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

Abstract

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 activation, it controls interactions between proteases and inhibitors, both protecting proteases from inhibition and, conversely, localizing inhibitors. The principal inhibitor of plasmin, α₂-antiplasmin, is cross-linked to fibrinogen and fibrin, inhibiting fibrinolysis. Our studies have shown that a second inhibitor, PAI-2, is also cross-linked to fibrinogen and fibrin, by either factor XIIIa or tissue transglutaminase. These inhibitors are both members of the serpin family but the cross-linking sites are quite unrelated. 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 α₂-AP and at position 83 and 86 in PAI-2, located in a loop between helices C and D. All cross-linking observed was to the Aα chain of fibrin(ogen). The two inhibitors did not compete for cross-linking sites. α₂-AP binds only to Lys 303 of the Aα chain and a 30-residue peptide based on the sequence around this Lys competed with fibrinogen for cross-linking to α₂-AP but not for cross-linking to PAI-2. PAI-2 was cross-linked to several Lys residues (but not Lys 303) in the Aα 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 to 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 covalently 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 being cross-linked. All three inhibitors achieve high local concentrations on fibrin, which they protect from lysis by t-PA, u-PA and plasmin. The inhibitors differ in their major sources in blood, with α₂-AP present at high concentrations in plasma, PAI-1 primarily in platelets, and PAI-2 a product of stimulated monocytes, giving them distinct and complementary roles in stabilizing fibrin in different physiological and pathological locations.