A Novel Mechanism of Plasmin-Induced Mitogenesis in Fibroblasts.

The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased ³H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.