Ongoing contact activation in patients with hereditary angioedema

Abstract

Hereditary angioedema (HAE) is predominantly caused by a deficiency in C1 esterase inhibitor (C1INH) (HAE-C1INH). C1INH inhibits activated factor XII (FXIIa), activated factor XI (FXIa), and kallikrein. In HAE-C1INH patients the thrombotic risk is not increased even though activation of the contact system is poorly regulated. Therefore, we hypothesized that contact activation preferentially leads to kallikrein formation and less to activation of the coagulation cascade in HAE-C1INH patients. We measured the levels of C1INH in complex with activated contact factors in plasma samples of HAE-C1INH patients (N=30, 17 during remission and 13 during acute attack) and healthy controls (N=10). We did not detect differences in enzyme-inhibitor complexes between samples of controls, patients during remission and patients during an acute attack. Reconstitution with C1INH did not change this result. Next, we determined the potential to form enzyme-inhibitory complexes after complete in vitro activation of the plasma samples with a FXII trigger. In all samples, enzyme-C1INH levels increased after activation even in patients during an acute attack. However, the levels of FXIIa-C1INH, FXIa-C1INH and kallikrein-C1INH were at least 52% lower in samples taken during remission and 70% lower in samples taken during attack compared to samples from controls (p<0.05). Addition of C1INH after activation led to an increase in levels of FXIIa-C1INH and FXIa-C1INH (p<0.05), which were still lower than in controls (p<0.05), while the levels of kallikrein-C1INH did not change. These results are consistent with constitutive activation and attenuated depletion of the contact system and show that the ongoing activation of the contact system, which is present in HAE-C1INH patients both during remission and during acute attacks, is not associated with preferential generation of kallikrein over FXIa.

Figure 1. Enzyme-inhibitory complexes in normal pool plasma after activation with ellagic acid or dextran sulphate.

The levels of inhibitory complexes were measured by ELISA after activation of normal pool plasma with either ellagic acid or dextran sulphate (DXS; Mr 500 000). Normal pool plasma was incubated for 25 minutes with either ellagic acid (0.25 mg/ml) or DXS (0.25 mg/ml) at 37^oC in Hepes-buffer (25 mM Hepes, 150 mM NaCl, pH = 7.5). The reaction was stopped with stop buffer containing STI and polybrene. The volume of plasma was 50% of total volume during the activation with ellagic acid or DXS, and diluted further in the assay. A.U. = Arbitrary units.

Figure 2. Enzyme-inhibitory complexes after activation with ellagic acid in samples from patients and healthy controls.

Levels of enzyme-inhibitory complexes were measured by ELISA in samples from patients with HAE-C1INH and healthy controls after activation with ellagic acid, without (black) and with (grey) addition of C1 esterase inhibitor (C1INH) after activation. Plasma was activated with ellagic acid (0.25 mg/ml) for 15 minutes, than C1INH or Hepes-buffer (25 mM Hepes, 150 mM NaCl, pH = 7.5) was added and after 10 minutes stop buffer containing STI and polybrene was added. The volume of plasma was 50% of total volume during the activation with ellagic acid, and diluted further in the assay. Significant differences are indicated in Table 3. The line represents the median value; A.U. = Arbitrary units.

Figure 3. Enzyme-inhibitory complexes after activation with dextran sulphate in samples from patients and healthy controls.

Levels of enzyme-inhibitory complexes were measured by ELISA in samples from patients with HAE-C1INH and healthy controls after activation with dextran sulphate (DXS; Mr 500 000), without (black) and with (grey) addition of C1 esterase inhibitor (C1INH) after activation. Plasma was activated with DXS (0.25 mg/ml) for 15 minutes, than C1INH or Hepes-buffer (25 mM Hepes, 150 mM NaCl, pH = 7.5) was added and after 10 minutes stop buffer containing STI and polybrene was added. The volume of plasma was 50% of total volume during the activation with DXS, and diluted further in the assay. Significant differences are indicated in Table 4. The line represents the median value; A.U. = Arbitrary units.