Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of the N-acetyl-␤-D-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian dermatan sulfates have ϳ10-fold and ϳ6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.Sulfated polysaccharides constitute a complex group of macromolecules known to possess a wide range of important biological properties. These anionic polymers are widespread in nature, occurring in a great variety of organisms. Marine invertebrates are a rich source of sulfated polysaccharides with novel structures (1-15).The ascidians (Chordata-Tunicata) are marine invertebrates covered by an external supportive tissue called the tunic, surrounding a body. The tunic contains large amounts of a unique high molecular mass sulfated ␣-L-galactan (1, 2, 5-7). Recently, we reported the occurrence of dermatan sulfate-like glycosaminoglycan in the body of this invertebrate (11, 13).Mammalian dermatan sulfate is an anticoagulant due to selective inhibition of thrombin by potentiating heparin cofactor II activity (16,17). Although of lower in vitro anticoagulant potency than heparin, it has efficacy in vivo with less hemorrhagic risk (18). Thus several authors have suggested using mammalian dermatan sulfate as an alternative antithrombotic polysaccharide (18 -21).In view of the increasing interest in the anticoagulant and antithrombotic actions of dermatan sulfate, we have characterized the fine chemical structure of dermatan sulfates extracted from the ascidian body and tested this compound in coagulation assays, including activation of heparin cofactor II. In addition, there is now more interest in therapeutics prepared from nonmammalian sources, thus reducing the risk of contamination with pathogenic agents.In the present work, we report that dermatan sulfates isolated from different ascidian species have distinguishable patterns and proportions of sulfate substitution. These ascidian dermatan sulfates (together with native and oversulfated mammalian dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, are a valuable tool to trace the relationship between structure versus anticoagulant activity of this glycosaminoglycan. Dermatan sulfates with potent anticoagulant potency and high heparin cofactor II activity have been found. EXPERIMENTAL PROCEDURESMaterials-Heparan sulfate from human aorta was extracted and purified as described previously (22). Chondroitin 4-sulfate fro...

Summary.We investigated a patient with a long-standing IgGk monoclonal gammopathy who developed severe haemorrhagic complications. At IgG concentrations of ϳ50 g/l the patient had severe bleeding associated with prolongation of the thrombin time, activated partial thromboplastin time, and reptilase time. Plasmapheresis resulted in improvement in the thrombin time and resolution of bleeding. Depletion of the IgG by absorption of plasma with protein G-Sepharose in vitro resulted in normalization of the thrombin time and reptilase time. The purified IgG bound to immobilized thrombin and immunoprecipitated human a-, b-and g-thrombin but not prothrombin, other vitamin K-dependent coagulation factors, or fibrinogen. Purified IgG at concentrations >1 × 10 ¹2 g/l decreased (ϳ50%) the rate of hydrolysis of a chromogenic substrate by thrombin. Addition of purified IgG to normal pooled plasma at concentrations >1 × 10 ¹2 g/l prolonged the thrombin time and activated partial thromboplastin time, but the reptilase time was prolonged only at IgG concentrations >1 g/l. This finding suggests that at low concentrations the IgG produces a specific antithrombin effect, but at higher concentrations it also affects fibrin polymerization; the combination of these effects probably produced clinical bleeding. This is the first report of a monoclonal antithrombin antibody associated with bleeding in a patient with multiple myeloma.

We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.

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The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-terminal amino acids of the mature functional protein. A positive TATA element is identified 43 nucleotides from the start codon. Pulse-field gel electrophoresis and hybridization with various cDNA probes and direct sequence data revealed the existence of a CEase-like gene. Partial sequence analysis of this gene from a human cosmid library and human genomic DNA showed a premature stop signal in exon 10, shortly after the codon for the active-site histidine. Both the functional gene and the CEase-like gene have a polyadenylation signal in the 3'-untranslated region. Thus, the complex gene structure for this intestinally active enzyme may provide in part a potential molecular explanation for the well-known heterogeneity of the intestinal absorption of cholesterol.