Separation of Plasma Thromboplastin Antecedent from Kallikrein by the Plasma α2-Macroglobulin, Kallikrein Inhibitor

Plasma thromboplastin antecedent (PTA, factor XI) is an important intermediate in the intrinsic coagulation system, and plasma kallikrein has been implicated as a mediator of the inflammatory process. Whereas their biologic activities are functionally distinct, their identity as separate entities in plasma has not been fully established, and the nature of their plasma inhibitors has not been completely characterized. A partially purified preparation containing the clotting, tosyl arginine methyl ester (TAMe) esterase and kinin-producing activities of these substances has been prepared by DEAE-cellulose chromatography of a Celite eluate obtained from acid-treated human plasma. These activities were not separable by acrylamide gel electrophoresis nor by isoelectric focusing, their pI being approximately 8.7. Human plasma alpha(2)-macroglobulin has been shown to inhibit the proteolytic activity of kallikrein and to inhibit partially its TAMe esterase activity. An alpha(2)-macroglobulin, PTA, kallikrein incubation mixture was separated by gel filtration chromatography. The alpha(2)-macroglobulin formed a high molecular weight complex with kallikrein and appeared in early chromatographic fractions. The PTA-clotting activity was not inhibited by the alpha(2)-macroglobulin; 64% of the initial PTA activity was isolated in later fractions free of kallikrein-induced kinin-like activity. In contrast, clotting, TAMe esterase, and kinin-forming activities were inhibited after gel filtration chromatography of an incubation mixture of these activities and partially purified C1 inactivator (C1 esterase inhibitor). Electrofocusing of an incubation mixture of an activated PTA, kallikrein preparation, and alpha(2)-macroglobulin resulted in the isolation of a PTA fraction free of kallikrein proteolytic activity, and with 4% of the original TAMe esterase activity. In this manner, activated PTA and plasma kallikrein have been shown to be distinct substances, and methods have been introduced for the further purification of active coagulation factor XI.     

STUDIES ON HUMAN PLASMA α2-MACROGLOBULIN-ENZYME INTERACTIONS : EVIDENCE FOR PROTEOLYTIC MODIFICATION OF THE SUBUNIT CHAIN STRUCTURE

Human plasma α₂-macroglobulin is an inhibitor of circulating proteases that function in hemostatic and inflammatory reactions but the biochemical nature of its interaction with these enzymes is not well defined. This investigation has found that α₂-macroglobulin is comprised of subunit chains of 185,000 molecular weight as analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Trypsin, thrombin, plasmin, and plasma kallikrein in amounts completely bound to α₂-macroglobulin attacked one region in the subunit chain producing a single derivative with a molecular weight of 85,000 indicating that hydrolysis occurred at or near the center of the parent chain. The proteolytic derivative was also identified in an α₂-macroglobulin preparation from plasma incubated with the plasminogen activator, urokinase. α₂-macroglobulin functionally capable of binding enzyme appeared to be required both for limiting tryptic hydrolysis and for confining the concentration dependent increase in the derivative chain to the 1st min of incubation since acid-denatured α₂-macroglobulin that failed to bind trypsin was extensively degraded. Three derivative chains resulted from the interaction of α₂-macroglobulin with chymotrypsin demonstrating the presence of at least two chymotrypsin susceptible regions in the precursor chain. Reduction of the α₂-macroglobulin-enzyme mixture was required for the identification of the derivative subunit chains establishing that these cleavage products were covalently linked to the parent molecule by disulfide bridges. Thus, α₂-inacroglobulin acts as a substrate for circulating proteases, a finding which may also pertain to the mechanism of action of other plasma enzyme inhibitors.     

Inhibitors of Kallikrein in Human Plasma

Human plasma was fractionated by ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration to determine which method would give the greatest number of clearly separable kallikrein inhibitory peaks. With G-200 gel filtration three peaks could be separated which were demonstrated to contain alpha(2)-macroglobulin, C1 inactivator, and alpha(1)-antitrypsin. No other kallikrein inhibitors could be identified. The fractions containing C1 inactivator and alpha(2)-macroglobulin appeared to be more effective against kallikrein than that containing alpha(1)-antitrypsin. A patient with hereditary angioneurotic edema was shown to have an abnormal C1 inactivator protein capable of interfering with kallikrein's biologic, but not its esterolytic activity. Heat-treated human plasma, a commonly used source of kininogen for experiments with kallikrein, was shown to have kallikrein inhibitory activity.     

SUBSTRATES OF HAGEMAN FACTOR : I. Isolation and Characterization of Human Factor XI (PTA) and Inhibition of the Activated Enzyme by α1-Antitrypsin

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α₁-chymotrypsin inhibitor, inter α-trypsin inhibitor, α₂-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α₁-antitrypsin, and it was absent in α₁-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α₁-antitrypsin.     

Catabolic Pathways for Streptokinase, Plasmin, and Streptokinase Activator Complex in Mice: IN VIVO REACTION OF PLASMINOGEN ACTIVATOR WITH α2-MACROGLOBULIN

The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. ¹²⁵I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and α₂-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either ¹²⁵I-plasminogen or ¹²⁵I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p′-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between α₂-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of α₂-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of ~ 2.5%). Substitution of human α₂-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37°C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol α₂-macroglobulin when activator complex was incubated at 37°C with α₂-macroglobulin for 40 min. Streptokinase transfer from activator complex to α₂-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the α₂-macroglobulin “bait region,” resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

Contribution of Plasma Protease Inhibitors to the Inactivation of Kallikrein in Plasma

Although l-inhibitor (l-INH) and α₂-macroglobulin (α₂M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in l-INH, or artificially depleted of α₂M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min⁻¹. This rate constant was reduced to 0.35, 0.30, and 0.06 min⁻¹, in plasma deficient respectively in l-INH, α₂M, or both inhibitors. Thus l-INH (42%) and α₂M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, ¹²⁵I-kallikrein (M_r 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of ¹²⁵I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M_r 370,000) with l-INH, while 48% formed a complex (M_r 850,000) with α₂M. After inactivation by l-INH-deficient plasma, >90% of the active ¹²⁵I-kallikrein was associated with α₂M. A similar proportion of the label was associated with l-INH in plasma deficient in α₂M. After inactivation by plasma deficient in both l-INH and α₂M, ¹²⁵I-kallikrein was found to form a complex of M_r 185,000. This latter complex, which may involve antithrombin III, α₁-protease inhibitor, and/or α₁-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either l-INH or α₂M, even after the addition of heparin (2 U/ml). These observations demonstrate that l-INH and α₂M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either l-INH or α₂M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.     

Studies on kallikrein-kinin system in plasma of patients with acute pancreatitis

The fact that esterolytic activity is significantly elevated in plasma of patients with acute pancreatitis and that it correlates with the serum amylase level was confirmed. No kinin activity, however, was detected in plasma. To characterize the esterolytic activity, patient and normal plasmas were chromatographed on Sephadex G-200 and the esterolytic and the kinin-forming activities of each fraction were examined. In the present study, it was speculated that during attacks of acute pancreatitis, kallikrein with amylase might be liberated into blood from the pancreas. It was confirmed that almost all of the kalli-krein liberated was combined with α₂-macroglobulin, and α₂-macroglobulin-bound kallikrein itself possessed kinin-forming activity.     

Cystic fibrosis α2-macroglobulin protease interaction in vitro

α₂-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native az-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control α₂-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsinbinding capacities of control and cystic fibrosis α₂-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-α₂-macroglobulin complexes.     

MECHANISMS OF ACTIVATION OF C''1 ESTERASE IN HEREDITARY ANGIONEUROTIC EDEMA PLASMA IN VITRO : THE ROLE OF HAGEMAN FACTOR, A CLOT-PROMOTING AGENT

The generation of C'1 esterase activity in siliconed plasma obtained from individuals with hereditary angioneurotic edema in remission tends to occur spontaneously, but can be hastened during its incubation with preparations of activated Hageman factor. This effect of activated Hageman factor could not be shown during its incubation with normal siliconed plasma, nor could consumption of normal serum inhibition of C'1 esterase be clearly shown. Soy bean trypsin inhibitor and heparin could impair this enhanced generation of C'1 esterase but neither inhibits the esterolytic function of C'1 esterase once formed. Trasylol was less effective in blocking this effect of activated Hageman factor. While the mechanism of the effect of activated Hageman factor upon C'1 activation remains obscure, it is apparent that some intermediate steps, possibly involving a kinin-forming system of plasma, may play a role.     

PLASMA PREKALLIKREIN: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION

The precursor of the kinin-forming enzyme, prekallikrein, was isolated from rabbit plasma protected from activation during preparatory procedures. Prekallikrein was shown to be a 4.5S γ₁-glycoprotein with an isoelectric point of 5.9 and a mol wt of 99,900. The proenzyme was activated at neutral pH by an enzyme from rabbit or human plasma we have termed prekallikrein activator (PKA) or by trypsin. Prekallikrein was activated by PKA by a process of enzymatic scission. This resulted in the appearance of two fragments; the larger of these possessed kallikrein activity.     

C'1 ESTERASE EFFECT ON ACTIVITY AND PHYSICOCHEMICAL PROPERTIES OF THE FOURTH COMPONENT OF COMPLEMENT

Highly purified C'1 esterase of human serum is capable of inactivating isolated fourth component of human complement (β_1E-globulin). Inactivation is accompanied by changes in electrophoretic and ultracentrifugal properties of β_1E-globulin. If non-sensitized sheep erythrocytes are present during the action of C'1 esterase on β_1E-globulin, a complex is formed consisting of cells and cytolytically active fourth component (EC'4). Thus, inactivation of β_1E-globulin by C'1 esterase appears to be preceded by a state of activation enabling β_1E-molecules to combine with cell membrane receptors. Acceptor groups appear to be present also in 7S γ-globulin and in β_1E-globulin itself, since C'1 esterase can induce the formation of β-β and of β_1E-7S γ-globulin complexes.     

Inactivation of Factor XIa by Plasma Protease Inhibitors: PREDOMINANT ROLE OF α1-PROTEASE INHIBITOR AND PROTECTIVE EFFECT OF HIGH MOLECULAR WEIGHT KININOGEN

Factor XIa is a plasma protease that, by activating Factor IX, plays an important role in the early phase of the intrinsic pathway of blood coagulation. Four plasma protease inhibitors, α₁-protease inhibitor, antithrombin III, C1-inhibitor, and α₂-plasmin inhibitor, have been reported to inactivate human Factor XIa, but their quantitative contribution to the inactivation of Factor XIa in plasma has not been fully assessed. Using purified systems, we observed that the second-order rate constants for the reaction of Factor XIa with α₁-protease inhibitor, antithrombin III, and CI-inhibitor were 4.08, 10, and 14.6 M⁻¹ min⁻¹ × 10³, respectively. The pseudo-first-order rate constants, at plasma concentration of the inhibitors, were 1.86 × 10⁻¹, 4.68 × 10⁻², and 2.4 × 10⁻² min⁻¹, respectively. These kinetic data predict that α₁-protease inhibitor should account for 68%, antithrombin III for 16%, and C1-inhibitor and the equipotent α₂-plasmin inhibitor each for 8% of the total inhibitory activity of plasma against Factor XIa. The rate of inactivation of Factor XIa in various plasma samples specifically deficient in inhibitors was consistent with these predictions.     

Immunochemical Studies of Plasma Kallikrein

A monospecific antibody against human plasma kallikrein has been prepared in rabbits with kallikrein further purified to remove gamma globulins. The antisera produced contained antikallikrein and also anti-IgG, in spite of only 8% contamination of kallikrein preparation with IgG. The latter antibody was removed by adsorption of antisera with either Fletcher factor-deficient plasma or with purified IgG. Both kallikrein and prekallikrein (in plasma) cross-react with the antibody with no apparent difference between the precipitation arcs developed during immunoelectrophoresis and no significant difference in reactivity when quantified by radial immunodiffusion.Kallikrein antibody partially inhibits the esterolytic and fully inhibits the proteolytic activity of kallikrein. In addition, the antibody inhibits the activation of prekallikrein, as measured by esterase or kinin release. The magnitude of the inhibition is related to the molecular weight of the activator used. Thus, for the four activators tested, the greatest inhibition is observed with kaolin and factor XII(A), while large activator and the low molecular weight prekallikrein activators are less inhibited.With the kallikrein antibody, the incubation of kallikrein with either plasma or partially purified C1 esterase inactivator results in a new precipitin arc, as detected by immunoelectrophoresis. This finding provides physical evidence for the interaction of the enzyme and inhibitor. No new arc could be demonstrated between kallikrein and alpha(2)-macroglobulin, or alpha(1)-antitrypsin, although the concentration of free kallikrein antigen decreases after interaction with the former inhibitor.By radial immunodiffusion, plasma from healthy individuals contained 103+/-13 mug/ml prekallikrein antigen. Although in mild liver disease, functional and immunologic kallikrein are proportionally depressed, the levels of prekallikrein antigen in plasma samples from patients with severe liver disease remains 40% of normal, while the functional kallikrein activity was about 8%. These observations suggest that the livers of these patients have synthesized a proenzyme that cannot be converted to active kallikrein.     

ASSOCIATION OF "NATURAL" ANTIBODIES TO GRAM-NEGATIVE BACTERIA WITH THE γ1-MACROGLOBULINS

"Natural" antibodies to representative species of Enterobacteriaceae were found to be principally associated with the γ₁-macroglobulin prepared from Cohn fraction III-I of pooled human plasma. The amount of antibodies present was estimated by measuring the bactericidal, hemagglutinating, mouse-protective, and opsonophagocytic activities of this fraction. Comparison with the antibody activity of γ₂-globulin of Cohn fraction II showed that up to 100 times more antibody activity on a weight basis was present in the macroglobulin fraction. These findings suggest that differences between natural and immune antibody result from the physicochemical properties of 2 different classes of immune globulin.     

Significance of Cross-Linking of α2-Plasmin Inhibitor to Fibrin in Inhibition of Fibrinolysis and in Hemostasis

When blood is clotted, α₂-plasmin inhibitor (α₂PI) is cross-linked to fibrin by activated fibrin-stabilizing factor (activated coagulation Factor XIII, plasma transglutaminase). The amount of cross-linked α₂-PI is proportional to the amount of α₂PI present at the time of clotting. Plasma from a patient with congenital deficiency of α₂PI was supplemented with various amounts of purified α₂PI. Clots were prepared from these plasmas and were suspended in plasma containing a normal concentration of α₂PI, and spontaneous clot lysis was observed. When the clot was formed in the presence of calcium ions and thereby allowing cross-linking to occur, the rate and extent of fibrinolysis were found to be inversely proportional to the concentrations of α₂PI present in the clot at the time of clotting. When the clot was formed in the absence of calcium ions so that no cross-linking occurred, the clot underwent fibrinolysis at similar rates, regardless of the concentrations of α₂PI in the clot. When the clot formed in the presence of calcium ions was squeezed and washed to remove unbound proteins before being suspended in plasma, the extent of fibrinolysis was also inversely proportional to the amount of α₂PI cross-linked to fibrin. Similar results were obtained when the clot was suspended in buffered saline instead of plasma. These observations suggest that spontaneous fibrinolysis is mainly carried out by plasminogen/plasminogen activator bound to fibrin, and this fibrinolysis caused by fibrin-associated activation of plasminogen was mainly inhibited by α₂PI cross-linked to fibrin. To further support this concept, α₂PI treated with activated fibrin-stabilizing factor and that had lost most of its cross-linking capacity was used in similar experiments. This modified α₂PI had the same inhibitory activity on plasmin as the native inhibitor, but gave significantly less inhibition of fibrinolysis in every experiment, particularly when the clot was compacted by platelet-mediated clot retraction or by squeezing. Thus, it was concluded that α₂PI cross-linked to fibrin plays a significant role in inhibition of physiologically occurring fibrinolysis. It is further suggested that the absence of cross-linked α₂PI contributes to accelerated fibrinolysis and hemorrhagic tendency in patients with congenital deficiency of fibrin-stabilizing factor.     

The contribution of α1-antitrypsin to the total antitrypsin activity of human serum. a study of two phenotype pi oo individuals

Comparison of the levels of α₁AT, α₂-M, inter α-AT, C₁ inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate α₁AT activities and two α₂-AT-deficient persons show that α₁AT contributes more than 90% of the total antitrypsin activity of normal plasma.AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of α₂-M is not assessed. C₁ inactivator and inter α₁-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

KHM-1, a novel plasmid-mediated metallo-beta-lactamase from a Citrobacter freundii clinical isolate

A novel gene, bla_KHM-1, encoding a metallo-β-lactamase, KHM-1, was cloned from a clinical isolate of Citrobacter freundii resistant to most β-lactam antibiotics. Escherichia coli expressing bla_KHM-1 was resistant to all broad-spectrum β-lactams except for monobactams and showed reduced susceptibility to carbapenems. Recombinant KHM-1 exhibited EDTA-inhibitable hydrolytic activity against most β-lactams, with an overall preference for cephalosporins.     

ENZYMATICALLY PRODUCED SUBUNITS OF PROTEINS FORMED BY PLASMA CELLS IN MICE : II. β2A-MYELOMA PROTEIN AND BENCE JONES PROTEIN

The relationship of Bence Jones protein (mol wt = 45,000) to a β_2A-myeloma protein (mol wt = 160,000) formed by the same mouse plasma cell tumor (MPC-2) was investigated. The β_2A-myeloma protein was split by treatment with papain and cysteine into fragments (S_20,w = 3.7S), similar in size to the Bence Jones protein (S_20,w = 3.6S). Two types of fragments with distinct antigenic groupings designated S and F, were present in the MPC-2 myeloma protein digest. These were partially separated by DEAE-cellulose chromatography. The Bence Jones protein was found to share antigenic determinants with S fragments from the MPC-2 β_2A-myeloma protein and with S fragments from γ-globulins. Physicochemical observations indicated, however, that the Bence Jones protein was not identical to the globulin fragments produced by treatment with papain and cysteine. Comparison of the S and F fragments from β_2A- and γ-globulins revealed that the antigenic features shared by the various globulins derived from plasma cells (γ- and β_2A-myeloma proteins, the range of normal γ-globulins) are largely properties of the S fragments, whereas the distinctive antigenic differences between the γ- and β_2A-myeloma proteins were properties which appeared in the F fragments of the molecules.     

Evidence for a Direct Stimulatory Effect of Prostacyclin on Renin Release in Man

The objectives of this investigation were: (a) to characterize the time and dose dependence of the effects of prostacyclin (PGI₂) on renin release in healthy men; (b) to define whether PGI₂-induced renin release is secondary to hemodynamic changes; (c) to determine the plasma and urine concentrations of 6-keto-PGF_1α (the stable breakdown product of PGI₂) associated with renin release induced by exogenous or pharmacologically enhanced endogenous PGI₂. Intravenous PGI₂ or 6-keto-PGF_1α infusions at nominal rates of 2.5, 5.0, 10.0, and 20.0 ng/kg per min were performed in each of six normal human subjects; in three of them, PGI₂ infusion was repeated after β-adrenergic blockade and cyclooxygenase inhibition. PGI₂, but not 6-keto-PGF_1α, caused a time- and dose-dependent increase of plasma renin activity, which reached statistical significance at 5.0 ng/kg per min and was still significantly elevated 30 min after discontinuing the infusion. Although combined propranolol and indomethacin treatment significantly enhanced the hypotensive effects of infused PGI₂, it did not modify the dose-related pattern of PGI₂-induced renin release.