Neutrophil-Derived Proteinase 3 Induces Kallikrein-Independent Release of a Novel Vasoactive Kinin

Abstract

The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH2-MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α1-antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B1 receptors, but did not bind to B2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B1 and B2 receptors as demonstrated using wild-type and B1 overexpressing rats as well as wild-type and B2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.

Met-Lys-bradykinin-Ser-Ser, a peptide produced by the neutrophil from kininogen, is metabolically activated by angiotensin converting enzyme in vascular tissue

“…The affinity of the PR3 kinin is marginally better at B 1 receptors (Fig. 1A), but not as strong as previously reported by Kahn et al [2]. Further, these authors reported that Met-Lys-BK-Ser-Ser activated phospholipase C in cells only at 1 µM, at variance with their own binding results, but possibly consistent with our affinity findings.…”

“…1B; table 1), as previously reported [2]. The affinity of the PR3 kinin is marginally better at B 1 receptors (Fig.…”

“…Kahn et al [2] stressed the activation of kinin B 1 receptors as a salient effect of Met-Lys-BK-SerSer. This 13-mer has a mediocre micromolar potency as a contractile agonist of the rabbit isolated aorta (Fig.…”

“…A new pathway of interest has been recently elucidated for kinin generation: the action of the neutrophil PR3 protease on high molecular weight kininogen releases a 13-mer, Met-Lys-BKSer-Ser also termed PR3-kinin [2]. Preliminary pharmacologic evaluation showed the absence of an affinity for recombinant B 2 receptors, a fair affinity for human B 1 receptors in a radioligand competition assay but a discrepant low potency to activate them (phospholipase C assay), and hypotensive effects in vivo mediated by both receptor subtypes, given that B 1 receptors were expressed in the animals [2].…”

“…Preliminary pharmacologic evaluation showed the absence of an affinity for recombinant B 2 receptors, a fair affinity for human B 1 receptors in a radioligand competition assay but a discrepant low potency to activate them (phospholipase C assay), and hypotensive effects in vivo mediated by both receptor subtypes, given that B 1 receptors were expressed in the animals [2]. The activation of the B 2 receptor at least must be accounted for by the generation of BK, Lys-BK and/or Met-Lys-BK, which are all known B 2 receptor agonists of nanomolar potencies at recombinant human or murine B 2 receptors [1], and it was found indeed that blood plasma generates BK, Met-Lys-BK and BK fragments from Met-Lys-BK-Ser-Ser [2].…”