Heparin was developed as a drug for clinical use 50 years ago by research groups in Toronto and Stockholm, headed respectively by Professors Charles H. Best and Erik Jorpes. Since then, the drug has been used for millions of patients and has been the subject of intensive clinical and laboratory study. There has been no question of the clinical effectiveness of this remarkable drug, but the results of studies on how the drug acts have often disappointed investigators. Undoubtedly, this is related to investigators’ assumptions which were found to be invalid by the original research teams. These teams recognized the chemical complexity of heparin and realized that the property of preventing blood from clotting in a test-tube was insufficient to explain clinical effectiveness.
Recent studies using new techniques have resolved the enigmas and now provide the clinician with enough basic knowledge to use the drug satisfactorily. I had the privilege of working with the late Gordon Murray in Toronto on the initial testing of the drugs effects in thrombosis; with the late A. F. Charles on the chemistry of the drug; and I discussed these problems with the late Professor Jorpes. I have recently published an extensive review of the total literature, together with a new overall view of what heparin is and does. The present article presents an organized synopsis of the knowledge of heparin that has been accumulated over the past 50 years. For detailed presentation of the observations and literature citations, the reader is directed to the first eight references. Initial Development of Commercial Heparin
In 1933, A. F. Charles and D. A. Scott established commercial sources and procedures for the preparation of heparin. This substance was shown to provide necessary insurance for successful vascular surgery and the prevention of intravascular clotting and venous thrombosis. Administration of the drug could be monitored by determination of clotting time. No evidence of bleeding was observed, even when the subjects blood was incoagulable for many hours. Charles and Scott crystallized heparin on a routine basis. This gave a basis for defining heparin free from other substances, and for making non-toxic preparations which could be used clinically. The drug entered quickly into the regimens for vascular surgery and prevention of venous thrombosis, and has been used continuously since. Additional important observations were made regarding the nature and action of heparin. It was shown to be a sulfated polysaccharide containing a variety of sulfuric and uronic acid groups which make heparin the strongest acid in biology with paradoxic physicochemical properties. Heparin prepared from different sources showed marked differences in relative inhibitory activities in different coagulation tests.
Heparin was shown by A. Fischer of Copenhagen to react with proteins, dyes, alkaloids, et cetera, and to change their properties, eg, inhibition or activation of enzymes. It showed a special affinity for toluidine blue and azure A, changing the blue to a reddish-purple. This metachromatic change was well-known to histologists as a characteristic of mast cells. Anaphylaxis in dogs resulted in the appearance in the circulation of large amounts of histamine and heparin, with disintegration of hepatic mast cells, thus demonstrating the close association of these and antigen-antibody reactions. Chemical sulfation of polysaccharides produced substances, heparinoids, with most of the properties of heparin apparent in some degree. Heparin and heparinoids were found to be taken up by the RES. Protamine was found to neutralize the anticoagulant action in vitro and in vivo, and its toxicity analyzed. Intravenous administration of certain heparin preparations was shown to cause thrombocytopenia in certain subjects. Occasional bleeding was observed clinically with heparin and was not related to degree of anticoagulation. Finally, the marked reduction in the incidence of postoperative thromboembolism with heparin treatment occurred with all dosage schedules, irrespective of maintenance of strict anticoagulation. During World War II, heparin became generally accepted in clinical use. An International Standard Heparin Preparation and pharmacopeia specifications were established.
Variation in Commercial Heparin
With the increasing demand for heparin, large amounts of the drug were produced by many manufacturers with uniformly satisfactory clinical results, suggesting a uniform product. However, from 1945 to 1965, a series of commercial preparations were analyzed for various chemical constituents and activity in various coagulation tests, and marked variability was observed even in such basic parameters as optical rotation, sulfate and amino-sugar values. Although the preparations met the requirements of the anticoagulant assay test of the US Pharmacopeia, et cetera, the unreliability of such assays was established by the 1970 international collaborative study on the assay of heparin. The WHO statistician found that simple replicate assays gave estimates so precise that an analysis of variance was meaningless, but when two different heparin preparations were compared, estimates by coagulation assay varied as much as 40 to 50 percent. This means that any two heparin preparations are not identical (as shown by coagulation in vitro), although apparently equally satisfactory clinically. Recent developments in theoretical and practical chemistry have made it possible to understand the nature of the drug. Electrophoresis in various gels on the micro scale, together with specific bacterial enzymes, nuclear magnetic resonance, and other techniques, have shown that a bottle of commercial heparin contains a large number of molecules differing in sulfate groups, uronic acids, amino sugars, and molecular weight. Fractionation by different procedures sequentially has shown over 120 different “heparins” in commercial heparin. The mix is not identical in different preparations, so individual batches can be fingerprinted. The differences in chemical structure mean that the individual entities in a given heparin preparation provide an array of negative charge patterns positioned on flexible molecules, which can provide specific matching to the less negative charge patterns on protein molecules. The individual “heparins” differ in biologic activities, so that commercial heparin preparations provide a bag of skeleton keys for a wide variety of biologic effects. Fractionation can change the relative proportion of components in the mix, but until it is established which components are clinically important, it is premature to recommend fractions for clinical use.
The Many Actions of Heparin and Heparinoids
Many investigators discovered new examples of the marked effect of heparin and heparinoids on proteins, cells, et cetera, and of the complexes formed with inorganic ions, alkylamines, alkaloids, dyes, biogenic amines, drugs, basic proteins, and plasma proteins. Recently, by incorporating such substances in an insoluble matrix, specific absorbents have been provided for heparin and, with heparin/heparinoid in the matrix, for specific proteins. Effects with over 50 enzymes have been reported for heparin and heparinoids, with activation of lipoprotein lipase, brain tyrosine hydroxylase, and DNA polymerase. Many enzymes are inhibited, and a large number of heparin/ heparinoids mean that a highly effective inhibitor can be found for any particular enzyme.
One well-known effect of heparin has been its antithrombin action. This has been shown to be caused by heparin activating the plasma protein antithrombin AT-III. Only one-third of commercial heparin combines with AT-III, and only a fraction of this actually activates. Active AT-III inhibits serine proteases (eg, thrombin; factors VII and IX to XIII; plasmin, urokinase, and kallinkrein.) When heparin and heparinoids are administered, the enzymes lipoprotein lipases, lecithinase, diamine oxidase, acid ribo-nuclease, beta-glycerophosphatase and procollagenase appear in the circulation. The release of diamine oxidase provides a break for fat absorption.
A great number of effects on cells and in animals have been reported. Heparin activates macrophages, increases B-lymphocyte migration, inhibits both T and B lymphocytes. Inhibition of sensitivity reactionshase been reported with many systems, eg, the complement system, anaphylaxis, antigen-antibody reactions, and glomerulonephritis. Modification of hormone actions in vitro and in vivo have been reported, although the effect on aldosterone production has now been shown to be caused by the antiseptic in commercial heparin solutions. A number of protective actions have been shown in animals against toxic bases, adverse drug reactions, toxic conditions (peritonitis, bums, et cetera), trauma and stress. For over 40 years, heparin has been administered intravenously and subcutaneously to millions of patients receiving other drugs without any report of adverse reaction.