Effects of PGI2 on the inactivation of thrombin, factor Xa, and plasmin by antithrombin-III and heparin.

The influence of PGI2 on the activity and on the inactivation of enzymes participating in blood coagulation (thrombin and Factor Xa) and fibrinolysis (plasmin) were investigated. According to the results PGI2 has no effect on the activity of Factor Xa and plasmin nor on the inactivation of these enzymes by antithrombin-III in the absence and presence of heparin at a concentration of PGI2 up to 400 micrograms/ml. An acceleration of the inactivation of thrombin by antithormbin-III was found in the presence of PGI2 within a concentration of 100-400 micrograms/ml without any effect on the heparin-accelerated inactivation of thrombin by antithrombin. We got similar results using clotting tests for the assay and the application of synthetic substrate for thrombin. This inactivation-accelerating effect of PGI2 on thrombin was only demonstratable at a concentration five magnitudes higher than that of the anti-aggregation effect on platelets.     

An Anti-plasmin Inhibitor,Eckol, Isolated from the Brown Alga Ecklonia kurome Okamura

The biological activities of eckol, a novel phlorotannin with a dibenzo-β-dioxine skeleton, were examined. Eckol inhibited the antiplasmin activity of a₂-plasmin inhibitor very efficiently (IC₅₀; 1.6 μg/ml) as well as those of α₂-macroglobulin and -antitrypsin. However, its inhibitory effect on the antithrombin III-heparin complex was very weak. Eckol also showed inhibitory activity on thrombin (IC₅₀; 12 μg/ml), but not on plasmin. Its inhibitory activity was reduced in whole human plasma, but at concentrations of above 200 μg/ml it enhanced urokinase-induced fibrinolysis in human plasma. Studies on the inhibitory spectra of several derivatives of eckol showed that the dibenzo-l,4-dioxane skeleton was necessary for inhibition of plasmin inhibitor. These observations suggest that eckol or its derivatives may be useful clinically for potentiating thrombolytic activity.     

Protamine, a substrate for thrombolytic agents and enzymes of similar specificity.

Protamine, a highly purified basic polypeptide of 4000 molecular weight containing 80–85% arginine, is a useful substrate for the assay of plasmin, activated plasminogen, and enzymes of similar specificity, e.g., urokinase, coagulation factor Xa, trypsin, and thrombin, and is also an excellent secondary substrate for activator assays of urokinase and streptokinase. The assays were performed manually, and also automated procedures for continuous multiple sample analyses were used. The relative sensitivities for various plasmin-like enzymes were: trypsin > plasmin > urokinase > factor Xa > thrombin. Using protamine with manual assay procedures, the amino-terminal groups of the enzyme-degraded protamine digestion products were detected and quantitated by the colorimetric ninhydrin or the fluorometric fluorescamine procedures, and using protamine with an automated system the ninhydrin method was used. Assigning the CTA casein assay for plasmin a nominal sensitivity of 0.1 (for 0.1 CTA unit of plasmin), the sensitivities of the various assay methods were casein, automated protamine, and manual protamine with ninhydrin, 0.1; manual protamine-trichloroacetic acid with fluorescamine, 0.005; and manual protamine direct fluorescamine, 0.0005. A unit of plasmin, based on the uptake of 1 μequiv base/min during hydrolysis of 0.4% protamine sulfate under standard conditions, is equal to approximately 1.7 × 10³ RFI units or 2.9 CTA units; or, 1 CTA unit of plasmin resulted in an average uptake of 0.346 μmol of base or equivalent bonds split per minute.     

Inhibitory effects of interleukin-6 on release of PGI2 by cultured human pulmonary artery smooth muscle cells

We evaluated the effect of interleukin-6 (IL-6) on the production of prostacyclin (PGI₂) by cultured human pulmonary artery smooth muscle cells (HPASMC). Incubation of these cells for up to 48 h with IL-6 led to a dose- and time-dependent decrease in the concentration of PGI₂ in the culture medium. The incubation of HPASMC with 10 μg/ml of lipopolysaccharide (LPS), 200 U/ml of IL-1β or 500 U/ml of TNFα for 24 hr significantly increased the concentration of PGI₂ in the medium. However, the addition of IL-6 to a medium containing LPS, IL-1β, or TNFα significantly inhibited the stimulatory effect of those substances on PGI₂ production. Such inhibition was closely related to the concentration of IL-6. IL-6 may counteract the roles of LPS and of other cytokines on the regulation of pulmonary vascular tension in endotoxin- and cytokine-mediated disorders such as sepsis and the acute respiratory distress syndrome (ARDS).     

Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 10⁶ cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.     

Release of prostaglandin I2-like activity from the rat aorta: Effect of captopril,furosemide, and sodium

The effect of captopril, furosemide, indomethacine and intake of sodium on the production of PGI₂-like material was studied in the rat aorta. Release of PGI₂-like material from these vessels was estimated by its ability to inhibit ADP-induced vessels was estimated by its ability to inhibit ADP-induced platelet aggregation. Pretreatment with indomethacin (15 mg/kg/day) reduced the capacity of the aorta to release PGI₂-like material. Pretreatment with captopril (10 mg/kg/day) had no effect. Intravenous furosemide (60 μg/ml plasma volume) increased the capacity of the aorta to inhibit by 28% (p<.025). The inhibitory capacity of aorta removed from rats on a low sodium diet did not differ from those on a high sodium diet. We conclude that the action of furosemide in reducing vascular tone may be related to stimulation of PGI₂ synthesis in blood vessels whereas the effect of captopril and sodiumin in reducing vascular tone may involve a mechanism unrelated to PGI₂ synthesis or may involve the synthesis of a prostaglandin other than PGI₂.     

Inactivation of factor Va by plasmin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1α on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI₂ directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI₂-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF_1α were without effect on plasma progesterone levels. The luteotrophic effect of PGI₂ was not due to alterations in circulating LH concentrations. An $\text{in vitro}$ experiment assessed the effects of either PGI₂ alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 μg PGI₂, 10 μg PGI₂, and 10 μg PGI₂ plus 5 ng LH averaged 99 ± 42, 353 ± 70, 152 ± 35, 252 ± 45, and 287 ± 66 ng/ml (n=4), respectively. Thus PGI₂ has luteotrophic effects on the bovine CL both $\text{in vivo}$ and $\text{in vitro}$.     

Prostaglandin levels in BL6 melanoma cells cultured in vitro: the effect of vitamin E succinate supplementation

Malignant murine melanoma (BL6-F10) cells convert arachidonic acid primarily to PGD₂, PGF_2α, PGE₂, PGI₂ in descending order of magnitude. Supplementation with 1–10 μg/ml vitamin E succinate resulted in a significant (P ≤ 0.05) decrease in PGD₂ levels at vitamin concentrations of 3,5,7 and 10 μg/ml respectively, while PGF_2α levels were significantly decreased at 1,3,5 (P ≤ 0.05), 7 and 10 μg/ml (P ≤ 0.01) vitamin E succinate. BL6-F10 cells supplemented with 7 and 10 μg/ml vitamin E succinate showed a marked increase in PGE₂ levels with a significant increase occurring at 10 μg/ml (P ≤ 0.025). PGI₂ levels followed a similar trend to PGE₂ with a significant increase (P ≤ 0.05) occurring at 10 μg/ml.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

EG-626: Not a thromboxane A2 antagonist, but a PGI2 potentiator in platelet aggregation

Intracerebroventricular administration of PGI₂ or PGE₂ reduced aconitine-induced cardiac arrhythmia in rats. PGF_2α had no antiarrhythmic effect under the same conditions. The ED₅₀ values of PGI₂ and E₂ were 0.25 μg/kg and 1.1 μg/kg, respectively. Central mechanisms may participate in the antiarrhythmic effect of these PGs.     

Prostacyclin-induced hypothermia: Involvement of central histamine H2-receptors

Some of the biological activities of prostacyclin (PGI₂) are known to be mediated through cyclic AMP (cAMP). The purpose of this study was to assess the involvement of histamine and serotonin receptors as well as cAMP in the PGI₂-induced hypothermia in conscious guinea pig. Intracerebroventricular administration of 50–500 μg/kg PGI₂ produced a dose-related hypothermia, whereas its stable metabolite 6-keto prostaglandin F₁α had an insignificant effect. Low central doses (10–50 μg/kg) of dibutyryl cAMP (DBC) were hyperthermic, but high doses (100–500 μg/kg) caused hypothermia. Theophylline and low doses of DBC given centrally attenuated the PGI₂-induced hypothermia. Mepyramine and methysergide did not antagonize the effects of PGI₂ or DBC. However, central administration of metiamide (10–100 μg/kg) attenuated the hypothermic responses to both PGI₂ and DBC. These results suggest that histamine H₂-receptors are involved in the hypothermia induced by PGI₂.     

Effect of heparin on thrombin inactivation by antithrombin-III.

The inactivation of thrombin by heat and by its physiological inhibitor, antithrombin-III, shows quite different dependence on heparin concentration. Heparin at 250 microgram/ml protects thrombin against heat inactivation, and thrombin behaves heterogeneously in this reaction. In the absence of heparin, the thermodynamic activation parameters change with temperature (deltaH+ = 733 kJ/mol and 210 kJ/mol at 50 and 58 degrees C respectively). When heparin is present, heat inactivation of the protected thrombin species proceeds with deltaH+ = 88 kJ/mol and is independent of temperature in the same range. On the other hand, heparin at 0.125-2.5 microgram/ml accelerates the thrombin-antithrombin-III reaction. Thrombin does not show heterogeneity in this reaction and the time courses at any heparin concentration and any temperature between 0 and 37 degrees C appear to follow first-order kinetics. Activation enthalpy is independent of heparin concentration or temperature, deltaH+ = 82-101 kJ/mol, varying slightly with antithrombin-III concentration and thrombin specific activity. Heparin seems to exert its effect by increasing activation entropy. On the basis of these data we suggest a mechanism of action of heparin in the thrombin-antithrombin-III reaction which accounts for all the important features of the latter and seems to unify the different hypotheses that have been advanced.     

Role of heparin in the interaction of serine proteinases with antithrombin III.

To characterize the mode of action of heparin, the kinetics of inhibition of thrombin, factor Xa, and plasmin by antithrombin III was studied without and in the presence of heparin. Following the concentration dependence of inactivation a linear dependence was found between the apparent first-order inactivation rate constant and the anti-thrombin III concentration. This behaviour is typical of enzyme-activator interaction. Values of kinetic constants of the inactivation reaction could be determined. Thus, heparin acts obviously as an activator of the enzymes and enhances their affinity for antithrombin III.     

Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E1 and E2, but not of prostacyclin and prostaglandin D2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE₁ and PGE₂ could abolish all types of aggregation studied, whereas prostacyclin (PGI₂) and PGD₂ were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE₁ and PGE₂ inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently.The lack of any hemostatic function of PGI₂ in chickens was also indicated by the absence of biosynthesis of endogenous PGI₂ in chicken aorta. PGI₂ was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.