In vitro reconstitution of kallikrein-kinin system and progress curve analysis

International audienceAbstract Human kallikrein-kinin system (KKS) is a proteolytic cascade with two serine-protease zymogen couples (Factor XII and prekallikrein (PK) and their activated forms, FXIIa, PKa, respectively), releasing bradykinin by cleavage of native high-molecular-weight kininogen (nHK) into cleaved HK. For KKS investigation in human plasma, this cascade is usually triggered on ice eventually by mixing with purified proteins. It has been established that purified FXIIa, PK, and nHK required a fixed order and timing for mixing protein on ice to ensure reproducibility of testing, we investigated the activation kinetics of both enzymes. The activation process of this in vitro minimal reconstitution of KKS was studied by progress curve analysis, in condition of high enzyme/substrate ratio and by using on natural rather than peptide substrates. FXIIa and PKa were found five-times less active on ice than at 37°C: kcat = 0.133 ± 0.034 and 0.0119 ± 0.0027 s−1, KM = 672 ± 150 and 115 ± 24 nM, respectively. The progress curve analysis of our in vitro KKS reconstitutions differed from a Michaelis–Menten mathematical simulation by a faster initial rate and a slower late rate. These two features were also observed ex vivo by using dextran sulfate-activated plasma and could reinforce the hypothesis of a maximal local effect (bradykinin release) and a minimal systemic consequence (PK preservation) in KKS activation process. Analyzing the complete curve of cold KKS activation would provide valuable information for ex vivo investigation of KKS in samples from patients presenting with hereditary angioedema and other inflammatory conditions

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

Angioedema 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.We 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.We 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.Circulating 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 (p14.4% HK cleavage for men; 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.As 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

HK molecular pattern analysis of AE patients.

<p><b>A</b>: High-molecular-weight kininogen (HK) molecular pattern of plasma samples (0.5μL/lane) from two nC1Inh-AE patients (W6, M6) less than 24h after the onset of an AE attack (Att) and at least two weeks after remission (Rem), analysed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.g001" target="_blank">Fig 1</a>. <b>B</b>: Same as for A, with plasma samples from three patients (K1–K3) with ACEi-AE and low kinin catabolism (L-Cat) and three patients (M7–M9) with nC1Inh-AE and normal kinin catabolism (N-Cat). N-HK concentrations were evaluated by the displayed linear regressions, kinin-forming activity was determined as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.ref026" target="_blank">26</a>], pathological values were labelled in <b>bold</b> font. densito: densitometry in 10⁷ arbitrary units; N-HK, LC-HK, cLC-HK: native chain, light chain, cleaved light chain of HK respectively.</p

HK concentration in samples from nC1Inh-AE patients taking oral contraception.

<p>High-molecular-weight kininogen (HK) molecular pattern of plasma samples from healthy women taking or not estrogen/progestin combination (OP; Healthy+OP, n = 32; Healthy-OP, n = 30) and from nC1Inh-AE patients taking OP and ≥2 months after OP withdrawal (nC1INh-AE+/-OP, n = 13). <b>A</b>: Analysis of plasma samples (0.5μL/lane) from six patients (O1–O6) and the corresponding figures. N-HK concentration of P1 and P2 was evaluated by the displayed linear regression, kinin-forming activity was determined as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.ref026" target="_blank">26</a>], pathological values were labelled in <b>bold</b> fonts. <b>B, C</b>: Box-plot displaying 5–95% range (<i>italic</i> fonts), median (<b>bold</b> fonts), and interquartile values of the plasma concentration of N-HK (B) and cleaved HK species (C), measured as in panel A. <b>D, E</b>: Details of the paired samples of panel B and C respectively analysed using the Wilcoxon matched-pairs signed rank test. densito: densitometry in 10⁷ arbitrary units; <i>NS</i>: non-significant; *: <i>p</i><0.05; **: <i>p</i><0.01; ***: <i>p</i><0.001; ****: <i>p</i><10⁻⁴ (non-parametric tests); N-HK, LC-HK, cLC-HK: native chain, light chain, cleaved light chain of HK respectively; nC1Inh-AE: angioedema with normal C1 inhibitor.</p

Threshold of N-HK concentration and HK cleavage.

<p><b>A, B</b>: ROC curve analysis between healthy men and nC1Inh-AE male patients of N-HK concentration (A) and HK molecular species (B), displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.g002" target="_blank">Fig 2B and 2C</a>, respectively. The arrows point to the dots corresponding to the maximum of the sum {sensitivity + specificity} and the corresponding thresholds. <b>C</b>: Figures associated with the ROC curve analysis shown in A and B along with their calculated 95% confidence levels (95% CI). <b>D, E, F</b>: as in A, B, C for indicators on women displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163958#pone.0163958.g002" target="_blank">Fig 2E and 2F</a>. AUC: area under the curve; PPV: predictive positive value; NPV: negative predictive value; N-HK: native single-chain of HK.</p

Quantification of HK molecular pattern in control male and female samples.

<p><b>A, B</b>: Purified human HK (20, 40, 80, and 110ng) and 0.5μL of plasma from five male healthy donors, (H1 to H5) (A), and five females (H6 to H10) (B) were subjected to SDS-PAGE. The signal of HK native chain (N-HK), light chain (LC-HK), and cleaved light chain (cLC-HK) were quantified by densitometry and N-HK concentration was evaluated by the displayed linear regression. <b>C, D</b>: Box-plot displaying 5–95% range (<i>italic</i> fonts), median (<b>bold</b> fonts), and interquartile values of plasma concentration of N-HK (C) and HK cleavage (D), measured as in panel A. densito: densitometry in 10⁷ arbitrary units; NS: non-significant; ****: <i>p</i><10⁻⁴ (Mann-Whitney-Wilcoxon test).</p