Helen Ritchie

Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aα chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with α2-antiplasmin (α2-AP), which is known to cross-link to lysine 303 of the Aα chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to α2-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-αC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aα chain, respectively. PAI-2 or α2-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aα chain. α2-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with α2-AP, and the two proteins utilized different lysines in the Aα chain. Therefore, PAI-2 and α2-AP can cross-link simultaneously to the α polymers of a fibrin clot and promote resistance to lysis.

Up-regulation of plasminogen activator inhibitor 2 (PAI-2) in response to α-lactalbumin

Abstract U937 cells, a monocyte-like cell line, are a potent source of plasminogen activator inhibitor 2 (PAI-2) particularly following stimulation with a variety of agents. Lactalbumin enzymatic hydrolysate (LEH) can be used as an alternative to fetal calf serum (FCS) during culture of cells. Here, we report an up-regulation of PAI-2 synthesis following treatment of U937 cells with LEH and purified α-lactalbumin. Secretion of PAI-2 antigen into the culture medium was found to increase over time following growth of U937 cells in medium containing LEH (1 %) compared to medium alone or containing FCS. The possibility that undigested lactalbumin was present in LEH was then examined. A potent increase in secreted and intracellular PAI-2 was seen following stimulation of U937 cells with purified α-lactalbumin. This effect was both dose- and time-dependent. Northern blotting confirmed that the up-regulation in PAI-2 was at the level of mRNA. Basal levels of PAI-2 were up-regulated by an analogue of CAMP, whereas there was no further effect on α-lactalbumin stimulated PAI-2. Lactalbumin is another agent that up-regulates the synthesis of PAI-2, an effect that influences the study of PAI-2 in cultured cells.

Characterization of Crosslinking Sites in Fibrinogen for Plasminogen Activator Inhibitor 2 (PAI‐2)

Abstract: PAI‐2 is a serpin that can be crosslinked to fibrin(ogen) via the Gln‐Gln‐Ile‐Gln sequence (residues 83–86). We have characterized the lysine residues in fibrinogen to which PAI‐2 is crosslinked by tissue transglutaminase and factor XIIIa. There was no competition with the crosslinking of α2‐antiplasmin, another inhibitor of fibrinolysis, which was specific for Lys 303 in the Aα chain. PAI‐2 was crosslinked to several lysine residues, all in the Aα chain, 148, 176, 183, 230, 413, and 457, but not to Lys 303. The contrast with α2‐antiplasmin was clear from studies with truncated fibrinogens and competition by peptides. This was confirmed and extended by mass spectrometry of peptides after protease digestion of crosslinked products, which identified the lysine residues to which the inhibitors were crosslinked. PAI‐2 remained active after cross‐linking and inhibited fibrin breakdown, even by two‐chain t‐PA. Thus, a second inhibitor of fibrinolysis, in addition to α2‐antiplasmin, is crosslinked to fibrin and protects it from lysis.

Local Increase in Pai-2 on Stimulation of Monocytes with Modified Ldl

Fibrin deposistion is a feature of inflammatory diseases including atherosclerosis. Fibrin is a component of the atherosclerotic plaque and it is thought to act as a chemoattractant for infiltrating cells, as a scaffold for migrating cells and as a lipid binding surface, therefore promoting accumulation in the artery wall. (reviewed inl) Thrombus formation at the atherosclerotic plaque leading to myocardial infarction is a major cause of death. The balance of the fibrinolytic and coagulation systems is therefore important in the local environment of the artery wall. Tissue factor (TF) is present in the atherosclerotic plaque2 and decreased fibrinolytic activity may be attributed to increased levels of plasminogen activator inhibitor types 1 (PAI-1) and/or 2 (PAI-2) leading to persistence of fibrin already deposited as a result of TF activity.