Mutant plasminogen in hereditary angioedema is bypassing FXII/kallikrein to generate bradykinin

Abstract

Hereditary angioedema (HAE) is characterized by recurrent localized edema in various organs, which can be potentially fatal. There are different types of hereditary angioedema, which include genetic deficiency of C1 inhibitor (C1-INH) and hereditary angioedema with normal C1-INH (HAEnCI). In HAEnCI patients mutations have been identified in the F12, PLG, KNG1, ANGPT1, MYOF, and HS3ST6 genes. The release of bradykinin from kininogen via the kallikrein-kinin system (KKS) has been shown to be the main mediator in HAE-FXII, but for HAE-PLG there are only first indications how the PLG mutations can result in bradykinin release. Here we identified in a multi-generation HAE-PLG family an additional F12 mutation, resulting in the loss of one F12 allele. There were no differences in the clinical presentation between HAE-PLG patients with and without the additional F12 mutation, thus we concluded that the kallikrein-kinin system is bypassed in HAE-PLG. Structural modeling and in vitro assays using purified proteins confirmed the PLG mutation c.988A>G; p.K330E to be a gain of function mutation resulting in an increased bradykinin release by direct cleavage of high molecular weight kininogen (HMWK). Thus, we can provide clinical and experimental evidence that mutant plasminogen in HAE-PLG is bypassing FXII/kallikrein to generate bradykinin.

Keywords: FXII; HAE-PLG; Hereditary angioedema (HAE); bradykinin; kallikrein-kinin system (KKS); normal C1-INH; plasminogen.

FIGURE 1

Pedigree of the large HAE-PLG family showing the inheritance of the HAE-causing PLG mutation c.988A>G; p.K330E and the F12 mutation c.1681-1G>A resulting in the loss of protein expression from the mutated allele.

FIGURE 2

Clotting activity of the F12 mutation c.1681-1G>A in combination with the c.-4C/T polymorphism in 31 unrelated individuals illustrating the loss of FXII protein.

FIGURE 3

Crystal structure of human plasminogen. The structure is shown in surface (left and middle) and cartoon (right) representations colored in grey with the Kringle domains 1-5 highlighted in light-orange. Cartoon presentations (right) indicate the ligand binding residues as orange sticks, and K330 (green) and the interacting amino acid D328 (orange) as well as the mutation E330 (red) as dots. Structural views of plasminogen (Protein Data Bank entry 4A5T) (Xue et al., 2012) were generated by using the PyMOL Molecular Graphics System, Version 2.4 (Schrödinger LLC).

FIGURE 4

Western blot after 22 h of incubations showing HMWK incubated alone, with wt plasminogen, and with mutant plasminogen (p.K330E) in the presence of proteinase inhibitor (left side of the blot) and without proteinase inhibitor (right side).

FIGURE 5

MALDI-TOF measurements for HMWK incubated with wt plasminogen and with mutant plasminogen (p.K330E) in the presence of proteinase inhibitor and without proteinase inhibitor, including the measurement of purified bradykinin as positive control (lower panel).