Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI

Abstract

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Keywords: Factor IX; Factor XII; intrinsic pathway; plasma kallikrein; prekallikrein.

Fig. 1.

PKa can initiate thrombin generation and fibrin formation independently of FXII. Plasmas deficient in proteins of the clotting cascade were assessed for thrombin generation and fibrin formation when initiated with PKa (0.797 to 581 nM). (A–F) Thrombin generation performed after recalcification with 16.7 mM CaCl₂ in the presence of 10 μM PLs. Thrombin generation in NPP (A), FXII-deficient plasma with CTI (B), FXI-deficient plasma (C), FXI-deficient plasma with CTI (D), FIX-deficient plasma (E), and FX-deficient plasma (F). Thrombograms show mean thrombin generated. n = 3. (G–K) Turbidimetric experiments were performed in plasma in the presence of 10 mM CaCl₂ and 10 μM PLs. Fibrin clot formation over time was measured by the change in absorbance at 340 nm in NPP (G), FXII-deficient plasma (H), FXI-deficient plasma (I), FIX-deficient plasma (J), and FX-deficient plasma (K). Data are presented as mean ± SD, n = 3.

Fig. 2.

PKa can selectively cleave FIX in a dose- and time-dependent manner. (A–D) The ability of PKa to cleave zymogen FX, FXI, prothrombin (FII), and FIX was determined by incubating reaction mixtures containing 0.5 mg/mL zymogen protein with a concentration series of PKa (2.39 to 581 nM), in the presence of 1.5 mM CaCl₂ and 10 μM PLs. Samples were incubated for 1 h at 37 °C before analysis by SDS-PAGE under reducing conditions. PKa titration incubated with FX (A), FXI (B), FII (C), and FIX (D). (E) The time dependency of FIX cleavage by PKa was assessed by incubating 0.5 mg/mL FIX with 65 nM PKa in the presence of 1.5 mM CaCl₂ for up to 240 min. Samples were assessed by SDS-PAGE under reducing conditions. (F) 5 nM PKa and 300 nM FIX protein were mixed in the presence of a variety of buffer conditions containing PLs (10 μM), CaCl₂ (2.5 mM), and ZnCl₂ (10 μM). Each of these conditions was assessed in the presence (blue bars) and absence (green bars) of 12 nM HK. Cleavage of chromogenic substrate S-2765 by samples was measured for 1 h. Data represent the rate of chromogenic substrate cleavage over 1 h, mean ± SD; n = 3. (G) 300 nM FIX protein was added to 0.726 to 5.81 nM PKa in the presence of CaCl₂ (2.5 mM) and ZnCl₂ (10 μM), followed by the addition of chromogenic substrate S-2765. Samples containing only PKa (0.726 to 5.81 nM) were run in parallel. Data represent PKa subtracted, mean ± SD; n = 3. In A–E, positions of molecular mass standards (in kDa) are shown to the left of each gel image.

Fig. 3.

PK(a) binds to FIX(a) with high affinity. Binding kinetics and affinities of the interaction of FIX and FIXa (± HK), and prothrombin (FII) with PK and PKa were studied by SPR using a Pioneer system. FIX (A and B) and FIXa (C and D) proteins were injected over a PK- or PKa-immobilized surface. Data are mean ± SEM; n = 3. (E–H) Experiments were also performed in the presence of HK. FIX was preincubated with HK and flowed over a PK-immobilized (E) or PKa-immobilized (F) surface. FIXa was preincubated with HK and injected over a PK-immobilized (G) or PKa- immobilized (H) surface. (I and J) Binding of FII to PK (I) and PKa (J). n = 1 for experiments in E–J.

Fig. 4.

The coagulation cascade. The intrinsic pathway is initiated after activation of FXII by contact with a negatively charged surface. FXIIa can activate PK to PKa, and PKa can activate more FXII. In the presence of a surface, reciprocal activation of FXII and PK to FXIIa and PKa is amplified by cofactor HK. FXII has intrinsic proteolytic activity that is able to activate PK without a surface (purple dotted arrows). Similarly, PK has proteolytic activity that can activate FXII, but this reaction requires a surface (purple dotted arrows). Following FXIIa activation, FXI is activated to FXIa, and FIX is activated to FIXa. The extrinsic pathway is initiated following tissue damage and exposure of tissue factor. Both pathways converge at the activation of FX to FXa and conversion of prothrombin to thrombin. Thrombin cleaves fibrinogen to fibrin, which polymerizes to form fibrin fibers and also activates FXIII, which introduces cross-links into the fibrin network, stabilizing it. Many reactions of the coagulation cascade require calcium (Ca²⁺) or PLs (yellow circles). We and others have demonstrated that PKa can directly activate FIX, bypassing FXI (red dashed arrow). Zymogens are indicated by roman numerals, and their activated forms end with an “a”.