Cryo-EM structures of human bradykinin receptor-Gq proteins complexes.

The type 2 bradykinin receptor (B2R) is a G protein-coupled receptor (GPCR) in the cardiovascular system, and the dysfunction of B2R leads to inflammation, hereditary angioedema, and pain. Bradykinin and kallidin are both endogenous peptide agonists of B2R, acting as vasodilators to protect the cardiovascular system. Here we determine two cryo-electron microscopy (cryo-EM) structures of human B2R-Gq in complex with bradykinin and kallidin at 3.0 Å and 2.9 Å resolution, respectively. The ligand-binding pocket accommodates S-shaped peptides, with aspartic acids and glutamates as an anion trap. The phenylalanines at the tail of the peptides induce significant conformational changes in the toggle switch W2836.48, the conserved PIF, DRY, and NPxxY motifs, for the B2R activation. This further induces the extensive interactions of the intracellular loops ICL2/3 and helix 8 with Gq proteins. Our structures elucidate the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins coupling of B2R.

Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10]Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumor Uptake.

Bradykinin B1 receptor (B1R) that is overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence, 68Ga-DOTA-linker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115, and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4-amino-(1-carboxymethyl)piperidine, respectively, as the linker were synthesized and evaluated. In vitro competition binding assays showed that the Ki values of SH01078, P03034, P04115, and P04168 were 27.8±4.9, 16.0±1.9, 11.4±2.5, and 3.6±0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of 68Ga-labeled SH01078, P03034, and P04115 in HEK293T::hB1R tumor was similar (1.96-2.17%ID/g) at 1 h postinjection. 68Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15±1.13%ID/g) and lower background activity, leading to a >2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over those of 68Ga-labeled SH01078, P03034, and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1-carboxymethyl)piperidine linker improved tumor visualization, and the resulting 68Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography.

Comparative studies of three 68Ga-labeled [Des-Arg10]kallidin derivatives for imaging bradykinin B1 receptor expression with PET.

UNLABELLED: Bradykinin B1 receptor (B1R) is a G-protein-coupled receptor that is overexpressed in a variety of cancers. B1R is not expressed in healthy tissues, making it an attractive cancer imaging marker. Previously, we reported selective uptake of (68)Ga-P03034 ((68)Ga-DOTA-dPEG2-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu) in B1R-positive (B1R+) HEK293T::hB1R tumor xenografts in mice. In this study, we compare (68)Ga-P03034 with (68)Ga-labeled P04158 ((68)Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic) and Z02090 ((68)Ga-DOTA-dPEG2-Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg) derived from 2 potent B1R antagonists, B9858 and B9958, respectively, for imaging B1R expression with PET. METHODS: Peptide sequences were assembled on solid-phase. Cold standards were prepared by incubating DOTA-conjugated peptides with GaCl3. Binding affinity was measured via competition binding assays using hB1R-expressing Chinese hamster ovary-K1 cell membranes. (68)Ga labeling was performed in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer with microwave heating and purified by high-performance liquid chromatography. Imaging/biodistribution studies were performed in mice bearing wild-type HEK293T (B1R-) and B1R+ HEK293T::hB1R tumors. RESULTS: P03034, P04158, and Z02090 bound B1R with high affinity, with Ki values at 16.0 ± 2.9, 1.5 ± 1.9, and 1.1 ± 0.8 nM, respectively. (68)Ga-labeled P03034, P04159, and Z02090 were obtained in greater than 50% decay-corrected radiochemical yields with more than 99% radiochemical purity. Biodistribution studies showed that all three (68)Ga-labeled tracers cleared rapidly from the blood and normal tissues, with excretion mainly via the renal pathway. At 1 h after injection, only the kidneys, bladders, and B1R+ HEK293T::hB1R tumors were clearly visualized in PET images. Uptake values of (68)Ga-labeled P03034, P04158, and Z02090 in B1R+ tumors were 2.17 ± 0.49, 19.6 ± 4.50, and 14.4 ± 1.63 percentage injected dose per gram, respectively. Uptake ratios of B1R+ to B1R- tumor, blood, and muscle were 6.23 ± 1.69, 5.72 ± 2.20, and 25.5 ± 13.1 for (68)Ga-P03034; 34.5 ± 10.5, 19.2 ± 8.21, and 66.1 ± 17.0 for (68)Ga-P04158; and 29.3 ± 9.68, 29.9 ± 5.58, and 124 ± 28.1 for (68)Ga-Z02090, respectively. CONCLUSION: All three (68)Ga-labeled B1R-targeting peptides generated specific and high-contrasted images of B1R+ tumors xenografted in mice. With significantly higher tumor uptake and target-to-nontarget ratios, (68)Ga-labeled P04158 and Z02090 are superior to P03034 for imaging B1R expression with PET.

(18)F-trifluoroborate derivatives of [des-arg(10)]kallidin for imaging bradykinin b1 receptor expression with positron emission tomography.

Bradykinin B1 receptor (B1R) is involved in pain and inflammation pathways and is upregulated in inflamed tissues and cancer. Due to its minimal expression in healthy tissues, B1R is an attractive target for the development of therapeutic agents to treat inflammation, chronic pain, and cancer. The goal of this study is to synthesize and compare two (18)F-labeled peptides derived from potent B1R antagonists B9858 and B9958 for imaging B1R expression with positron emission tomography (PET). Azidoacetyl-B9858 2 and azidoacetyl-B9958 3 were synthesized by a solid-phase approach and subsequently clicked to ammoniomethyl-trifluoroborate (AmBF3)-conjugated alkyne 1 to obtain AmBF3-B9858 and AmBF3-B9958, respectively. AmBF3-B9858 and AmBF3-B9958 bound B1R with high affinity, with Ki values at 0.09 ± 0.08 and 0.46 ± 0.03 nM, respectively, as measured by in vitro competition binding assays. (18)F labeling was performed via an (18)F-(19)F isotope exchange reaction. The radiofluorinated tracers were obtained within a synthesis time of 30 min and with 23-32% non-decay-corrected radiochemical yield, >99% radiochemical purity, and 43-87 GBq/µmol specific activity at the end of the synthesis. PET imaging and biodistribution studies were carried out in mice bearing both B1R-positive (B1R(+)) HEK293T::hB1R and B1R-negative (B1R(-)) HEK293T tumors. Both tracers cleared rapidly from most organs/tissues, mainly through the renal pathway. High uptake in B1R(+) tumors ((18)F-AmBF3-B9858: 3.94 ± 1.24% ID/g, tumor-to-muscle ratio 21.3 ± 4.33; (18)F-AmBF3-B9958: 4.20 ± 0.98% ID/g, tumor-to-muscle ratio 48.6 ± 10.7) was observed at 1 h postinjection. These results indicate that (18)F-AmBF3-B9858 and (18)F-AmBF3-B9958 are promising agents for the in vivo imaging of B1R expression with PET.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins.

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A.

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL). The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL. The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds. The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.

Prediction of band profiles of mixtures of bradykinin and kallidin from data acquired by competitive frontal analysis.

The competitive adsorption isotherms of two closely related peptides, bradykinin and kallidin, were measured by frontal analysis on a Zorbax SB-C18 microbore column. An aqueous soluton at 20% acetonitrile (0.1% TFA) was used as the mobile phase. The competitive isotherm data were fitted to four different models: Langmuir, Bilangmuir, Langmuir-Freundlich, and Toth. These data fitted best to a Bilangmuir isotherm model. The influence of the pressure on the retention factors of the two peptides was found to be small and was not investigated in detail. The band profiles of large samples of the single components and of their mixtures were recorded. The overloaded profiles calculated using either the equilibrium-dispersive or POR model are in excellent agreement with the experimental profiles in all cases. Our results confirm that the competitive isotherm data derived from mixtures may suffice for a reasonably accurate prediction of the band profiles of all mixtures of the two components, provided their composition is close to 1/1.

The kallikrein-kinin system in humans.

1. Kinin peptides are implicated in many physiological and pathological processes, including the regulation of blood pressure and sodium homeostasis, inflammation and the cardioprotective effects of preconditioning. In humans, the plasma and tissue kallikrein-kinin systems (KKS) generate bradykinin and kallidin peptides, respectively. 2. We established methodology for the measurement of bradykinin and kallidin peptides and their metabolites in order to study the function of the plasma and tissue KKS in humans. 3. Bradykinin peptides were more abundant than kallidin peptides in blood and cardiac atrial tissue, whereas kallidin peptides were predominant in urine. The levels of kinin peptides in tissue were higher than in blood, confirming the primary tissue localization of the KKS. 4. Angiotensin-converting enzyme inhibition increased blood levels of bradykinin and kallidin peptides. 5. Blood levels of kallidin peptides were suppressed in patients with severe cardiac failure, indicating that the activity of the tissue KKS is suppressed in this condition. 6. Bradykinin peptide levels were increased in the urine of patients with interstitial cystitis, suggesting a role for these peptides in the pathogenesis and/or symptomatology of this condition. 7. Cardiopulmonary bypass, a model of activation of the contact system, activated both the plasma and tissue KKS. 8. Measurement of individual bradykinin and kallidin peptides and their metabolites gives important information about the operation of the plasma and tissue KKS and their role in physiology and disease states.

Probing the topological arrangement of the N- and C-terminal residues of bradykinin for agonist activity at the B1 receptor.

The conformational features of H-Lys-Arg-Ado-Ser-Pro-Phe-OH (Ado = 12-aminododecanoic acid), a des-Arg(9) analogue of Lys-bradykinin, have been determined by high-resolution NMR in the presence of a zwitterionic lipid environment. The analogue is the most active member of a series of analogues designed to probe the topological arrangement of the N- and C-termini required for agonistic activity at the B1 kinin receptor. A novel computational procedure for the utilization of NOE constraints from cis and trans configurational isomers is illustrated. Only with this computational methodology could the structural features of the N-terminus of the peptide be determined. Using radical-induced relaxation of the (1)H NMR signals, we measured the topological orientation of the peptide with respect to the zwitterionic lipid interface. The results indicate that the long, alkyl chain of the Ado amino acid imbeds into the lipid surface. The structural features of the C-terminus of the B1-selective analogue consist of a well-defined turn. Although removed from a standard beta-turn, required for activity at the B2 kinin receptor, the topological orientation of the side chains of the des-Arg(9) compound are surprisingly similar to those previously observed for beta-turn-containing bradykinin analogues. Therefore, we attribute the high B1 receptor selectivity, observed upon removal of Arg(9) from bradykinin, solely to the loss of a charged amino acid and not to altered structural features.

Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B1 kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B1 receptors.

Blood pressure regulation by the kallikrein-kinin system.

1. The kallikrein-kinin system has a significant role in regulating arterial blood pressure. 2. Reduced formation of the kinin compontents may cause hypertensive diseases. This is because of the fact that this system is responsible for vasodilatation, reduction in total peripheral resistance, natriuresis, diuresis, increasing renal blood flow and releasing various vasodilator agents. 3. Reduced kinin-kallikrein generation in hypertensive subjects may also be associated with genetic and environmental defects. 4. The kallikrein-kinin system when administered to hypertensive patients can lower their raised blood pressure to normotensive levels. 5. The mode of action of angiotensin-converting enzyme inhibitors principally may be dependent on the kinin system protection.

Bradykinin receptors in mouse and rat isolated superior cervical ganglia.

1. The ability of bradykinin and its analogues to depolarize rat and mouse superior cervical ganglia was studied by use of in vitro grease-gap recording techniques, and the ability of antagonists selective for bradykinin receptor subtypes to block their effects was examined. 2. Bradykinin (3 microM) depolarized ganglia from both species, although the magnitude of the maximal response was less in mouse (15 +/- 5%, n = 7) than rat tissue (33 +/- 6%, n = 7), relative to muscarine (1 microM). 3. Interleukin 1 beta (30 u ml-1 for 18 h at 37 degrees C) increased the depolarization caused by bradykinin (3 microM) in mouse ganglia from 15% to 54% (P < 0.001, n = 12). Responses to the B1 receptor agonist, [des-Arg10]-kallidin (3 microM) were similarly potentiated but this was only detected after inhibition of peptidase activity with 10 microM captopril (4% to 35%, n = 5). 4. In ganglia from both species the rank order of agonist potency was bradykinin = [Lys0]-bradykinin >> [des-Arg10]-kallidin. However, like responses to [des-Arg10]-kallidin in mouse tissue, both the potency of bradykinin and the maximal depolarization achieved (EC50 = 912 nM; 80%, n = 11) was enhanced following inhibition of angiotensin converting enzyme with 10 microM captopril (EC50 = 50 nM; 135%, n = 4). 5. Responses to bradykinin were selectively antagonized by the B2 receptor antagonist, Hoe 140 but not by the B1 antagonist, [Leu8]-bradykinin1-8. From Schild analysis the pA2 value for Hoe 140 in mouse tissue was 9.65, although the slope of the regression line was significantly greater than unity, indicating non-competitive kinetics (slope = 1.88 +/- 0.18, n = 9). The depolarization caused by [Lys0]-bradykinin was also antagonized by Hoe 140 (3 nM).6. Thus the predominant bradykinin receptor in mouse superior cervical ganglia is compatible with a B2 subtype. Furthermore the depolarizations caused by B1 and B2 agonists in this tissue can be increased following exposure to interleukin l beta, and by blocking peptide degradation with captopril.

Determination of trace impurities of peptides and alkaloids by capillary electrophoresis-ion spray mass spectrometry.

Two different mixtures have been analyzed by CE-UV-MS using selected ion monitoring (SIM) conditions to evaluate whether this technique can detect trace impurities in such mixtures. The first mixture consisted of two bioactive peptide analogues which included Lys-bradykinin (kallidin) and Met-Lys-bradykinin. The presence of 0.1% Lys-bradykinin was detected by SIM CE-MS but not by CE-UV at the 0.1% level as it migrated from the capillary column prior to the major component, Met-Lys-bradykinin. The second mixture consisted of two antibacterial alkaloids, berberine and palmatine. The presence of 0.15% palmatine was detected by CE-UV and SIM CE-MS at the 0.15% level as it migrated from the capillary column following the major component, berberine. These results suggest that SIM CE-MS offers the necessary separation efficiencies and sensitivity to provide a complementary analytical determination of trace components in such sample mixtures.

Purification of a vasoactive peptide related to lysyl-bradykinin from trout plasma.

Incubation of plasma from the steelhead trout, Oncorhynchus mykiss with porcine pancreatic glandular kallikrein generated bradykinin-like immunoreactivity. The primary structure of the immunoreactive peptide was established as: Lys-Arg-Pro-Pro-Gly-Trp-Ser-Pro-Leu-Arg. This sequence shows two amino acid substitutions (Phe6-->Trp and Phe9-->Leu) compared with mammalian lysyl-bradykinin (kallidin). Bolus intraarterial injection of the purified peptide produced a strong and sustained vasopressor response in the unanaesthetized trout. The data demonstrate that the kallikrein-kinin system predates the appearance of tetrapods and suggest a role for this system in cardiovascular regulation in fish.

Arachidonic acid release and inositol lipid metabolism in response to bradykinin and related peptides in human endometrial cells in vitro.

Prostaglandins of the 2 series are known to play a role in the regulation of menstruation and implantation but, more recently, other vasoactive peptides have been considered as potential regulators of these endometrial processes. The aim of the present study was to investigate the action of the potent vasoactive peptide bradykinin and the structurally related peptide, kallidin, on endometrial function by examining their effect on phosphoinositide hydrolysis and arachidonic acid release from endometrial cells in vitro. Primary cultures of endometrial glands and stromal cells were prelabelled with [14C]-arachidonic acid (AA) or [3H]-inositol to monitor arachidonic acid release and inositol phosphate accumulation respectively. Bradykinin and kallidin stimulated a dose and time-dependent release of arachidonic acid from stromal cells which, with 100 nmol/L bradykinin, was 30-150% above basal release and maximal at 5 min. Glands were less responsive; 100 nmol/L bradykinin (at 5 min) caused a release of AA of 30-69% above basal level. Bradykinin also stimulated a dose dependent increase in inositol monophosphate production. The maximum response with stromal cells was 8- to 10-fold and with glands, 2-fold (1 and 100 nmol/L bradykinin, respectively). Kallidin was equipotent to bradykinin with respect to both AA and inositol phosphate accumulation. The bradykinin analogue des Arg bradykinin (which acts through the B1 receptor) released AA from stromal cells but did not alter phosphoinositide hydrolysis, suggesting that these two cellular responses are mediated by different receptors (B1 and B2 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of bond cleavages at kallidin release by tissue kallikrein: cleavage of two peptide bonds in a single enzyme-substrate complex?

The kinetics of the release of kallidin, L- and KL-chains from bovine L-kininogen by porcine tissue kallikrein were followed and individual kinetic constants for cleavage of the Met-360 and the Arg-370 bond determined. The results suggest that both these bonds in L-kininogen r are hydrolyzed "simultaneously" without appearance of a free singly-nicked intermediate. Kallidin release in the human analogous system is also compatible with such a mechanism.