Increased fibrinolysis-induced bradykinin formation in hereditary angioedema confirmed using stored plasma and biotechnological inhibitors

Abstract

Objective We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 μl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors. Results Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h. Plasma samples from HAE-1/-2 patients responded earlier to tPA than those from controls, as previously reported with whole blood. Lanadelumab inhibited iBK formation induced by Kontact-APTT and tPA. A highly specific plasmin inhibitor, DX-1000, abolished tPA-induced iBK formation in plasma but had no effect against Kontact-APTT, confirming the role of fibrinolysis in tPA-induced kinin formation. The anti-lanadelumab neutralizing antibody M293-D02 reversed the inhibitory effects of lanadelumab. Frozen plasma is a suitable material for measuring iBK formation kinetics, with possible applications such as investigating the effect of rare disease states on the kallikrein–kinin system and monitoring the effect of HAE prophylactic treatments. Electronic supplementary material The online version of this article (10.1186/s13104-019-4335-8) contains supplementary material, which is available to authorized users.

Measurement of Bradykinin Formation and Degradation in Blood Plasma: Relevance for Acquired Angioedema Associated With Angiotensin Converting Enzyme Inhibition and for Hereditary Angioedema Due to Factor XII or Plasminogen Gene Variants

“…While plasmin is a known activator of normal FXII ( 31 ), the kinetics was particularly intense and rapid for tPA stimulation in FXII T328K heterozygotes ( Figure 3D ). Under the same experimental conditions, the plasma of HAE-C1-INH patients also released iBK faster than the controls in response to tPA, but the concentration did not exceed 300 ng/ml ( 21 ), whereas values in the HAE-FXII group were larger after 10 or 20 min of incubation ( Figure 3D ). The exhaustion of iBK formation later in the 2 h incubation period is specific to HAE-FXII plasma, suggesting the consumption of a factor critical for kinin production.…”

“…We previously reported that iBK formation was negligible in either whole blood or plasma incubated at 37°C without stimulus both in healthy donors and HAE patients sampled during remission (HAE-C1-INH type I or II) ( 20 , 21 ). This may be paradoxical because HK is spontaneously and progressively cleaved in the blood of patients with HAE-C1-INH ( 15 , 16 ) or HAE-nC1-INH ( 17 ).…”

“…In addition and unexpectedly, the early effect of recombinant tissue kallikrein (KLK-1) was enhanced in HAE-PLG. From the beginning of our investigations of HAE, we have kept monitoring the KLK-1 stimulation as an alternate pathway of kinin formation independent of the contact system and have not found profiles different from controls in HAE-C1-INH ( 20 , 21 ). Lys-BK, formed by KLK-1 mainly from low molecular weight kininogen, fully cross reacts with the applied BK EIA and is a direct B 2 receptor agonist ( Figure 1 ).…”

“…The tPA concentration was reproduced from Molinaro et al ( 19 ) and within the range reached in plasma during therapeutic fibrinolysis. Biotechnological inhibitors used in the mechanistic analysis of iBK formation were previously exploited ( 20 , 21 ) and are listed in Table 2 .…”

“…In all experiments dealing with BK EIA, the reactions were stopped by adding 1 ml of cold ethanol, as described ( 21 ). After further incubation on ice-water for an additional period of at least 60 min, the plasma/ethanol mixtures were cleared of precipitated proteins by centrifugation (13,000 g, 1 min), evaporated to dryness (Speed Vac apparatus) and stored at −80°C until used for BK determination.…”