Protein Engineering, Vol. 14, No. 6, 427-438,
June 2001
© 2001 Oxford University Press
Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada
Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an
in vitro
clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV
2
, KIV
7
, KIV
9
Cys (which lacks the seventh unpaired cysteine) and KIV
10
which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV
10
to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV
10
is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase
in vitro
fibrin clot lysis time by ~43% at a concentration of 10 µM KIV
10
. The ability of the KIV
10
mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp
70
to Arg abolished binding to both lysine–Sepharose and plasmin-modified fibrinogen, while the Trp
70
Phe and Arg
35
Lys substitutions each resulted in decreased binding to these substrates. None of the KIV
10
mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV
7
contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV
10
. However, apo(a) KIV
7
had no effect on fibrinolysis
in vitro
. Apo(a) KIV
2
and KIV
9
Cys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis
in vitro
.