EFFECT OF PLASMINOGEN-ACTIVATOR (UROKINASE), PLASMIN, AND THROMBIN ON GLYCOPROTEIN AND COLLAGENOUS COMPONENTS OF BASEMENT-MEMBRANE

Abstract

Tumor cells traverse basement membranes (BM) during the metastatic process. Penetration of the BM may involve proteolysis by enzymes directly or indirectly associated with tumor cells. This study evaluated the role of the serine proteases urokinase (plasminogen activator), plasmin and another regulatory protease, .alpha.-thrombin, in the degradation of the BM. Homogeneously pure enzyme preparations were incubated with isolated components of BM and with whole human amnion BM. The BM components consisted of acid-extracted type IV collagen, pepsin fragments of collagen type IV, laminin and fibronectin. Collagen type V (.alpha.A.alpha.B) associated with the peri-BM zone was also studied. Digestion of the BM components was performed at 25.degree. C using matched activity for the different enzymes. Urokinase failed to significantly degrade fibronectin or any of the other BM components. Plasmin and thrombin cleaved fibronectin and laminin into multiple specific fragments but did not produce a major cleavage of acid-extracted type IV collagen, pepsinized type IV collagen or .alpha.A.alpha.B (type V) collagen. .alpha.-Thrombin selectively degraded only the MW 400,000 chain of laminin; plasmin degraded both laminin chains. Digestion of laminin by the serine proteases was time and concentration dependent. A variety of normal and neoplastic cells were tested for the presence of laminin-degrading proteases. Macrophages, polymorphonuclear leukocytes and metastatic tumor cells contained a significant laminin-degrading activity. The activity was enhanced by the addition of plasminogen. Type V collagen was cleaved by thrombin and plasmin at 35.degree. C but not at temperatures below 33.degree. C. Following treatment of whole-amnion BM with any of these enzymes, EM demonstrated preservation of the lamina densa. Immunohistology studies indicated that laminin, but not type IV collagen, was removed from the whole BM by plasmin treatment. These BM components are poor substrates for plasminogen activators and plasmin alone is not sufficient to completely degrade the whole BM. Plasmin, generated through the action of plasminogen activator, may play a significant role in the degradation of noncollagenous components of the BM.