A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools.

Intimatan (dermatan 4,6-O-disulfate) prevents rethrombosis after successful thrombolysis in the canine model of deep vessel wall injury.

INTRODUCTION: Intimatan (dermatan 4,6-O-disulfate), a heparin cofactor II (HCII) agonist, inhibits both the fluid phase and thrombus bound thrombin. The efficacy of Intimatan as an adjunctive anticoagulant during thrombolysis was evaluated in the canine model of arterial injury. MATERIALS AND METHODS: After forming an occlusive thrombus in the right carotid artery (RCA), twenty-one dogs were administered recombinant tissue plasminogen activator (rt-PA) intra-arterially to achieve thrombolysis in the presence of either 0.9% NaCl or Intimatan (9 mg/kg bolus+300 mug/kg/min i.v. infusion). Next, the left carotid arteries (LCA) of the same animals were injured in the presence of either Intimatan or 0.9% NaCl. RESULTS: The incidence of RCA rethrombosis between the Intimatan and control groups was 2/9 and 8/12, respectively. The quality of RCA blood flow, i.e., patency score (Scale of 0-3, i.e., no flow to high flow, respectively), was 2.3+/-0.4 (Intimatan) versus 0.9+/-0.4 (0.9% NaCl). The incidence of primary thrombosis was determined among the groups as 0/9 (Intimatan) versus 7/12 (0.9% NaCl); the patency score was 2.8+/-0.1 (Intimatan) versus 0.9+/-0.4 (0.9% NaCl). Intimatan resulted in a >90% ex vivo inhibition of gamma-thrombin-induced platelet aggregation whereas 0.9% NaCl had no inhibitory effect. Clot-bound thrombin activity was reduced significantly by Intimatan. Intimatan induced <2-fold change in aPTT and bleeding time (BT) when corrected for the 0.9% NaCl group. CONCLUSIONS: Intimatan significantly reduces the incidence of both primary and secondary arterial thrombosis while maintaining a high-grade vessel patency score with only moderate increases in BT and aPTT.

Effects of antithrombin and heparin cofactor II levels on anticoagulation with Intimatan.

Heparin is the current mainstay drug for anticoagulation during cardiac surgery, but it requires normal levels of antithrombin (AT) for optimal anticoagulation. Heparin anticoagulation may be less effective in cardiac surgical patients because of decreased AT levels due to the prolonged heparin therapy. Therefore, other anticoagulants that would work well in AT deficient patients may be more desirable. One such agent currently being evaluated is Intimatan, which catalyzes heparin cofactor II (HCII) dependent inhibition of thrombin. In the current in vitro study we examined the effects of Intimatan (20 microg/ml), heparin (0.25 U/ml), or both drugs in combination on thrombin generation in plasma with decreasing levels of AT, HCII or both cofactors, using a novel method based on the continuous measurement of thrombin generation. For the study, we collected blood samples from healthy volunteers, isolated platelet poor plasma by centrifugation and mixed it with AT, HCII, or AT-HCII deficient plasma samples to achieve different levels of AT, HCII and AT-HCII. Thrombin generation was inhibited equally well with heparin or Intimatan when the level of their respective cofactors was within the normal range. With decreasing levels of AT or HCII, heparin and Intimatan became less effective in thrombin inhibition, respectively. With the absence of both cofactors, neither agent alone or in combination had any effect on thrombin generation. We conclude that Intimatan may be an effective adjunct to heparin therapy under low AT conditions.

Selective and sustained inhibition of surface-bound thrombin activity by intimatan/heparin cofactor II and its relevance to assessing systemic anticoagulation in vivo, ex vivo and in vitro.

We compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001. We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.

Anticoagulant and antithrombin effects of intimatan, a heparin cofactor II agonist.

Surface-bound thrombin, which is resistant to inhibition by heparin/antithrombin III (/AT), plays a key role in vessel wall disease. In contrast, surface-bound thrombin is not resistant to inhibition by heparin cofactor II (HCII) and its acceleration of its inhibitory effect by dermatan sulfate. However, the potential use of dermatan sulfate to prevent thrombus formation in vivo is limited by its low specific activity, which in turn, necessitates excessively high doses when given on a gravimetric basis. Recently, a novel HCII agonist, Intimatan, has been synthesized by site-specific sulphation of highly purified dermatan sulfate comprising primarily of L-iduronic acid-4-O-sulphated N-acetyl-D-galactosamine, yielding a 4, 6-O-disulphate compound on the galactopyranose ring with a lower molecular weight, higher solubility, and specific activity than its parent, dermatan sulfate. In this study, we compared the abilities of Intimatan with its parent compound, dermatan sulfate, and with heparin to affect coagulation and to inhibit surface-bound thrombin both in vitro and in vivo, to determine if Intimatan demonstrates a better potential than either other compound in preventing thrombus formation in vivo. Intimatan prolonged the activated partial thromboplastin time (APTT) more effectively than either dermatan sulfate or heparin at comparable antithrombin concentrations. This activity was attributed to the more selective action of Intimatan against surface-bound thrombin in vitro. Intimatan also inhibited thrombin bound to an injured vessel wall surface in vivo more effectively than heparin, i.e., when measured in injured carotid arteries of rabbits injected with Intimatan or with heparin at the time of injury. We conclude that Intimatan effectively inhibits surface-bound thrombin, thereby exhibiting better anticoagulant and antithrombin properties than heparin and dermatan sulfate.