Plasminogen binds to plasmin-modulated factor Xa by Ca2+- and C-terminal lysine-dependent and -independent interactions

Abstract

SummaryPlasminogen binding to receptors involves both C-terminal lysine-dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca2+-enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca2+may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1–77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 2:1 plasminogen:FXa33/13 stoichiometry for both, which were proportionately enhanced by Ca2+. Only Lys-Pg demonstrated significant Ca2+-independent binding to FXa33/13. In the presence of Ca2+, weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1–3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1–3 binding to FXa33/13 had both Ca2+-dependent and -independent components. The principal KMd for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca2+, eACA inhibited FXa33/13 binding to K1–3 by 30%, but eliminated binding in the absence of Ca2+. These studies suggest that Ca2+-dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1–77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.

Rivaroxaban and apixaban induce clotting factor Xa fibrinolytic activity

“…The corresponding ligand blot (Fig. A) showed preferential binding of [ 125 I]Pg to the 33‐kDa component of Xa33/13 as compared with FXaβ, as previously observed . In the presence of DOACs, however, [ 125 I]Pg associated prevalently with FXaβ, as Xa33/13 was not produced.…”

“…However, a role for other cofactors that may participate in the initial production of plasmin has been underinvestigated. Recently, the use of lower t‐PA concentrations within the physiological range revealed that FXa and other clotting factors act as auxiliary t‐PA cofactors . These are cleaved by plasmin more effectively than fibrin, providing early C‐terminal lysine‐mediated t‐PA enhancement in the context of clotting.…”

“…Regardless of whether FX aβ is produced when bound to PL , clotting function is maintained, but Xa40 and Xa33/13 have negligible clotting activity in plasma (data not shown). Gla, γ‐carboxyglutamic acid‐containing domain ( PL ‐binding site); I, active site inhibitor; ▲, plasmin cleavage site determined by N‐terminal amino acid sequencing; ▵, plasmin cleavage site inferred by N‐terminal amino acid sequencing and lack of Pg binding ; ◊, inferred from electrophoretic mobility and Pg binding ).…”

Does plasmin have anticoagulant activity?

“…Plasmin-mediated cleavage of FXa exposes a Plg binding site16,32 and inhibits coagulation. In the presence of anionic phospholipid, FX and FXa enhance Plg activation by tPA33,34 to plasmin. Thus, plasmin cleavage of FX not only inhibits coagulation but also enhances fibrinolysis.…”

Plasmin‐mediated proteolysis of human factor IXa in the presence of calcium/phospholipid: Conversion of procoagulant factor IXa to a fibrinolytic enhancer

“…Plasmin cleavage of FX/FXa has been extensively studied 8‐13,19 . Circulating FX consists of a light chain (residues 1‐139) and a heavy chain (residues 143‐448) linked by a single disulfide bond; the tripeptide Arg‐Lys‐Arg that connects the light and heavy chains is proteolytically removed during the biosynthetic process 20 .…”