Contribution of conserved lysine residues in the alpha{2}-antiplasmin C terminus to plasmin binding and inhibition

alpha 2 -antiplasmin is the physiological inhibitor of plasmin and is unique among the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn410-Lys464 and the lysine residues (Lys418, Lys427, Lys434, Lys441, Lys448 and Lys464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C-terminus of alpha[SUB]2[/SUB]-antiplasmin, we have systematically and sequentially mutated the C-terminal lysines and studied the effects on the rate of plasmin inhibition and measured binding affinity with plasmin via surface plasmon resonance. We have determined that the C-terminal lysine (Lys464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C-terminus of alpha[SUB]2[/SUB]-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between alpha[SUB]2[/SUB]-antiplasmin and plasmin. When the C-terminus of alpha[SUB]2[/SUB]-antiplasmin was removed, binding affinity to active site-blocked plasmin remains high suggesting additional exosite interactions between the serpin core and plasmin.