Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions

Abstract

BackgroundAngioedema without wheals (AE) is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh), but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases.ObjectivesWe wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker.MethodsWe retrospectively investigated high molecular weight kininogen (HK) cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis.ResultsCirculating native HK plasma concentrations were similar in the healthy men (interquartile range: 98–175μg/mL, n = 51) and in healthy women (90–176μg/mL, n = 74), while HK cleavage was lower (p<0.001) in men (0–5%) than women (3–9%). Patients exhibited lower native HK concentration (p<10−4; 21–117μg/mL, n = 31 for men; 0–84μg/mL, n = 41 for women) and higher HK cleavage (p<10−4; 10–30% and 14–89%, respectively) than healthy donors. Pathological thresholds were set at: <72μg/mL native HK, >14.4% HK cleavage for men; <38μg/mL; native HK, >33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13) or two weeks after AE attack (n = 2), HK cleavage was not fully restored, suggesting its use as a post-attack assay.ConclusionAs a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management.

2D-LC–MS/MS to Measure Cleaved High-Molecular-Weight Kininogen in Human Plasma as A Biomarker for C1-Inh-Hae

“…In addition, receiver operator curve in Supplementary Figure 3 supports above claim as well (the area under the curve is 1.0 and p < 0.0001). These results are consistent with the previous reports that patients with C1-INH-HAE exhibit a dysregulated contact system between attacks and thus exhibit elevated cHMWK and bradykinin in plasma [12,[14][15][16][17]24]. Overall, we have demonstrated that the reagent-free LC-MS assay is able to accurately measure the plasma 46 kDa cHMWK concentration in the presence of a large amount of endogenous HMWK.…”

“…The peptide sequence was further blasted against the protein database to ensure it has no overlap with the hypothetical chymotrypsin peptides derived from other human proteins except HMWK. In previous reports, western blotting has been a method of choice to measure the relative abundance of the total HMWK and cHMWK as a CI-INH-HAE biomarker [12,24]. In contrast, our LC-MS assay measures the absolute quantitation of cHMWK (without normalized to total HMWK) with an aim to explore whether cHMWK level alone is correlated with C1-INH-HAE disease.…”

“…The resulting cleaved kininogen (cHMWK) contains two chains linked together by an interchain disulfide bond, the heavy chain (66 kDa) containing domains 1-3 and the light chain (56 kDa) containing domains 5-6. The 56 kDa light chain in cHMWK (denoted 56 kDa cHMWK) can be further cleaved by plasma kallikrein at the N-terminal por-2D-LC-MS/MS to measure cleaved highmolecular-weight kininogen in human plasma as a biomarker for C1-INH-HAE Guodong (Figure 1) [9][10][11][12]. The bradykinin released from HMWK acts as a key mediator of pain, inflammation, edema and angiogenesis [13].…”

“…The bradykinin released from HMWK acts as a key mediator of pain, inflammation, edema and angiogenesis [13]. Dysregulated plasma kallikrein proteolysis of HMWK can result in accumulation of bradykinin, which induces vasopermeability resulting in the edematous attacks associated with C1-INH-HAE [12,[14][15][16][17]. Therapeutic intervention by inhibition of plasma kallikrein has led to acute and prophylactic treatments of C1-INH-HAE [18][19][20][21].…”

“…As a result, there is considerable interest in developing sensitive and reliable assays to measure the level of cHMWK as a potential biomarker for C1-INH-HAE [10,[24][25]. Due to the lack of appropriate immunoassay reagents to specifically detect cleaved products, western blotting has currently been utilized to measure the cHMWK concentration for a variety of angioedema conditions and correlate it with drug efficacy in the early stage of clinical study [12,[18][19][20]24]. It represents a challenge to validate the western assays since they are generally semiquantitative, variable and of low sample throughput.…”