Background: Stabilized fibrin can contribute to metastasis by protecting circulating tumor cells and by enhancing their sustained adherence to blood vessel walls for extravasation out of vessels increasing the likelihood of metastasis. Alpha 2-antiplasmin (α2-AP) inhibits fibrinolysis by plasmin and may promote metastasis by stabilizing fibrin. Alpha 2-AP can be cleaved by fibroblast activation protein-α (FAP) and this promotes faster incorporation of α2-AP into fibrin and inhibits dissolution of fibrin by plasmin. FAP expression by breast cancer cells can increase levels of matrix metalloproteinases (MMPs), which can also mediate fibrinolysis. I hypothesize that FAP will enhance α2-AP inhibition of fibrinolysis and increase the protective effects of fibrin for tumor cells. Methods: A fibrin degradation assay that measures the D-dimer was used to determine if FAP increased inhibition by α2-AP of plasmin-mediated fibrinolysis and the effects of α2-AP on plasmin-independent fibrinolysis. Proteases responsible for plasmin-independent fibrinolysis were identified by using protease inhibitors, flow cytometry, and zymography. Co-immune precipitations of α2-AP with MMP-9 and MT1-MMP were used to determine the affinity binding interaction α2-AP to either proteinase. To determine if α2-AP could directly inhibit MMPs that cause fibrinolysis an activity assay was performed using purified proteases and a fluorogenic substrate for MMPs. Results: FAP expression did not enhance α2-AP inhibition of plasmin-dependent fibrinolysis in these experiments. However, high FAP expression correlated with increased plasmin-independent fibrinolysis. Interestingly, α2-AP protected fibrin from the plasmin-independent degradation. Marked inhibition of the plasmin-independent fibrin proteolysis by inhibitors of MMPs suggested it was mediated by MMPs. Further investigation revealed that these cells express high levels of MMP-9 and MT1-MMP. Co-immune precipitation assays of α2-AP with MMP-9 and MT1-MMP showed that α2-AP binds to MT1-MMP but not the MMP-9. Moreover, á2-AP significantly inhibited the proteolytic activity of purified MT1-MMP but not purified MMP-9. Conclusions: FAP on breast cancer cells does not enhance α2-AP inhibition of plasmin under the conditions used in this study. However, α2-AP can protect fibrin from degradation by MMPs through specific inhibition of MMPs such as MT1-MMP. Alpha 2-AP inhibition of MMPs and plasmin fibrinolysis may contribute to the fibrin preservation around cancer cells and create an environment conducive for invasion and metastasis of breast cancer.
|Commitee:||Haun, Randy, Nakagawa, Mayumi, Suva, Larry, Ware, Jerry|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Biology, Cellular biology, Oncology|
|Keywords:||Alpha-2 antiplasmin, Breast cancer, Fibrin, Fibrinolysis, Metatasis, Mt1-mmp, Plasmin-independent fibrinolysis|
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