Mark T. Yates

Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech.

Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.

Antioxidant activity and serum levels of probucol and probucal metabolites.

Publisher Summary Probucol [bis(3,5-di- tert -butyl-4-hydroxyphenylthio)propane] is a marketed cholesterol-lowering agent that has been shown to be a potent antioxidant. The antioxidant activity of probucol inhibits the formation of oxidatively modified low density lipoproteins (LDL) and reduces atherosclerosis in animals. The uniqueness of probucol is its association with LDL; about 70% of the plasma probucol resides in LDL particles. It has been hypothesized that such association directly protects LDL from oxidation inside the arterial wall. Similar to all chain-breaking antioxidants, probucol is oxidized, consumed, and depleted during oxidation. This chapter describes the in vivo metabolic pathway of probucol as it undergoes oxidation. Three major metabolites, spiroquinone, diphenoquinone, and bisphenol are discussed. Bisphenol is also an antioxidant, being oxidized to diphenoquinone when acting as such. Therefore, the antioxidant activity is continuously regenerated in vivo. The chapter describes the assay for the antioxidant activity of probucol in LDL and whole serum and the analytical procedures for the determination of probucol and probucol metabolites in serum. Determination of these metabolites in serum may provide insight as to the mode of action of probucol in inhibition of atherosclerosis.

Diet and HDL Metabolism: High Carbohydrate vs. High Fat Diets

An increased level of plasma low density lipoproteins (LDL) is a major risk factor in the development of coronary artery disease (1). Conversely, plasma high density lipoproteins (HDL) and apolipoprotein A-I, the major protein constituent of HDL, are inversely related to the disease (2-4). In an attempt to reduce the incidence of heart disease, a number of organizations have suggested that dietary fat intake be reduced to 30% of calories (5). Since the protein intake of the U.S. population is approximately 20%, the resultant diet would result in an increase in dietary carbohydrate (CARB). Although high CARB diets reduce LDL-cholesterol, they also produce fasting hypertriglyceridemia, hyperinsulinemia (6) and decreased levels of HDL-cholesterol (6-8), all of which may be associated with increased risk of heart disease. Lipoprotein lipase (LpL) and hepatic triglyceride lipase are key enzymes in lipoprotein metabolism (9). LpL catalyzes the hydrolysis of chylomicron triacylglycerols and very low density lipoproteins (VLDL). The lipoprotein substrates for H-TGL are less well defined, although most evidence suggests that the enzyme plays a role in the catabolism of remnant lipoproteins (intermediate density lipoproteins) and in the degradation of HDL, particularly the HDL subfraction corresponding to HDL2.The purpose of the present study was to determine whether the effect of a high CARB diet on plasma lipids, and lipoproteins is correlated with changes in the activity of LpL and H-TGL. The results show that the dietary CARB-induced changes in HDL-cholesterol and HDL2, is significantly correlated to changes in LpL activity whereas H-TGL activity does not vary on a high CARB diet.

Design, synthesis and antithrombin activity for conformationally restricted analogs of peptide anticoagulants based on the C-terminal region of the leech peptide, hirudin

Synthetic peptides cyclized via disulfide linkages have been synthesized as conformationally restricted analogs of a novel class of antithrombotic peptides that inhibit fibrinogen cleavage by binding to a non-enzymatic site on thrombin. Several conformational models for these inhibitors have been considered and cyclic analogs were synthesized to test their validity. Compounds designed on an alpha-helical model yielded several cyclic analogs that retained antithrombin activity. [D-Cys58, Cys61]-hirudin54-65, 5, and [D-Cys60, Cys63]-hirudin54-65, 6, had IC50 values of 26 and 30 microM, respectively, in an in vitro clot assay compared with a value of 3.7 microM for the linear hirudin54-65.

Comparison of hirudin and hirudin PA C-terminal fragments and related analogs as antithrombin agents

Hirudin PA54-66 and related hirudin fragment analogs were synthesized and assessed for their inhibition of thrombin-induced fibrin-clot formation in plasma. Pro58 and Ala63-Tyr64 modifications in the hirudin sequence resulted in increased antithrombin potency, whereas Asp62, Ala63 and Tyr64 individual substitutions each resulted in a loss of potency.

C-terminal peptide alcohol, acid and amide analogs of desulfato hirudin54-65 as antithrombin agents.

Analogs of the antithrombin peptide hirudin54-65 with C-terminal modifications have been synthesized in order to examine the requirements for alpha-thrombin inhibition. The C-terminal residue, Gln65, could be replaced with L-amino acids or amino alcohols with neutral or charged hydrophilic side chains without greatly affecting the peptides antithrombin potency as determined by inhibition of thrombin-induced clot formation in human plasma in vitro. Derivatives with D- or L-amino carboxamides at position 65 had significantly reduced potency, but still retained activity. Deletion of residue 65 with conversion of residue 64 to the amide or alcohol derivative resulted in a three-fold loss of potency. In addition to these results the solid-phase synthesis of peptide alcohols via direct displacement of p-nitrobenzhydrylideneisonitroso resin attached peptides with the desired C-terminal amino alcohol is reported.

The C-terminal binding domain of hirullin P18: Antithrombin activity and comparison to hirudin peptides

Hirullin P18 is a 61‐amino acid hirudin‐related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C‐terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C‐terminal fragment acetyl‐hirullin P1840–61 possesses an antithrombin potency similar to that of acetyl‐desulfatohirudin45–65. Additionally, like the hirudin fragment analog, it inhibits fibrin‐clot formation by binding to a non‐catalytic site on thrombin. Sequential shortening of the hirullin P18 C‐terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P1854–61 and hirudin59–65 have substantial differences, the C‐terminal functional domain represented by hirullin P1850–61 appears to be comparable to hirudin55–65 in terms of its functional role in antithrombin activity.

Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site

Hirudin is a 65 amino acid anticoagulant peptide produced in the leech. The single polypeptide is cross-linked by three disulfide linkages in the NH2 terminal half of the molecule. A peptide corresponding to the COOH terminus (residues 45-65) was synthesized utilizing lysine 47 as a specific residue to conjugate to thyroglobulin as a carrier for raising antibodies in mice. Using an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the major antigenic domain(s) was located between residues 52-65. The COOH terminal residues Ile-59, Tyr-63, and Leu-64 are crucial for maintaining the antigenic structure. The NH2 terminal region (residues 45-52) that is proximal to the carrier protein, however, was not immunoreactive. A possible mechanism by which antibodies recognize the COOH terminal region of the synthetic peptide and the strategy for raising such antibodies are discussed.

Total synthesis of human plasma apolipoprotein C-II

Abstract Apolipoprotein C-II (apoC-II), a protein of 79 amino acid residues present in very low density lipoproteins, enhances the lipoprotein lipase (LpL)-catalyzed hydrolysis of triacylglycerols transported in plasma triglyceride-rich lipoproteins. To elucidate the structure-activity relationship of this activator protein, the complete amino acid sequence of apoC-II has been synthesized by the solid-phase method with Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 10% yield by a combination of ion-exchange and preparative high-performance liquid chromatography (HPLC). The purified peptide had the expected amino-terminal sequence and amino acid composition. Synthetic and native apoC-II were indistinguishable by cochromatography on analytical HPLC, peptide mapping of tryptic digest, radioimmunoassay, and activation of LpL with both artificial and lipoprotein substrates.

Diet and High-Density Lipoprotein Metabolism

High levels of plasma low-density lipoprotein (LDL) cholesterol constitute a major risk factor for coronary heart disease. Conversely, plasma high-density lipoprotein (HDL) cholesterol and apolipoproteins A-I and A-II, the major protein constituents of HDL, are inversely related to the disease. One approach to lowering LDL and increasing HDL-cholesterol is by dietary means. The recommended diet is low in saturated fat, total fat, and cholesterol. Since the protein content of most diets is relatively constant, a low-fat diet would result in an increase in dietary carbohydrate. The present studies were designed to determine the effect of dietary fatty acids and carbohydrate on lipoprotein metabolism. All studies were performed with normal subjects under metabolic ward conditions with solid-food diets.