Comparing Pathways of Bradykinin Formation in Whole Blood From Healthy Volunteers and Patients With Hereditary Angioedema Due to C1 Inhibitor Deficiency

Abstract

Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood. We applied various forms of activation to fresh blood obtained from 10 healthy subjects or 10 patients with hereditary angioedema (HAE-1 or −2 only) to investigate kinin formation. An enzyme immunoassay for BK was applied to extracts of citrated blood incubated at 37°C under gentle agitation for 0–2 h in the presence of activators and/or inhibitory agents. Biologically active kinins in extracts were corroborated by c-Fos accumulation in HEK 293a cells that express either recombinant human B2 or B1 receptors (B2R, B1R). Biological evidence of HAE diagnostic and blood cell activation was also obtained. The angiotensin converting enzyme inhibitor enalaprilat, without any effect per se, increased immunoreactive BK (iBK) concentration under active stimulation of blood. Tissue kallikrein (KLK-1) and Kontact-APTT, a particulate material that activates the contact system, rapidly (5 min) and intensely (>100 ng/mL) induced similar iBK generation in the blood of control or HAE subjects. Tissue plasminogen activator (tPA) slowly (≥1 h) induced iBK generation in control blood, but more rapidly and intensely so in that of HAE patients. Effects of biotechnological inhibitors indicate that tPA recruits factor XIIa (FXIIa) and plasma kallikrein to generate iBK. KLK-1, independent of the contact system, is the only stimulus leading to an inconsistent B1R stimulation. Stimulating neutrophils or platelets did not generate iBK. In the HAE patients observed during remission, iBK formation capability coupled to B2R stimulation appears largely intact. However, a selective hypersensitivity to tPA in the blood of HAE patients suggests a role of plasmin-activated FXIIa in the development of attacks. Proposed pathways of kinin formation dependent on blood cell activation were not corroborated.

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

“…It offers a different point of view because this incriminated vasoactive mediator is short-lived and will accumulate only if its formation rate exceeds that of its breakdown. No significant spontaneous release of kinin was previously seen in whole blood or plasma sampled during remission from HAE-C1-INH patients upon incubation at 37°C but in vitro activation of fibrinolysis induced in these patients an earlier and brisker production of BK than that seen in normal controls ( 20 , 21 ). The formation of immunoreactive BK (that includes Lys-BK) induced by contact system activation or by recombinant tissue kallikrein (KLK-1) did not differ between the 2 groups.…”

“…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 ).…”

“…Incubation with tPA (169 nM) cleaves (activates) all the plasminogen in control or HAE-FXII plasma samples in 10 min ( Figure 6C ), a short period during which little iBK is formed in healthy donor plasma ( Figure 3D ). This suggests that activation of FXII is the limiting step for kinin formation since iBK formation in tPA-stimulated control blood is dependent on FXIIa ( 20 ). Accordingly, little or none of the FXII heavy chain (52 kDa, open arrow) is seen in response to tPA in controls.…”

“…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 .…”

“…The BK EIA fully reacts with C-terminal BK fragments that have no biological activity (e.g., des-Arg 1 -BK) ( 20 ) whereas Lys-BK is approximately equipotent to BK at the human B 2 receptor ( 25 ). Verifying the agonist status of representative extracts was based on the detection of c-Fos accumulation in cells that expressed the recombinant human B 2 receptor ( 20 ) (methods, Supplementary Material ).…”