Antiangiogenic Forms of Antithrombin Specifically Bind to the Anticoagulant Heparin Sequence

The serpin, antithrombin, has been found to possess potent antiangiogenic properties in addition to being an important physiological anticoagulant. The anticoagulant native form of antithrombin has an intact surface-exposed loop, the reactive center loop (RCL)1, containing a reactive site that is recognized by the target proteases. In contrast, the antiangiogenic activities are exerted by conformationally changed forms of the protein, i.e. cleaved, latent and prelatent antithrombin (1-5). Cleaved antithrombin is formed by proteolytic cleavage in the RCL, which leads to the insertion of the N-terminal part of this loop as a new strand in the center of a large central β-sheet, called β-sheet A, of the inhibitor. Latent antithrombin is formed by mild heat treatment, which gives a conformation similar to cleaved antithrombin, although the RCL is intact. The latent and cleaved forms have lost their ability to inhibit proteases (6, 7). Prelatent antithrombin is formed as an intermediate in the conversion from native to latent antithrombin in the presence of stabilizing ions and possesses both anticoagulant and antiangiogenic properties (3, 8). The antiangiogenic forms of antithrombin have been found to inhibit angiogenesis in several in vivo models and to inhibit tumor growth in several mouse models (1-3, 9). Mechanisms of the antiangiogenic actions include inhibition of FGF-2 and VEGF-induced proliferation of endothelial cells (2, 9). Moreover, latent and cleaved antithrombin down-regulate several proangiogenic genes and upregulate several antiangiogenic genes (10), suggesting that these antithrombin forms have direct signaling functions. The interaction of heparin or heparan sulfate with native antithrombin plays an essential role in activating the inhibitory potential of antithrombin, by increasing the rate of attack of the inhibitor on its target proteases, partially due to a bridging mechanism and partially due to an allosteric mechanism (11). In the bridging mechanism, protease-inhibitor complex formation is enhanced through the binding to the same heparin chain. The allosteric mechanism is achieved by a global conformational change, induced by the binding of a specific pentasaccharide sequence (12), and is accompanied by a 40% enhancement in tryptophan fluorescence (13). Heparin fractions that comprise this pentasaccharide sequence are denoted high-affinity heparin (HAH) and those that lack it are called low-affinity heparin (LAH). It has recently been shown that the heparin-binding site is required for the antiangiogenic activities of latent and cleaved antithrombin, suggesting that heparin/heparan sulfate is involved in mediating these activities (9). An essential question is therefore whether the antiangiogenic antithrombin forms have different heparin sequence specificities than native antithrombin. In this work, the interactions of HAH, LAH and two synthetic pentasaccharides with cleaved, latent and native antithrombin were studied by fluorescence measurements and by an affinity matrix method. The heparin preference of latent and cleaved antithrombin and the mechanism of heparin interactions with these antithrombin forms were characterized and compared with those of native antithrombin. Latent and cleaved antithrombin were found to have specificity for the same pentasaccharide sequence as native antithrombin, although with approximately 30-fold lower affinities. Furthermore, the interaction with longer heparin chains differed, because latent and cleaved antithrombin repelled heparin in what is in native antithrombin denoted the extended heparin binding site. This information may be useful in defining the type of heparin or heparan sulfate that is used by the antiangiogenic antithrombin forms to mediate antiangiogenic activity. Moreover, since treatments with various heparin forms have been found to reduce the mortality rate of cancer (14), information on the sequences that interact with the different antithrombin forms may be of importance for achieving the optimal desired effects on angiogenesis as well as coagulation during such treatments.