Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.

SummaryMDL 28,050 is a decapeptide antithrombin agent that inhibits a-thrombin-induced fibrin clot formation by binding to a non-catalytic site on α-thromhin. It is the result of chemical and structural optimization of a functional domain of the leech anticoagulant, hirudin. In contrast to the contention that the polyanionic nature of this C-terminal functional domain governs its interaction with α-thrombin, systematic study of this region has shown the importance of the lipophilic residues for providing the functionality necessary foi potent binding to a-thrombin. The development of MDL 28,050 and other effective antithrombin agents are outlined through the description of the structure-activity relationships (SAR) for these peptides. These peptides are effective in a variety of in vitro and in vivo models of thrombosis.

Probucol (1) and probucol analogues with the substitutions at the disulfide-linked carbon (2, 3) and an additional substitution at a tert-butyl of each phenolic ring (4) were tested for their ability to lower total serum cholesterol and prevent aortic atherosclerosis in modified Watanabe heritable hyperlipidemic (WHHL) rabbits and to inhibit Cu2(+)-induced lipid peroxidation of isolated plasma low-density lipoproteins (LDL). After 84 days of feeding 1% of each compound in rabbit chow, probucol was effective in lowering serum cholesterol, whereas 2-4 were not. The concentration of drug in serum and LDL was 2 greater than 1 greater than 3 greater than 4. Probucol and analogues prevented Cu2(+)-induced oxidation of LDL in vitro to an extent that directly related to their concentrations in LDL. The decrease in lipid oxidation was directly correlated with the inhibition of both oxidized-LDL-induced cholesteryl ester synthesis in cultured macrophages and to the inhibition of aortic atherosclerosis in vivo. These results show that the antioxidant activity of probucol and analogues is directly related to their concentration in LDL, which may explain their pharmacological activity in reducing atherosclerosis.

Low denfity lipoprot¢ins (LDL) oxidati~ly modified by maeropha~s have been shown to tm atherogeni¢ in ~ vivo studio, W= studied the potential role of nitric oxide (NO), a free r-adi~l produt:ed by maerophag=;, in LDL modifi~tion, Human LDL (I mg/ml) were incubated with mouse Ixritom:al ma¢rophag~ in Ham's F-IO medium. The ¢¢1ls w=r¢ then stimulat~ by interferon.y and tumor n¢¢rosi= fa=or-¢ to inerea~ their production of NO from t.3 to 12.2 tam in 24 h. us mcatsured by nitrite. Lipid l~roxidation of LDL, as m~sured by thio~rbitudc acid.tractive materials (";'BARS). was reduexd in stimulated c¢{l~ in a time.dependent manner. At 24 h, the d=rcar¢ ~s about 2"/%. In tl;¢ preu:n¢¢ of an NO syntha~ inhibitor (N".aminophomoarginin¢). the Ilcncration of NO was diminished and tim proration a~init LDL lipid pcroaidation wa~ reverted, The extent of LDL protein modifiemtion was al~ assessed by examining its electropho~tic mobility. It wall found that macrophage NO reduced the change in LDL ¢lectromobility, These data ladS=ate that the production of NO may inlfibit the oxidative modification of LDL with cytokine,stimulatcd macrophag¢*, We suggest that NO plays a protective role in limiting macrophage.indutu=d LDL modifl=ation, [15,16], In the presence of an antioxidant, such as probucol, the formation of foam ~lls is inhibited both in vitro and in vivo [17][18][19][20][21]. However, the sources and species of those free radicals responsible for the oxidation of LDL are not well established, The superoxid¢ anion (Of) produced by macrophages is known to be involve, sin~ the pretreatment of macrophages with superoxid¢ dismuta~e (SOD) attenuates LDL lipid peroxidation [22]. Macrophages have been shown to produce the nitric oxide (NO) radical via nitric oxide synthase. This enzyme converts arginine into citrulline and is indued by cytokin, treatment [23,24], Although NO can neutrali~ Oi-to form a stable peroxynitrit¢ anion (ONOO') [25], under some conditions th¢ peroxynitrit¢ can also rapidly decompose to form a strong oxidant with reactivity similar to the hydroxyl radical In addition. NO generation in vivo may lead to the mobilization of iron with a subsequent increase in the level of reactive oxygen slzt¢ics [2?]. These observation= could ¢~plain the cytotoxi¢ action of NO that has been demonstrated in a numL~r of studies [28,29]. The purpose of the present study was to dctermin, the ,ff~t of NO production by macrophagcs on LDL lipid [mroxi. datioa. To this end we incubated LDL with macrophages in which the synthesis of NO was induced with interf¢ron-7 and tumor necrosis factor-~, and followed the extent of oxidative modification of LDL. ,i, Nitric oxide .¢k.tztha~'e inhibitor (MDL lO0.24tt) $-Mcthylisothio~emi~ar "b~tde hydroio,Jid¢ (17,8 8. 0.0?fi tool) was added to L-ly£n¢ monohydrate (9,3 $, 0,076 tool) in water (t00 ml) and adjusted to pH 9.5 with I N NaOH, The r~gtion mi~tu~ wa= h~t~ and stirred at 40"C for 30 h. The solution was then cooled to room teml~rature and acidified to pH 4.0 with acetic acid. F~vianic acid (...

No abstract