it has been found that the application of certain fish epidermal preparations to wounds appears to act quite generally in stimulating all observed phases of the healing process . the complex mixture of pharmacologically active components in the preparation thus appears to contain a balanced combination of stimulatory agents which influence the entire healing process . this is supported by both histological and biochemical observations . many of the active constituents of the preparation have now been isolated and their modes of action in healing have been documented . these provide a clear indication of how the preparation can function to stimulate healing . it has been observed that ariid catfish , when threatened or injured or otherwise shocked , secrete a proteinaceous , gel - like mixture from unicellular glands , which are frequently termed club cells . this material , together with some additional epidermal components , is collected by gentle mechanical scraping , or suction , from the epidermal surfaces . the collected material has a gel - like consistency and will be referred to as epidermal gel . the gel is immediately chilled or frozen to limit microbial growth . the total gel preparation may be used for application to wounds , but further fractionation and purification of components is commonly employed to provide a safer , more viable preparation for use on wounds . shocking of the fish can result from thermal shock , physical abrasions or neural stimulation . the catfish as well as many additional fish species , have club cells in the epidermis . epidermal preparations from these other species will contain some components analogous to the major constituents of the ariid catfish secretion , but these will be more difficult to obtain in a form separated from the bulk of epidermal materials . fractionation and purification of a soluble fraction of the ariid epidermal gel is by homogenization of the total gel with dilute saline , followed by centrifugation . the soluble gel fraction is sterilized by membrane filtration or ultrasound treatment . soluble protein fraction may be stored either frozen , preferably at - 80 ° c . soluble gel fraction may be applied directly to wounds to enhance healing . the soluble gel may be fractionated further to yield active proteins and lipids which have effects on wound healing as described below . these are active individually in enhancing various steps of the wound healing processes . they may be recombined to reconstitute general activity . a summary of some active constituents of soluble gel which have been identified , to date , as affecting specific healing characteristics as identified below ( a through g ) is as follows : a . clotting factor . a high molecular weight protein ( 500 , 000 g / m ) catalyzes conversion of blood clotting factor x to xa . active factor xa then catalyzes production of thrombin and subsequently the formation of the fibrin clot . the soluble gel clotting factor is a calcium dependent serine protease with specificity similar to that of normal blood proteases causing activation of factor x . b . lectin . a galactose specific lectin protein ( 200 , 000 molecular weight ) accounts for about 2 % of the soluble protein . this protein causes agglutination of human types a , b , and o red blood cells . it is capable of recognition of galactose moieties on the surface of cells and may aid in wound closure and adhesion of injured surfaces . c . lytic enzymes . the gel contains a number of hydrolytic components . there is an active hemolytic protein which induces lysis of red blood cells . proteolytic activity is very low in the crude preparation , but is noted in partially purified preparations , indicating removal of endogenous protease inhibitors . there are at least four esterases present in the preparation . phosphatase activity is present and will be discussed in detail later . no lipase activity is measurable . d . phosphorylases . a family of phosphorylases is present in the soluble gel protein mixture . these include ( a ) a tyrosine phosphorylase , ( b ) phosphorylase ( s ) which appear to generally phosphorylate many of the gel proteins or added plasma membrane proteins at serine , threonine and tyrosine residues , and ( c ) an enzyme which phosphorylates phosphatidyl inositol . e . platelet activating factors . the epidermal preparation contains a family of 1 - 0 - alkyl - 2 - sn - acetyl glycerolphosphatidyl choline molecules , with alkyl chain lengths varying from 15 to 24 carbons , which stimulate activation of platelets . these are present at levels 5000 times those commonly found in animal sources . f . prostaglandins . the epidermal gel preparation contains low levels of prostaglandins , and in addition is able to induce formation of high levels of prostaglandins via stimulation of endothelial cells or epidermal cells . g . smooth muscle contraction . the epidermal preparation contains at least two components which stimulate contraction of smooth muscle . one is blocked by cyclooxigenase inhibitors , and the other by atrophine . the benefits of an externally added clotting factor in aiding wound healing are readily apparent . the first step in wound healing is clotting . clot formation initiates a number of important metabolic events which stimulate cell metabolism and initiate cell proliferation . primary among these is platelet activation . the high level of platelet activating factors found in the epidermal preparation also rapidly induces platelet activation in a wound area . among the results of this activation are secret from the platelets of serotonin , platelet derived growth factor , and other biochemicals . serotonin aids vasoconstriction , shutting down the flow of blood to the wound area . platelet derived growth factor ( pdgf ) is both chemotactic and mitogenic for fibroblasts , thus it aids in promoting the movement of these collagen producing cells into the wound area and induces their proliferation . the products of the fibroblasts are major components needed for tissue repair . platelet activation also stimulates hydrolysis of phospholipids and release of inositol phosphates . these inositol compounds regulate calcium levels in the cells and alter cell metabolism . phosphorylase action directed by the epidermal preparation has stimulatory activity at several levels in cell metabolism . direct phosphorylation of phosphatidyl inositol to the di - and triphosphate forms stimulates as described above . in addition , an endogenous inhibitor protein of phospholipase a2 in epidermal membranes is phosphorylated , resulting in deactivation of the inhibitor and expression of the lipase activity . the lipase catalyzes release of arachadonic acid . arachadonic acid is rapidly converted to prostaglandins which regulate many cellular processes in the wound area . for example , prostaglandins affect inflammatory responses , pain responses , migration of cells into the wound area , angiogenesis and rates of cell proliferation . all of these processes are key steps in wound healing . lytic enzymes from the epidermal secretion may play important roles in break down of damaged cells . no definite role has yet been established for the lectin present in the epidermal secretion . lectins have in general been postulated to serve as important elements in cellular adhesion . the epidermal gel preparation does promote a rapid adhesion of wound tissues . the lectin is a likely candidate for this action as it may recognize and bind galactose moieties on adjacent cell surfaces to give the noted adhesion . although a preferred form of our invention has been herein disclosed , it is to be understood that the present disclosure is by way of example and that variations are possible without departing from the subject matter coming within the scope of the following claims , which subject matter we regard as our invention .
a method of producing a wound healing preparation comprising shocking a club cell producing creature such as an ariid catfish to stimulate secretion of the contents of such club cells , collecting the secreted materials by collecting epidermal materials of the creature as a gel .