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.
This publication used a Bradykinin EIA kit (EK-009-01) from Phoenix Pharmaceuticals.
Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn(2+). We examined the Zn(2+) requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/10(6) HUVEC) is seen with no added Zn(2+) in HEPES-Tyrode buffer containing gelatin versus 16--32 microM added Zn(2+) in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 +/- 1.8 nM and a maximal binding of 1.08 +/- 0.26 x 10(7) sites/cell (means +/- SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn(2+) on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/10(6) HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.
This publication used a Bradykinin peptide from Phoenix Pharmaceuticals.
Zhao Y, Qiu Q, Mahdi F, Shariat-madar Z, Røjkjaer R, Schmaier AH. Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation. Am J Physiol Heart Circ Physiol. 2001;280(4):H1821-9.
|009-04||[Des-Pro2]-Bradykinin (Human, Rat, Mouse)||5 mg||$95|
|009-05||[Hyp3]-Bradykinin (Human, Rat, Mouse)||500 µg||$31|
|009-06||[Lys0, D-Phe8, Des-Arg9]-Bradykinin (Human, Rat, Mouse)||500 µg||$107|
|009-07||[Ac-Lys0, (β-D-Nal7, Ile8, Des-Arg9]-Bradykinin (Human, Rat, Mouse)||1 mg||$85|
|009-09||[Des-Arg9, Leu8]-Bradykinin (Human, Rat, Mouse)||5 mg||$67|
|009-10||[Thi5, D-Phe7, Thi8]-Bradykinin (Human, Rat, Mouse)||500 µg||$54|
|009-11||[D-Arg0, Hyp3, D-Phe7]-Bradykinin (Human, Rat, Mouse)||500 µg||$54|
|009-12||[Tyr8]-Bradykinin (Human, Rat, Mouse)||100 µg||$213|
|009-13||[D-Arg0, Hyp3, Thi5, D-Phe7, Thi8]-Bradykinin (Human, Rat, Mouse)||500 µg||$72|
|009-15||[D-Arg0, Hyp2, Hyp3, Thi5, D-Phe7, Thi8]-Bradykinin (Human, Rat, Mouse)||500 µg||$67|