0° to 5° C., (b) separate and recover a solid precipitate by centrifugation, homogenize and again extract with dilute aqueous acid at from 0° to 5° C., (c) separate solid precipitate by centrifugation and recover aqueous phase, (d) filter aqueous phase and dialyze against dilute aqueous acid at from 0° to 5° C. to produce a concentrated retentate containing the hemostatic agent; said dilute aqueous acid being at a concentration of from 0.05 to 0.1 molar.

Claim 19

A method as in claims 10, 11, 12 and 13 wherein the dilute acid used for extraction and dialysis is acetic acid.