Apoprotein(a), (apo[a]), the specific antigen of lipoprotein(a) (Lp[a]>, consists of structural domains (a serine protease unit, kringles 4 and 5) with marked homology to those of the corresponding domains in plasminogen. In this study, we have investigated the impact of this unique structural mimicry on the binding and activation of plasminogen by fibrin-bound tissue-type plasminogen activator at the plasma-fibrin interface. We found that the total amount of plasmin generated on the surface of fibrin was decreased in the presence of high concentrations of Lp (

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P1) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P1, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P1-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P1. These findings demonstrate that sustained S1P1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.

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It is well established that tissue-type plasminogen activator (t-PA) binds to the D region of fibrin(ogen) and that two distinct CNBr fragments of fibrinogen (FCB), FCB-2 and FCB-5, comprising parts of this region, stimulate plasminogen activation by t-PA. In the present work, ligandbinding studies were performed to characterize the interactions between t-PA and the corresponding fibrin regions using a well defined model of a fibrin surface and both FCB-2 and FCB-5 in liquid and solid phase. Binding isotherms showed a characteristic Langmuir adsorption saturation profile. The dissociation constants determined for the binding of t-PA to immobilized FCB-2 (& = 0.70 2 0.10 nM) and FCB-5 (& = 0.47 * 0.08 nM) were of the same order of magnitude as the Kd for fibrin binding (Kd = 1 ? 0.2 nM). The specificity of the binding was demonstrated by the ability of soluble FCB-2 and FCB-5 to inhibit t-PA binding to solid-phase fibrin (K, = 3.3 pM and 6.4 pM, respectively). The binding of t-PA to fibrin and to immobilized FCB-2 was partially inhibited by the lysine analogue 6-aminohexanoic acid (K, = 123 * 47 pM and 364 pM, respectively) but was not modified by carboxypeptidase B, thus indicating involvement of internal lysine residues. Removal of lysine residues by treatment with, successively, plasmin and carboxypeptidase B, produced only a partial inhibition of t-PA binding, thus confirming the existence of both a lysine-dependent and a lysine-independent mechanism of binding of t-PA to both fibrin and FCB-2. In contrast, the binding of t-PA to F C B J was not significantly affected by 6-aminohexanoic acid. Altogether, these data indicate that the mechanism of binding of t-PA to fibrin involves mainly a lysine-independent interaction with the D region which is contributed by sequences present in FCB-5 and FCB-2; contribution to binding by a lysine-dependent interaction was detected only in FCB-2 and is probably of minor relevance as suggested by the limited effect of 6-aminohexanoic acid.Fibrinolysis, the plasmin-induced degradation of a fibrin clot that preserves blood vessel flow, is finely regulated at several levels. Among them, fibrin itself triggers its own degradation by offering specific binding sites for tissue-type plasminogen activator (t-PA) and plasminogen on its surface. The formation of this ternary complex [l] affects the K,,, of the plasminogen activation by t-PA. In the absence of fibrin, In contrast to fibrin, fibrinogen is a soluble protein that does not stimulate plasminogen activation significantly [5]. However, plasmin-generated fibrin degradation products [6] and CNBr fragments of fibrinogen (FCB) stimulate plasminogen activation by t-PA [7]. Based on these observations, it has been suggested [8] that stimulatory site(s) buried on fibrinogen are unmasked by conformational changes induced either by thrombin, by plasmin or by CNBr cleavage of fibrinogen. Indeed, with the use of the monoclonal antibodies anti-(Aal48 -160) and anti-(y312-324), raised against amino-acid sequences derived from FCB-2 and FC...