The Antiangiogenic Agent Neovastat (Æ-941) Stimulates Tissue Plasminogen Activator Activity

Abstract

The plasminogen activator/plasmin system represents a key component of the proteolytic machinery underlying angiogenesis. In this work, we investigated the effect of Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent that is currently in Phase III clinical trials, on tissue and urokinase plasminogen activator activities. We found that in vitro, Neovastat at 100 microg/ml markedly stimulates t-PA-mediated plasmin generation, while it slightly inhibits the generation of plasmin mediated by uPA. The stimulatory effect of Neovastat on t-PA activity was markedly increased by a heat treatment, resulting in a 15-fold increase in the rate of activation of plasminogen. Neovastat did not directly stimulate the activity of t-PA or plasmin towards exogenous substrates, suggesting that its effect requires the presence of plasminogen. Accordingly, kinetic analysis showed that Neovastat increases both the k(cat) of t-PA as well as its affinity for plasminogen by 10-fold. The stimulation of t-PA activity by Neovastat was also correlated with a direct interaction of Neovastat with plasminogen as monitored by the surface plasmon resonance technology. Overall, these results identify Neovastat as a potent stimulator of t-PA-dependent activation of plasminogen, further emphasizing its pleiotropic mechanism of action on several molecular events involved in angiogenesis.

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“…Paradoxically, shark cartilage extract (including AE-941) also has fibrinolytic activity. 145,146 Nevertheless, fibrinolysis and anticoagulation may also reduce tumor cell metastasis. 147,148 Shark cartilage extracts are pleiotropic, having multiple phenotypic activities.…”

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“…These findings suggest that an increase in either PLAT activity or expression levels is beneficial, possibly due to the overstimulation of plasmin generation by PLAT that induces the degradation of the pro-angiogenic fibrin matrix, resulting in the inhibition of angiogenesis (71,72). Moreover, Gingras et al (72,73) reported that Neovastat, an inhibitor of angiogenesis derived from marine cartilage, specifically stimulates PLAT-dependent plasmin generation through an increase in the affinity of the enzyme towards plasminogen.…”

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“…Neovastat inhibits CAM vascularization and Matrigel-induced angiogenesis [88]; it competitively suppresses the VEGF-dependent phosphorylation of VEGFR, the proliferation of endothelial cells, and VEGF-induced vascular permeability in mice [89]. Neovastat could also inhibit MMP-2 [90] but stimulate tissue plasminogen activator (tPA) [91] and activate caspase-mediated apoptotic pathways in endothelial cells [92]. In addition, neovastat has significant antitumor and antimetastatic activity [93].…”