Thrombin-induced release of active basic fibroblast growth factor-heparan sulfate complexes from subendothelial extracellular matrix.
The angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM-bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti-bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.
Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel.
M
M
Benezra
I
I
Vlodavsky
R
R
Ishai-Michaeli
G
G
Neufeld
R
R
Bar-Shavit
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D002417:Cattle; D002455:Cell Division; D003315:Cornea; D004727:Endothelium / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D019763:Heparin Lyase; D006497:Heparan Sulfate / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D000990:Antithrombin III; D016222:Fibroblast Growth Factor 2; D019763:Heparin Lyase; D006497:Heparan Sulfate; D006629:Hirudins; D011133:Polysaccharide-Lyases; D013917:Thrombin