Abstract
In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-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-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-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 Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.
Highlights
Fibrinogen is a 340-kDa plasma protein and is a symmetrical dimer containing 29 disulfide bonds
Cross-linked plasminogen activator inhibitor 2 (PAI-2) Co-localized with the A␣ Chains of Fibrinogen—Cross-linking of PAI-2 to the ␣ chains of fibrinogen was examined by immunoblotting for the A␣ chain and for PAI-2
The addition of tissue transglutaminase (tTG) generated higher molecular mass cross-linked PAI-2 at 210, 240, and 270 kDa, which was consistent with PAI-2 that was cross-linked to ␣ polymers
Summary
Transglutaminase-mediated Cross-linking of PAI-2 and ␣2-AP—Fibrinogen for routine cross-linking reactions was purchased from Kabi (Sweden). Peptides were synthesized in-house using a Pioneer peptide synthesizer (PE Biosystems) and were confirmed by mass spectrometry These peptides were incorporated into cross-linking reactions at a final concentration of 1 mM. Enzymatic Digestion of Cross-linked Samples under Denaturing Conditions—Fibrinogen (Kabi, 2 mg) was cross-linked using 300 g of guinea pig liver transglutaminase and 2.5 mM CaCl2 in the absence or presence of 800 g PAI-2 or ␣2-AP for 2 h at 37 °C. 6 M fibrinogen was cross-linked to 6 M PAI-2 or ␣2-AP in the presence of 2.5 mM CaCl2, 600 nM tTG or 600 nM FXIIIa, and 0.4 units/ml thrombin for 2 h before electrophoresis on a 4 –12% NuPage acrylamide gel (Novex) under reducing conditions. All experiments were carried out at least five times except the gel filtration and trypsin digestion analysis of cross-linked samples, which was performed on two separate occasions
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