Matrix Localization of TFPI-2/MSPI Involves Arginine-Mediated Ionic Interactions with Heparin and Dermatan Sulfate: Heparin Accelerates the Activity of TFPI-2/MSPI Towards Plasmin.

Human tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI),a Kunitz-type serine protease inhibitor,inhibits plasmin,trypsin,chymotrypsin,plasma kallikrein,cathepsin G,and factor VIIa-tissue factor complex. The mature protein has a molecular mass of 32-33 kDa,but exists in vivo as two smaller,underglycosylated species of 31 and 27 kDa. TFPI-2/MSPI triplet is synthesized and secreted by a variety of cell types that include epithelial,endothelial,and mesenchymal cells. Because the majority (75-90%) of TFPI-2/MSPI is associated with the extracellular matrix (ECM),we examined which components of the ECM bind TFPI-2/MSPI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan sulfate. Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains,as evidenced by cross-competition experiments. However,binding affinity for TFPI-2/MSPI with heparin was 250-300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Binding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by glucose,mannose,galactose,6-aminohexanoic acid,or urea,suggesting that arginine-mediated ionic interactions participate in the GAG binding of TFPI-2/MSPI. This supposition was supported by the observation that only NaCl or arginine could elute the TFPI-2/MSPI protein triplet from an ECM derived from human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin,suggesting that proper disulfide pairings and conformation are essential for matrix binding. To determine whether heparin modulates the activity of TFPI-2/MSPI,we determined the rate of inhibition of plasmin by the inhibitor with and without heparin and found that TFPI-2/MSPI is more active in the presence of heparin. Collectively,our results demonstrate that conformation-dependent arginine-mediated ionic interactions are responsible for the TFPI-2/MSPI triplet binding to fibroblast ECM,heparin,and dermatan sulfate and that heparin augmented the rate of inhibition of plasmin by TFPI-2/MSPI.