Regulation of fibrinolytic activity by localization of inhibitors to fibrin(ogen)

Fibrin plays a key role in fibrinolysis, acting not only as a substrate but also as a regulator of activity. As well as stimulating plasminogen activa tion, it controls interactions between proteases and inhibitors, both prote cting proteases from inhibition and, conversely, localizing inhibitors. The principal inhibitor of plasmin, alpha(2)-antiplasmin, is cross-linked to f ibrinogen and fibrin, inhibiting fibrinolysis. Our studies have shown that a second inhibitor, PAI-2, is also cross-linked to fibrinogen and fibrin, b y either factor XIIIa or tissue transglutaminase. These inhibitors are both members of the serpin family but the cross-linking sites are quite unrelat ed. Cross-links are formed between glutamine residues in the inhibitors and lysine residues in fibrin(ogen). The Gln residues involved are at position 2 in the N-terminus of alpha(2)-AP and at position 83 and 86 in PAI-2, loc ated in a loop between helices C and D. All cross-linking observed was to t he A alpha chain of fibrin(ogen). The two inhibitors did not compete for cr oss-linking sites. alpha(2)-AP binds only to Lys 303 of the A alpha chain a nd a 30-residue peptide based on the sequence around this Lys competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to P AI-2. PAI-2 was cross-linked to several Lys residues (but not Lys 303) in t he Aa chain, as shown by tryptic digestion and mass spectrometry. PAI-2 was cross-linked to Lys 148, 176, 183 and 467 by tissue transglutaminase and t o Lys 148, 176, 230 and 413 by factor XIIIa. The activity of PAI-2 was not affected by cross-linking, so that this is a mechanism whereby it can be co valently bound to fibrinogen and retained in a fibrin clot, without loss of activity towards u-PA and two-chain t-PA. PAI-1, the other major inhibitor of fibrinolysis, also binds to fibrin but we find no evidence for its bein g cross-linked. All three inhibitors achieve high local concentrations on f ibrin, which they protect from lysis by t-PA, u-PA and plasmin. The inhibit ors differ in their major sources in blood, with alpha(2)-AP present at hig h concentrations in plasma, PAI-1 primarily in platelets, and PAI-2 a produ ct of stimulated monocytes, giving them distinct and complementary roles in stabilizing fibrin in different physiological and pathological locations. (C) 2000 Harcourt Publishers Ltd.