Some side effects of heparin, heparinoids, and their antagonists Effects of heparin, heparinoids, and their antagonists, other than actions on blood coagulation and lipemia clearing, are described. Many, if not all, of these properties are probably related to the unusual structure and high-charge density of these macromolecules. Some may have therapeutic utility. William W. Coon, M.D., and Park W. Willis, III, M.D. Ann Arbor, Mich. Departments of Surgery and Medicine, University of Michigan Since its discovery fifty years ago, and Astrup56 demonstrated that these salts heparin has been extensively used as an partially dissociate according to mass law anticoagulant and, in recent years, as an as long as the product remains in solution; agent to facilitate more rapid clearing of this has been confirmed by Jaques.100 Com­ lipemic plasma. These functions of heparin plexing with heparin has been observed have been adequately reviewed in the re­ not only with proteins and peptides (silk cent past. This monograph lists some of peptone, gelatin, clupein, salmine, his tones the side effects of heparin which are not from thymus and blood, hemoglobin, egg so well known. Some may ultimately be albumin, casein, serum proteins, liver pro­ shown to be directly related to the anti­ teins, thromboplastins, enzymes, and so coagulant effect of heparin but, if so, this forth) but also with organic bases (benzi­ association has not yet been proved. dine, quinine, brucine, and piperidine) and Several of these effects have been observed basic dyes (thionine, toluidine blue, and only in vitro, and to our knowledge studies others). Although optimal combination of to document whether or not they persist in heparin with protein occurs near the iso­ vivo have not yet been carried out. A few electric point, it is possible for heparin to may have therapeutic applications. Knowl­ combine with proteins on the alkaline side edge of others may aid in the correct inter­ of their isoelectric point. Dissociation of pretation of extraneous effects which might these compounds is markedly affected by occur during heparin therapy. pH and ionic strength.10o Jaques has made reference to the difficulty of confirmation Formation of "complexes" of reports by others regarding the inhibi­ One of the earlier known side effects tory effect of heparin on certain proteins of heparin was demonstration of its ability because reproduction of relative concen­ to form stable salts with many pro­ trations of heparin, the protein under teins.31 , 54, 172 Over thirty years ago, Fischer study, of other proteins, pH, ionic strength, and so forth is often difficult. The early Supported in part by the Parke-Davis Surgical Research Fund and the Michigan Heart Association. work on complex formation by heparin 106 Received for publication Jan. 25, 1966. has been summarized by Jorpes. Studies 379 380 Coon and Willis Clinical Pharmacology and Therapeutics of charged polyglucose derivatives add to of affinity between heparin and other our understanding of the mechanism of anionic polysaccharides have been attrib­ effect of heparin and heparinoids. Macro­ uted to differences in structure of these cationic substances with enzymatic activity several molecules. Their association with (hyaluronidase, ribonuclease, and lyso­ cations in solution is thought to be of elec­ zyme) are reversibly inhibited by synthetic trostatic rather than covalent type. The sulfated polysaccharides; these enzymes affinity of acid mucopolysaccharides for can be reactivated by addition of other calcium decreases in the order: heparin, cationic agents such as sodium ion or pro­ chondroitin sulfate B, chondroitin sulfate tamine. Highly substituted sulfates are the A, and hyaluronic acid.24 most effective enzyme inhibitors, while The effect of heparin upon cellular cal­ sulfates of similar molecular size but lower cium has been studied in vitro in Ehrlich charge density have less inhibitory activity. ascites tumor cells labeled in vivo with 4li The product with highest branching and isotopic Ca ; no change in calcium content charge density is about five times as active of cells was observed when cells were in­ as heparin in inhibiting ribonuclease. These cubated in solutions containing up to 0.6 derivatives also precipitate other basic pro­ mg. heparin per milliliter.205 Heparin does teins (protamine, an ACTH preparation, increase Rb86 outflow from the diaphrag­ cytochrome C, and serum albumin) from matic muscle of rats, an effect similar to aqueous solution at low pH and salt con­ that produced by reduction in concentra­ centration.15l tion of calcium ions. 28 Potassium exchange The electric charges of heparin prepara­ is affected in a similar manner.86 tions are proportional to their content of Heparin is a potent inhibitor in vitro of ester sulfate; cataphoric mobility is of the fumarase,57 lysozyme,115 and tissue ribonu­ order of 17 to 19 x 10-5 cm.2 V-I sec-1 at cleases and desoxyribonucleases.128, 178, 207 pH 2 to 8; heparin apparently has the Significant inhibition of acid and alkaline strongest electric charge of any organic ribonucleases of hepatic origin has been compound normally found in the human obtained one hour after heparin injection body.219 in vivo in white mice.129 Heparin, but not Macroglobulins isolated from patients hyaluronic acid or polyglutamic acid, in­ with macroglobulinemia are precipitable hibits adenylic deaminase in vitro; no by heparin but not by chondroitin sul­ inhibition of phosphoenolpyruvic kinase, fate. 149 Heparin and heparinoids precipitate hexokinase, phosphoglyceric mutase, or fibrinogen, particularly at low tempera­ enolase has been observed!' Heparin is a tures.9. 174. 194. 204 A stoichiometric relation­ nonspecific and apparently competitive ship exists between heparin and fibrino­ inhibitor of hyaluronidases of diverse gen65 during the process of precipitation, origins.77,95 Both inhibitor and substrate as it does for other heparin-protein com­ compete for the same energy sites; an in­ plexes.56 Beta liproproteins are also pre­ crease in ionic strength decreases the cipitated by heparin.25 inhibitory effect of heparin and chondroitin Heparin, along with other anionic poly­ sulfate B.95 Serum beta-glucuronidase saccharides, acts as an ion exchanger.51 . activity is decreased 43 to 64 per cent in 182, 183, 211 The order of increasing cation human postheparin sera.14 Beta-glucuroni­ affinity, when acid mucopolysaccharides dase shows a two thousand fold greater are treated as ion exchangers, varies affinity for heparin than for phenol­ for heparin, heparin monosulfate, and phthalein .beta-glucuronide. 27 Nonspecific chondroitin sulfate.45 The order of increas­ stimulation of tyrosine-alpha ketoglutarate ing cation affinity for heparin is: sodium, transaminase activity in vivo by heparin, potassium, magnesium, strontium, barium, chondroitin sulfate, and a number of other and calcium; the differences in order agents has been observed. This stimulation Volume 7 Side effects of heparin 381 Nllmber3 is dependent upon intact adrenal glands to molecular size.32 Heparin has been or upon the presence of hydrocortisone.138 shown to dissociate the insulin complex in Heparin is thought to function in an ion­ vitro, freeing insulin from its basic pro­ exchange capacity in its complexing with tein. 76 Heparin also complexes with histamine. In vitro, heparin binds hista­ kallidin; the complex may be split with mine4, 12, 167 and also apparently inhibits compound 48/80. 217 Heparin and dextran release of histamine from cells to plasma.44 sulfate accelerate plasma kinin formation Histamine is selectively bound by heparin in vitro, as reHected by increased oxytocic in the presence of equivalent amounts of activity, pOSSibly by blocking the effect of cadaverine, serotonin, or epinephrine but an inhibitor; protamine sulfate and hexadi­ is displaced by multivalent ions in prefer­ methrine bromide but not toluidine blue ence to univalent ions,119 One mole of block kinin formation both in the presence heparin will bind 21.5 to 22.0 moles of his­ and absence of heparin but do not inter­ tamine.119,2l6 However, heparin, even in fere with uterine response to preformed high doses, has little effect on the actions kinin. G Hexadimethrine apparently inhibits 26 of histamine in vivo. , 132 kinin formation by an inhibition of Hage­ Heparin inhibits gastric secretion from man factor which normally plays a role Heidenhain pouches in dogs after stimula­ in kinin formation by activating a kinin­ tion by food or by exogenously adminis­ forming enzymeY tered histamineY' However, whether this Heparin also complexes with trypsin; the suppression of gastric secretion is secon­ heparin-trypsin complex may be disso­ dary to complexing with histamine in vivo ciated by acidification to pH 3 for 30 is not yet known. Heparin, chondroitin minutes with complete recovery of tryptic sulfate, and sodium polyhydromannuronic activity. Heparin does not inhibit chymo­ acid sulfate, but not hyaluronic acid, in­ trypsin.94 Although heparin completely pro­ hibit pepsin in vitro. Chondroitin sulfate, tects rats against LD99 doses of trypsin which markedly reduces the number of intravenously, this effect may be related gastric ulcers in Shay rats, has been shown to the anticoagulant action of heparin, to inhibit pepsin in vivo as well.135 blocking the coagulant properties of The nonspecific complexing of heparin trypsin. 43 with cationic macromolecules has been used In the past few years, the relationship experimentally to protect mice