Matrix localization of tissue factor pathway inhibitor-2/matrix-associatedserine protease inhibitor (TFPI-2/MSPI) involves arginine-mediated ionic interactions with heparin and dermatan sulfate: Heparin accelerates the activity of TFPI-2/MSPI toward plasmin

Human tissue factor pathway inhibitor-2 (TFPI-2)/ matrix-associated serine protease inhibitor (MSPI), a Kunitz-type serine protease inhibitor, inhibit s plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G, and facto r VIIa-tissue factor complex. The mature protein. has a molecular mass of 3 2-33 kDa, but exists in vivo as two smaller, underglycosylated species of 3 1 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 extrace llular matrix (ECM), we examined which components of the ECM bind TFPI-2/MS PI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan su lfate, Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains, as evidenced by cross-competit ion experiments. However, binding affinity for TFPI-2/MSPI with heparin was 250-300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Bin ding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by gl ucose, mannose, galactose, 6-aminohexanoic acid, or urea; suggesting that a rginine-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 f rom human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin, suggesting that proper disulfide pairings and conformation are essential fo r matrix binding. To determine whether heparin modulates the activity of TF PI-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 conformatio n-dependent arginine-mediated ionic interactions are responsible for the TF PI-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 . (C) 1999 Academic Press.