Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase

The degradation of extracellular matrix (ECM) adhesive glycoproteins, fibro nectin (FN), thrombospondin (TSP) and von Willebrand factor (vWF): by human leukocyte cathepsin G and elastase, and by plasmin or thrombin, was analys ed by immunoblotting after incubation of physiologic doses of the proteases with confluent human umbilical vein endothelial cells. Elastase induced an almost complete disappearance of intact FN, TSP, and vWF from the ECM at 0 .02 units/ml within 5 minutes of incubation at 37 degrees C. Plasmin (0.2 u nits/ml) was also active on all three substrates, whereas cathepsin G (0.2 units/ml) had a preferential effect on TSP. Most remarkably, these degradat ions occurred with no apparent change in endothelial cell morphology, as sh own by phase-contrast microscopy. In contrast, thrombin (0.2 units/ml) had no apparent proteolytic action on ECM glycoproteins, where it induced cell retraction and rounding, The release of adhesive glycoproteins from the ECM was accompanied by the detection of proteolytic fragments in the condition ed medium. Kinetic studies indicated that proteolysis started within minute s and proceeded for at least 1 hour. TSP was extremely sensitive to degrada tion by all enzymes except thrombin, whereas VWF released from the ECM was more resistant to proteolysis than constitutively secreted VWF, and FN was poorly degraded by plasmin. Our results indicate that serine proteinases, l ocally produced during inflammation and/or thrombolysis, can release extrac ellular matrix components and generate proteolytic fragments with potential biological activities. (C) 2000 Elsevier Science Ltd. All rights reserved.