Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth. (J. Clin. Invest. 1995.

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that cytosolic phospholipase A2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase.

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinasetype plasminogen activator [scuPA(32kDa)J and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either 13-globin or mouse immunoglobulin. This modification resulted in a >100-fold improvement in the level of protein expression.The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis.Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.Acute thrombotic occlusion of a major epicardial coronary artery results in myocardial infarction, the single most common cause of death in industrialized societies. The use of thrombolytic therapy in patients with acute myocardial infarction has resulted in a significant reduction in mortality (1-3). At its present stage of development, however, thrombolytic therapy is limited by (i) significant bleeding at high doses (intracranial hemorrhage causes stroke or death in 0.1-0.5% of patients who receive plasminogen activators)(1-4), (ii) the failure to restore blood flow in 20%o of patients or the fact that thrombotic reocclusion after cessation of therapy occurs in 15-25% of patients (5), and (iii) the lag between initiation of therapy and clot lysis, which averages about 60 min (5).These limitations have prompted generation (by recombinant DNA technology) of hundreds of plasminogen activator mutants, which have produced, at best, only modest improvements in thrombolytic efficacy. Most investigators have either rearranged (or duplicated or deleted) various plasminogen activator functional domains or altered their posttranslational modification (6-10). We and others (11-16) have pursued an alternative strategy, the generation ofchemical conjugates or recombinant molecules with domains for both plasminogen activator activity and high-affinity fibrin binding [conferred by a monoclonal antibody (17) that binds to fibrin, the principal component of a thrombus, but not fibrinogen, its circulating precursor]. Here we report the generation and characterization (in vitro and in vivo) of a recombinant molecule that is fibrin selective by two different mechanisms. It combines a high-affinity anti-fibrin antibody, 59D8, with a low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)], a fibrin-selective plasminogen activato...

Acanthamoeba profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1 .4 x 104 M-'cm -'. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis . These antibodies react with a 11,700 M r polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria . Antibody-binding assays indicate that^-2% of the ameba protein is profilin and that the concentration of profilin is^-100 pmol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions : an unbound fraction previously identified by Reichstein and Korn ( 1979, / . Biol . Chem ., 254 :6174-6179) and a tightly bound fraction . Purified profilin from the two fractions is identical by all criteria tested . The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCI, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCI with 2 MgCl2 . By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C .-H ., and T. D. Pollard, 1982, J. Cell Biol . 94 :213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions . Profilin is a small protein, first isolated from mammalian lymphoid tissue (2), that has attracted considerable attention as a possible regulator of actin polymerization in nonmuscle cells. An initial study (2) suggested that mammalian profilin binds tightly to actin, completely blocking actin polymerization: spleen and thymus extracts yielded a 1:1 complex of profilin with actin that would not polymerize except under harsh conditions ; and urea was required to dissociate the complex . Later, Blikstad et al. (1) found that a 2:1 molar excess of mammalian brain profilin completely prevented actin polymerization in 2 mM CaC12, 10 mM NaCl, but Grumet and Lin (6) found that mammalian profilin purified by another method inhibited, but did not prevent, the polymerization of muscle actin in MgC12. In recent experiments, Markey et al. (10) were able to polymerize the mammalian actin-profilin complex in MgCl, . Several different types of nuclei stimulated polymerization .Slightly smaller profilins with molecular weights of about 12,000 have been isolated from Acanthamoeba (16) and Physarum (13) without urea and found to inhibit the initial rate, but not the extent, of muscle actin polymerization in 2 mM MgC12 even at two-to threefold molar excess. More detailed studies by Tseng and Pollard (23) and Tobacman and Ko...

BACKGROUND Although the indirect thrombin inhibitor heparin and the more potent direct inhibitor hirudin are useful in preventing thrombosis, a substantial opportunity remains for improving the thrombus selectivity of thrombin inhibitors. METHODS AND RESULTS To explore the effect of targeting an antithrombin to the surface of a clot, we covalently linked recombinant hirudin to the Fab' (or IgG) of a monoclonal antibody (59D8) that selectively binds to an epitope on fibrin that becomes exposed only after thrombin cleaves fibrinopeptide B. Antibody-coupled hirudin bound to an immobilized peptide of the fibrin beta-chain amino-terminal sequence and inhibited the peptidolytic activity of thrombin more efficiently than free hirudin. Thrombin inhibition dependent on binding to immobilized fibrin monomer was enhanced 1100-fold (P < .0001). Hirudin-59D8 Fab' was 10 times more effective than hirudin in inhibiting fibrin deposition on experimental clot surfaces in fibrinogen solution (P < .0001) and human plasma (P < .0001). The more effective inhibition of thrombin by the conjugate was supported by significantly diminished concentrations of fibrinopeptide A in the plasma supernatant of the clot (P = .0001). Inhibition of clotting by an uncoupled mixture of hirudin and 59D8 Fab' was indistinguishable from that by hirudin alone, indicating that the conjugate's greater inhibitory activity was due to the covalent linkage between antibody and hirudin. CONCLUSIONS Fibrin-targeted hirudin (in comparison with unmodified hirudin) significantly reduces fibrin deposition on the surface of experimental clots.