Patterns of C1-Inhibitor/Plasma Serine Protease Complexes in Healthy Humans and in Hereditary Angioedema Patients

Abstract

C1-inhibitor (C1-INH) is an important regulator of the complement, coagulation, fibrinolytic and contact systems. The quantity of protease/C1-INH complexes in the blood is proportional to the level of the in vivo activation of these four cascade-like plasma enzyme systems. Parallel determination of C1-INH-containing activation complexes could be important to understand the regulatory role of C1-INH in diseases such as hereditary angioedema (HAE) due to C1-INH deficiency (C1-INH-HAE). We developed in-house ELISAs to measure the concentration of complexes of C1-INH formed with active proteases: C1r, C1s, MASP-1, MASP-2, plasma kallikrein, factor XIIa, factor XIa, and thrombin, as well as to determine total and functionally active C1-INH. We measured the concentration of the complexes in EDTA plasma from 6 healthy controls, from 5 with type I and 5 with type II C1-INH-HAE patients during symptom-free periods and from five patients during HAE attacks. We also assessed the concentration of these complexes in blood samples taken from one C1-INH-HAE patient during the kinetic follow-up of a HAE attack. The overall pattern of complexed C1-INH was similar in controls and C1-INH-HAE patients. C1-INH formed the highest concentration complexes with C1r and C1s. We observed higher plasma kallikrein/C1-INH complex concentration in both type I and type II C1-INH-HAE, and higher concentration of MASP-1/C1-INH, and MASP-2/C1-INH complexes in type II C1-INH-HAE patients compared to healthy controls and type I patients. Interestingly, none of the C1-INH complex concentrations changed significantly during HAE attacks. During the kinetic follow-up of an HAE attack, the concentration of plasma kallikrein/C1-INH complex was elevated at the onset of the attack. In parallel, C1r, FXIIa and FXIa complexes of C1-INH also tended to be elevated, and the changes in the concentrations of the complexes followed rather rapid kinetics. Our results suggest that the complement classical pathway plays a critical role in the metabolism of C1-INH, however, in C1-INH-HAE, contact system activation is the most significant in this respect. Due to the fast changes in the concentration of complexes, high resolution kinetic follow-up studies are needed to clarify the precise molecular background of C1-INH-HAE pathogenesis.

Keywords: C1-inhibitor; HAE attack; activation; hereditary angioedema; kinetic follow-up; serine protease.

FIGURE 1

Purification of Berinert P (C1-inhibitor) by anion exchange chromatography. A black rectangle indicates the collected pure fractions representing the middle of the main peak. Blue, red, green and brown lines depict the absorbance at 280 nm, the absorbance at 254 nm, the gradient, and the specific conductivity, respectively. Removed impurities are indicated by labels on the chromatogram, and by arrows on the gel (insertion). SDS-PAGE was performed under reducing conditions. The marker (GE Healthcare Low Molecular Weight marker) is composed of 97, 66, 45, 30, 20.1, and 14.4 kDa proteins.

FIGURE 2

Mean concentration of protease/C1-INH complexes in C1-INH-HAE patients and controls. Comparison of the concentration of protease/C1-INH complexes relative to active C1-INH concentration (C1-INH_a) (A), and pattern of individual protease/C1-INH complexes (B). M1 = MASP-1, M2 = MASP-2, KK = Kallikrein, and TR = Thrombin.

FIGURE 3

Mean concentration of protease/C1-INH complexes in C1-INH-HAE patients during attack and in symptom-free period. Comparison of total C1-INH concentration (C1-INH_t), active C1-INH concentration (C1-INH_a); and concentrations of C1r-, C1s-, MASP- 1-, MASP- 2-, kallikrein-, FXIIa-, FXIa-, and thrombin/C1-INH complexes in patients with C1-INH-HAE in symptom-free periods and during HAE attacks.

FIGURE 4

Kinetic follow-up of protease/C1-INH complexes during an angioedematous attack. Kinetics of total C1-INH concentration (C1-INH_t), active C1-INH concentration (C1-INH_a) and concentrations of C1r-, C1s-, MASP- 1-, MASP- 2-, kallikrein-, FXIIa-, FXIa-, and thrombin/C1-INH, complexes during an HAE attack of a type I C1-INH-HAE patient. Severity of angioedematous symptoms were assessed by the patient in a visual analog scale (VAS, 0–100%). Symptom-free periods are marked with green, prodromal phase with yellow and HAE attack phase with orange colors.