The molecular details linking integrin engagement to downstream cortactin (Ctn) tyrosine phosphorylation are largely unknown. In this report, we show for the first time that Fer and Ctn are potently tyrosine phosphorylated in response to hydrogen peroxide (H 2 O 2 ) in a variety of cell types. Working with catalytically inactive fer and src/yes/fyn-deficient murine embryonic fibroblasts (fer DR/DR and syf MEF, respectively), we observed that H 2 O 2 -induced Ctn tyrosine phosphorylation is primarily dependent on Fer but not Src family kinase (SFK) activity. We also demonstrated for the first time that Fer is activated by fibronectin engagement and, in concert with SFKs, mediates Ctn tyrosine phosphorylation in integrin signaling pathways. Reactive oxygen species (ROS) scavengers or the NADPH oxidase inhibitor, diphenylene iodonium, attenuated integrininduced Fer and Ctn tyrosine phosphorylation. Taken together, these findings provide novel genetic evidence that a ROS-Fer signaling arm contributes to SFK-mediated Ctn tyrosine phosphorylation in integrin signaling. Lastly, a migration defect in fer DR/DR MEF suggests that integrin signaling through the ROS-Fer-Ctn signaling arm may be linked to mechanisms governing cell motility. These data demonstrate for the first time an oxidative link between integrin adhesion and an actin-binding protein involved in actin polymerization.
In the present study, we assessed the binding of recombinant forms of apolipoprotein(a) [r-apo(a)] to plasminogen. Apo(a)-plasminogen interactions were demonstrated to be lysine-dependent, as they were abolished by the addition of epsilon-aminocaproic acid. Binding of r-apo(a) and plasma-derived Lp(a) to Glu-plasminogen was assessed in solution using a mutant form of recombinant plasminogen [Plg(S741C)] labeled at the active site with 5'-(iodoacetamido)fluorescein. High-affinity binding of apo(a) to plasminogen was observed with the 17-kringle r-apo(a) (Kd = 20.1 +/- 3.3 nM) as well as with plasma-derived Lp(a) (Kd = 5.58 +/- 0.08 nM). Binding studies using various truncated and mutant forms of r-apo(a) demonstrated that sequences within apo(a) kringle IV types 2-9 and the strong lysine binding site (LBS) in apo(a) kringle IV type 10 are not required for high-affinity binding to plasminogen. In all cases, the binding stoichiometry for the apo(a)-plasminogen interaction was determined to be 1:1. Binding data obtained using a 17-kringle r-apo(a) derivative lacking the protease-like domain (17KDeltaP; Kd = 3158 +/- 138 nM) indicate that sequences within the protease-like domain of apo(a) mediate its interaction with LBS in plasminogen. We determined that r-apo(a) and plasminogen bind to distinct sites on plasmin-modified fibrinogen with the concentration of plasminogen binding sites exceeding the concentration of r-apo(a) sites by a factor of 10. Furthermore, r-apo(a) is capable of inhibiting the binding of plasminogen to plasmin-modified fibrinogen surfaces, an effect which we show is attributable to the formation of a solution phase apo(a)/plasminogen complex which exhibits a greatly reduced affinity for plasminogen binding sites on plasmin-modified fibrinogen. The results of this study provide new insights into the mechanism by which apo(a) and Lp(a) may inhibit fibrinolysis, thus contributing to the atherothrombotic risk associated with this lipoprotein.
Abstract-Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [ 14 C]serotonin and suspended in Tyrode's solution, to ADP and the thrombin receptor-activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 mol/L) or Lp(a) (up to 0.1 mol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 mol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 mol/L 17K r-apo(a) increased aggregation from 15Ϯ4% to 58Ϯ6%, release of [14 C]serotonin from 9Ϯ3% to 36Ϯ6%, and formation of thromboxane A 2 , measured as its stable metabolite thromboxane B 2 , from 7Ϯ1 to 29Ϯ5 ng/10 9 platelets (nϭ3; PϽ0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 mol/L. Purified Lp(a) (0.25 to 0.1 mol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 mol/L) and low density lipoprotein (0.025 to 0.1 mol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo (
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