Isidore Danishefsky

Evidence for a heparin-induced conformational change on antithrombin III☆

Abstract The effect of heparin on the conformation of antithrombin III (AT-III) was investigated. Solvent perturbation difference spectroscopy shows that the binding of heparin to AT-III results in exposure of two tyrosine residues and a partial burial of a tryptophan residue. The occurrence of a conformational change suggested by this study is also substantiated by circular dichroism (CD) findings in the aromatic and peptide regions. The data in the peptide region show that heparin produces a decrease in the β-structure of AT-III, with a compensatory increase in random coil.

Investigations on the chemistry of heparin. I. Desulfation and acetylation

Abstract Desulfation of heparin with 0.06 M anhydrous methanolic HCl resulted in the formation of a polysaccharide containing equimolar amounts of sulfur and nitrogen, wherein the amino groups are free. When this was N-acetylated and again submitted to methanolysis, an additional sulfate per tetrasaccharide was cleaved yielding a product containing nitrogen, sulfur, acetyl, and methoxy in the ratio of 2:1:2:2. The rate of hydrolysis in 1 N H2SO4 of the glycosidic linkages of this product is appreciably higher than that of heparin. This desulfated, N-acetylated derivative would, therefore, be more suitable for graded hydrolysis studies than heparin itself, since the conditions required for hydrolysis of the latter also bring about excessive destruction of the uronic acid moiety.

Synthesis of heparin-sepharoses and their binding with thrombin and antithrombin-heparin cofactor

Abstract Heparin was linked covalently to Sepharose by a reaction between a uronic acid-carboxyl group of heparin and aminohexyl-Sepharose. The characteristics of this material in binding thrombin and antithrombin-heparin cofactor, was compared with those of heparin-aminoethylbromide method (heparin-Sepharose I). Although all the preparations bind thrombin and antithrombin-heparin cofactor they differ in their binding properties and purification efficiency. Highly purified antithrombin heparin cofactor was isolated by adsorption on heparinaminohexylsepharose and desorption with 1.0 M NaCl. The greatest purification of thrombin is obtained by absorption on heparin-Sepharose I and successive elution with 0.3 M and 0.4 M NaCl. Heparin-Sepharose prepared from partially degraded heparin has a relatively low affinity for thrombin and antithrombin-heparin cofactor.

Preparation of heparin-linked Agarose and its interaction with plasma

Abstract Heparin was linked covalently to Agarose by a reaction between aminoethyl Agarose and heparin in the presence of a water-soluble carbodiimide. Treatment of plasma with this product resulted in prolongation of coagulation and partial thromboplastin times. Assays of the treated plasma revealed that it was deficient in factors IX and XI. Elution of the heparin-linked Agarose with salt solutions yielded fractions containing these factors. Similar studies with serum indicated that heparin-cofactor is also bound to the immobilized heparin.

Studies on the metabolism of heparin

Abstract Heparin-S 35 was injected into dogs, and the properties of the radioactive material excreted in the urine were studied. It was found that after administration of small doses, all the label is excreted as inorganic sulfate. On the other hand, after injection of larger amounts the urine contains an appreciable portion of the S 35 associated with heparin and other mucopolysaccharide material. It is concluded that as a result of metabolic degradation the sulfate linkages of heparin are cleaved. However, the capability of the animal to perform this hydrolysis is limited so that when excessive amounts are administered, a significant portion is excreted unchanged or only partially desulfated.

Effect of heparin modification on its activity in enhancing the inhibition of thrombin by antithrombin III

Studies were conducted to determine the effect of modifying specific functional groups of heparin on its antithrombin III-enhancing activity. The derivatives employed were heparin methyl ester, heparinylglycine and N-desulfated heparin. The carboxyl-modified derivatives increase the rate of inhibition of thrombin by antithrombin III, although not to the same extent as heparin. N-Desulfated heparin is devoid of any activity. Heparin methyl ester is more potent than heparinylglycine in activating antithrombin III, as exhibited by its immediate effect on the thrombin-fibrinogen reaction. However, heparinylglycine is the more effective of the two, in increasing the rate of thrombin deactivation by antithrombin III. The results indicate that although free carboxyl groups of heparin are not crucial for its binding to antithrombin III, they are important for the combination of the latter with thromobin. In contrast, N-sulfates are critical for the interaction of heparin with antithrombin III.

Studies Made With Radioactive Heparin in Humans

The present study was conducted in order to gain more insight into the disposition and metabolism of heparin after intravenous injection in humans. The major pharmacologic effects of heparin, i.e., anticoagulant and clearing action, are well established, and although almost nothing is known concerning its metabolism, advantage may be taken of its clinical properties. Nonetheless, an understanding of the metabolism and, ultimately, the exact mechanism of the action of heparin may show the way for additional applications or possible limitations of this substance. Since heparin is a comparatively complex molecule which can interact with various body constituents, the problem of its chemical determination is extremely difficult and in many cases unreliable. This problem is further complicated by the fact that a number of substances

Heparin derivatives prepared by modification of the uronic acid carboxyl groups

Abstract Heparin derivatives in which the carboxyl groups of the uronic acid are modified, were synthesized by mild procedures whereby the sulfate groups remained intact. It was found that esterification of the carboxyl group resulted in complete loss of anticoagulant activity. However, heparinyl glycine and heparinyl aminomethanesulfonic acid which contain free acidic groups had considerable effects in prolonging blood clotting. Other bulkier units which may modify the conformation of heparin, or products in which there are no free carboxyl groups had little anti-coagulant effects.

Tryptophan residue at the heparin binding site in antithrombin III

Abstract Chemical modifications have demonstrated that the ultraviolet difference spectrum produced when heparin interacts with antithrombin III is due primarily to changes in the tryptophan environment. This is based on the observation that this spectrum could be abolished by treatment of antithrombin III with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide but not with tetranitromethane. The tryptophan-modified antithrombin III is still capable of binding to thrombin even when it has lost 85% of heparin cofactor activity. A marked decrease in reactivity of tryptophan residues is observed when modification is carried out in the presence of heparin. Evidence is presented that tryptophan is in the heparin binding site.

Requirement of free carboxyl groups for the anticoagulant activity of heparin

The uronic acid carboxyl groups of a heparin fraction with high anticoagulant activity, were converted to the methyl ester by treatment with diazomethane. The product obtained after purification did not have the characteristic activity of heparin in accelerating the inhibition of thrombin or factor Xa, by antithrombin. Esterification also abolished the binding of heparin to antithrombin as measured by changes in the intrinsic fluorescence. It is concluded that free carboxyl groups are essential for the activity of heparin.