Human Alpha-2 Plasmin Inhibitora2 plasmin inhibitor (a2 -PI) is a single-chain glycoprotein and is one of the major serine proteinase inhibitors circulating in plasma. Physiologically, it is the predominant inhibitor of plasmin and it therefore plays a significant role in the specific inhibition of fibrinolysis. The role of a2 -PI in fibrinolysis is three fold: covalent inhibition of plasmin; interference with the binding of plasminogen to fibrin; and factor XIIIa catalyzed cross-linking of a2-PI to fibrin. Rapid inactivation of plasmin proteolytic activity occurs through a two-step process. The inhibitor first forms a reversible complex with plasmin which is sub-sequently followed by the formation of a covalent, enzymatically inactive, complex with the catalytic site in plasmin. a2-PI also functions by interfering with the binding of plasminogen to fibrin, effectively slowing the activation of plasminogen by fibrin-bound plasminogen activator. The interference in binding ultimately delays the initiation of fibrinolysis. Covalent cross-linking of a2-PI to the a-chains of fibrin which is mediated by factor XIIIa, protects crosslinked fibrin clots from plasmin degradation and thereby markedly stabilizes the fibrin clot against fibrinolysis. Failure to protect the fibrin clot from rapid dissolution before injured vessels can be restored results in a bleeding tendency described in patients with a deficiency in a2-PI or factor XIII.
The structure of a2-PI consists of three functionally important regions. A reactive site is located at Arg-364 that forms a covalent bond with the plasmin active site. A high affinity plasminogen-binding site located within the last 20 COOH-terminal amino acids is responsible for binding the NH2 -terminal kringle structures of plasmin(ogen). An endogenous partially degraded form (non-plasminogen-binding form) of a2-PI lacking this COOH-terminal region makes up about 30% of the circulating a2-PI found in normal plasma. Lastly, the cross-linking site in a2-PI is located in the NH2 -terminal part of the molecule at Gln-2.
a2plasmin inhibitor is prepared from fresh frozen plasma by a combination of ion exchange, affinity, and gel filtration chromatography steps. Our purification selects exclusively for the native plasminogen-binding form. Purified a2 plasmin inhibitor is supplied in 50 mM potassium phosphate, 7.5 mM KCl, 0.075 mM EDTA, pH 7.4 and should be stored at -70 C. Purity is assessed by SDS-PAGE analysis and plasmin inhibition assay.