Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Inhibition of human renin by synthetic peptides derived from its prosegment

The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.     

Highly potent and specific inhibitors of human renin

We have designed and synthesized a series of small peptides containing a perfluoroalkyl ketone group at the C-terminal position of the angiotensin I sequence as inhibitors of human renin. From this series of compounds, 8 and 10 showed strong inhibition of human renin (IC₅₀ = 3 × 10⁻⁹, 7 × 10⁻⁹ M, respectively). Compound 10 did not inhibit pepsin and cathepsin D at 10⁻⁴ M. Comparison of the IC₅₀ of compound 8 and compound 11 (8.7 × 10⁻⁷ M) demonstrated the marked effect of the perfluoropropyl group on the potency of inhibition on renin, presumably due to the strong electron-withdrawing effect causing the ketone in 8 to exist predominantly as the hydrate — thus mimicking the tetrahedral transition state during hydrolysis of the scissile Leu¹⁰—Val¹¹ amide bond.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

Direct inhibitory effect of atriopeptin III on renin release in primate kidney

Patterns of in vitro renal renin release and the ability of atriopeptin to directly inhibit renin release have been examined in the rat, rabbit, and dog, but have been unstudied in the primate kidney. Accordingly, we examined renin release from superficial renal cortical slices of the squirrel monkey (Samiri sciuresus). The average age of the 5 animals was 10.2 +/- 2.5 yr at the time of study. Renin release was stimulated significantly by the beta-adrenergic agonist isoproterenol in concentrations of 10(-5) M (1.67-fold) and 10(-4) M (1.84-fold). Isoproterenol-induced renin release was inhibited by atriopeptin III (ANP, 2 X 10(-8) M) and the adenylate cyclase inhibitor dideoxadenosine (DDA, 10(-5) M). Similarly, the incubation of the superficial cortical slices with arachidonic acid (10(-3) M) resulted in a 4-fold increase in tissue renin release which was blocked by the calcium ionophore A23187 (17 X 10(-6) M) and ANP; interestingly, DDA did not block arachidonic acid-induced renin release. These results suggest that ANP exerts a direct inhibitory effect on B-adrenergic and arachidonic acid-induced renin release in the primate kidney. Further, the inhibitory action of A23187 on renin release suggests, as in other species, an integral role for intracellular calcium in the renin release process. These patterns of renin release in primate kidney are similar to those observed in the rodent kidney in vitro.     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

The effect of prostaglandin synthesis inhibition on the direct stimulation of renin release from rabbit renal cortical slices

Prostaglandins have been hypothesized to have several mechanistic functions in sympathetically mediated release of renin. The rabbit renal cortical slice system was chosen to examine the prostaglandin dependency of renin release directly stimulated by either a direct adenylate cyclase activator, forskolin, or a beta-agonist, isoproterenol. In this study, we demonstrate that with forskolin (1 X 10(-5) M) or isoproterenol (1 X 10(-6) M), renin release was elevated 2-3 fold above control, and that this increase was shown to accompany a substantial increase in the tissue levels of cAMP (19.5 fold and 3.5 fold respectively). We also demonstrate that the increase in renin release produced by these compounds was not inhibited by cyclooxygenase inhibitors, indomethacin (25 microM) or eicosatetraynoic acid (30 micrograms/ml), nor was it inhibited by the selective prostacyclin synthesis inhibitor, U-51605 (30 micrograms/ml). Each of these inhibitors was demonstrated to block the synthesis of prostaglandins in the cortical slices at the concentrations used. Thus we propose that prostaglandins do not play a role in the induction of renin release resulting from elevated cyclic nucleotide levels or beta-adrenergic stimulation.     

Selective inhibition by secosteroids of 5 alpha-reductase activity in human sex skin fibroblasts.

The effects of 5,10-secoestra-4,5-diene-3,10,17-trione (Compound I) and 5,10-seco-19-norpregna-4,5-diene,3,10,20-trione (Compound II) on the 5 alpha-reductase activity and on the androgen receptors of normal human sex skin fibroblasts were investigated. The Vmax and Km of the transformation of testosterone to 5 alpha-reduced products was 387 pg/microgram DNA/30 min and 234 X 10(-9)M, respectively. When the inhibitors were introduced in the assay, the 5 alpha-reductase activity was markedly reduced, Compound I being a less potent inhibitor than Compound II. At 15 min, the inhibition was greater than at 30 and 60 min. The Ki for Compound I was 1.60 x 10(-6)M with a Vmax of 83 to 553 pg/microgram DNA/30 min. For Compound II, the Ki was 0.53 x 10(-6)M with a Vmax of 70 to 340 pg/microgram DNA/30 min. The inhibition was of the noncompetitive type. Studies with androgen receptors showed that Compound I had a lower affinity for the receptors than Compound II. The ID50 for 3H-DHT and 3H-T for Compound I were 42.9 x 10(-7)M and 8.6 x 10(-7)M, respectively, whereas for Compound II, they were 10.6 x 10(-7)M and 4.8 x 10(-7)M.     

Effect of atriopeptin III on renin release in vitro

The ability of atriopeptin III (AP) to directly inhibit renal renin release has not been resolved. This issue was examined in a series of experiments performed in a system of rat renal cortical slices (dry weight 1.91 mg) in which the goal was to explore the effects of AP on renin release induced by cyclic AMP (cAMP)-coupled stimuli or by agents which are believed to decrease intracellular calcium (Cai). Concentration response relationships were initially established for all test agents. The cAMP stimuli utilized were isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (3 X 10(-4) M); each of these agents produced a significant increase in renin release in the system (with isoproterenol a 59% increase, with forskolin 37%, and with dibutyryl cAMP 52%). The addition of AP (2.09 X 10(-8) M, a minimum inhibitory concentration derived from preliminary studies) significantly blunted these increases; in the case of the dibutyryl cAMP-stimulated renin release, the inhibition was partial as a significant 25% increase in renin occurred in the presence of AP. The addition of the calcium channel blocking agent diltiazem (10(-4) M) resulted in a significant increase in renin release (364 to 567 ng X mg-1, p less than .05) which was not blocked by the addition of AP. Similarly, TMB-8 (0.6 X 10(-4) M), another agent thought to lower Cai, also resulted in increased renin release (455 to 810 ng X mg-1), p less than .01) which was also unaffected by the addition of the AP. In summary, these results show that AP is capable of partially inhibiting renin release in vitro, particularly renin release coupled to cAMP action. In contrast, renin release induced by a decline in Cai appears to be unaffected by the addition of AP.     

A potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

The in vitro binding of [3H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a Ki of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [3H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the KD was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [3H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [3H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [3H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.     

1-Amino-2-phenylethylphosphonic acid: an inhibitor of L-phenylalanine ammonia-lyase in vitro

L(-)-, and D(+)-enantiomers of 1-amino-2-phenylethylphosphonic acid (PheP), a phosphonic analogue of phenylalanine, inhibit the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) of potato tuber tissue in vitro. The apparent type of inhibition depends on concentration of PheP; as the concentration of D-PheP is raised from 10(-5) M to 2.5 X 10(-3) M, the type of inhibition shifts from competitive through mixed and non-competitive to uncompetitive. L-PheP exerts either a competitive or mixed-type inhibition at low (10(-6)-10(-5) M) or moderate (5 X 10(-5)-2 X 10(-4) M) concentration. Ki for the concentration range of competitive inhibition were 6.5 X 10(-6) M, 5.3 X 10(-5)M and 1.6 X 10(-5) M for L-, D-, and D,L-PheP, respectively. These Ki values are valid for a relatively narrow range of L-Phe concentration (0.2-4 mM) as L-phenylalanine ammonia-lyase does not follow the Michaelis-Menten kinetics of the reaction.     

Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin.

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.     

Detection and measurement by high-performance liquid chromatography of malondialdehyde crosslinks in DNA

Val-D-Leu-Pro-Phe-Phe-Val-D-Leu, a specific inhibitor of aspartate proteinases of the pepsin type, was synthesized. Its bonding to activated 6-aminohexanoic acid-Sepharose 4B afforded an affinity support suitable for the purification of human, porcine, and chicken pepsin, human gastricsin, and bovine cathepsin D. These enzymes bind to the support over the pH range 2-5 at 0-1.5 M concentration of NaCl. A buffer at pH greater than or equal to 6, low ionic strength, and containing 20% dioxane can serve as a general desorption agent. The proteinases were isolated from the crude extracts by a single-step procedure in a high degree of purity and in yields exceeding 70%; human pepsin, however, was not separated from human gastricsin. The support does not show any binding capacity for rat plasma renin at pH 7.4 and for some cysteine endopeptidases (cathepsin B, H, and L) at pH 3-5. The cathepsin D preparations isolated by affinity chromatography on the new support and on pepstatin-Sepharose were of the same degree of purity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequences, and specific activity.     

Interaction of mouse submaxillary gland renin with a statine-containing, subnanomolar, competitive inhibitor

The interaction between mouse submaxillary gland renin and a statine-containing, iodinated substrate analog inhibitor was studied. The compound, 1 (Boc-His-Pro-Phe-(4-iodo)-Phe-Sta-Leu-Phe-NH2, Sta = (3S,4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid), a statine-containing analog of the renin substrate octapeptide, was a competitive inhibitor of cleavage of synthetic tetradecapeptide renin substrate by mouse submaxillary gland renin, with a Ki of 6.2 x 10(-10) M (pH 7.2, 37 degrees C). Titration of the partial quenching of the tryptophan fluorescence of the enzyme by 1 revealed tight binding with a dissociation constant less than 3 nM and a binding stoichiometry of one mole 1 per mole enzyme. The time course of tight binding of 1 to mouse renin appeared to be fast, with kON greater than or equal to 1.3 x 10(6) s-1 M-1. The UV difference spectrum generated upon binding of 1 to mouse renin had two prominent features: a strong, broad band that had a minimum at 242 nm with delta epsilon (242) = -19,500 cm-1 M-1, and a triplet of enhanced bands centered at 286 nm with delta epsilon (286) about +1100 cm-1 M-1. The strong, broad, negative band was similar to the difference between the UV absorbance of 1 in methanol and in 0.1 M citrate phosphate pH 7.2. A structure-activity correlation for analogs of 1 showed some moieties of 1 that are important for potent inhibition of mouse renin. The inhibition data for these compounds versus human kidney renin suggested that the solution of the crystal structure of 1 bound to mouse renin will provide useful information for the design of inhibitors of human kidney renin.     

Species differences in the effect of isoproterenol and pindolol on renin release in vitro.

The effects of selective beta adrenergic receptor stimulation with isoproterenol (3 X 10(-8) M) and of beta adrenergic blockade with pindolol (3 X 10(-5) M) on the renin release in vitro were investigated in incubated canine and rat kidney slices. Bioassay was used to measure the renin content of the tissue samples and incubation media; renin content in the canine incubation medium was measured also by radioimmunoassay. Isoproterenol in a concentration of 3 X 10(-8) M brought about a significant increase in the renin content of the incubation media as well as the tissue slices obtained from canine kidney, however, there was no change in these parameters under similar conditions if rat kidneys were incubated. Pindolol, on the other hand, in a concentration of 3 X 10(-5) M caused a significant decrease in the renin release from as well as in the renin content of the rat kidney slices, while canine kidney slices failed to respond to the same dose of the drug. The differences between the two species is suggested to be due to the differences in basal renin levels.     

Structure-conformation relationships of synthetic peptide inhibitors of human renin studied by resonance energy transfer and molecular modeling

The structure-conformation relationships of a series of angiotensinogen6-13 (ANG6-13, His-Pro-Phe-His-Leu-Val-Ile-His) congeners substituted by Nin-For-Trp (Ftr), D-Ftr or Trp at the N-terminus, Tyr at the C-terminus and Phe psi[CH2NH]Phe at the P1-P'1 cleavage site (i.e. Leu10-Val11) were studied using resonance energy transfer coupled with molecular modeling of the peptide conformation using macromolecular energy refinement and dynamics simulation. Average end-to-end intramolecular distances (r) of the peptides in solution were determined by fluorescence spectroscopy. For example, Ac-Ftr-Pro-Phe-His-Phe psi[CH2NH]Phe-Val-Tyr-NH2 (U-70714E) gave an average intramolecular donor (Tyr)-acceptor (Ftr) distance of 16.3A in aqueous solution. This experimental value was consistent with a distance of 17.9 A determined by molecular modeling of U-70714E to a human renin 3-D structure (developed from known homologous aspartyl protease inhibitor X-ray crystallographic data) followed by simulation of the solution phase conformation of the peptide. An extended backbone secondary structure of U-70714E is suggested from these studies and the relationship(s) of structure-conformation to structure-activity was explored by analysis of several congeners of U-70714E, a potent (IC50 = 3.0 X 10(-9)M) inhibitor of human renin in vitro.     

Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.