Concentrations Of Epsilon-aminocaproic Acid Research Articles

Population Pharmacokinetics of Epsilon-Aminocaproic Acid in Infants Undergoing Craniofacial Reconstruction Surgery

Background Understanding the clinical pharmacology of the antifibrinolytic epsilon-aminocaproic acid (EACA) is necessary for rational drug administration in children. The aim of this study is to determine the pharmacokinetics (PKs) of EACA in infants aged 6–24 months undergoing craniofacial reconstruction surgery. Methods Cohorts of six infants were enrolled sequentially to one of the three escalating loading dose–continuous i.v. infusion (CIVI) regimens: 25 mg kg−1, 10 mg kg−1 h−1; 50 mg kg−1, 20 mg kg−1 h−1; 100 mg kg−1, 40 mg kg−1 h−1. Plasma EACA concentrations were determined using a validated high-performance liquid chromatography-tandem mass spectrometry assay. A population non-linear mixed effects modelling approach was used to characterize EACA PKs. Results Population PK parameters of EACA were estimated using a two-compartment disposition model with weight expressed as an allometric covariate and an age effect. The typical patient in this study had an age of 38.71 weeks and a weight of 8.82 kg. PK parameters for this typical patient were: pre-/postoperative plasma drug clearance of 32 ml min−1 (3.6 ml kg−1 min−1), inter-compartmental clearance of 42.4 ml min−1 (4.8 ml min−1 kg−1), central volume of distribution of 1.27 litre (0.14 litre kg−1), and peripheral volume of distribution of 2.53 litre (0.29 litre kg−1). Intra-operative clearance and central volume of distribution were 89% and 80% of the pre-/postoperative value, respectively. Conclusions EACA clearance increased with weight and age. The dependence of clearance on body weight supports weight-based dosing. Based on this study, a loading dose of 100 mg kg−1 followed by a CIVI of 40 mg kg−1 h−1 is appropriate to maintain target plasma EACA concentrations in children aged 6–24 months undergoing these procedures.

Therapeutic Plasma Concentrations of Epsilon Aminocaproic Acid and Tranexamic Acid in Horses

Antifibrinolytic drugs such as epsilon aminocaproic acid (EACA) and tranexamic acid (TEA) are used to treat various bleeding disorders in horses. Although horses are hypofibrinolytic compared to humans, dosing schemes have been derived from pharmacokinetic studies targeting plasma concentrations in humans. We hypothesized therapeutic plasma concentrations of antifibrinolytic drugs in horses would be significantly lower than in humans. Our objective was to use thromboleastography (TEG) and an in vitro model of hyperfibrinolysis to predict therapeutic concentrations of EACA and TEA in horses and humans. Citrated plasma collected from 24 random source clinically healthy research horses. Commercial pooled human citrated plasma with normal coagulation parameters was purchased. Minimum tissue plasminogen activator (tPA) concentration to induce complete fibrinolysis within 10 minutes was determined using serial dilutions of tPA in equine plasma. Results used to create an in vitro hyperfibrinolysis model with equine and human citrated plasma, and the minimum concentrations of EACA and TEA required to completely inhibit fibrinolysis for 30 minutes (estimated therapeutic concentrations) determined using serial dilutions of the drugs. Estimated therapeutic concentrations of EACA and TEA were significantly lower in horses (5.82; 95% CI 3.77-7.86 μg/mL and 0.512; 95% CI 0.277-0.748 μg/mL) than in humans (113.2; 95% CI 95.8-130.6 μg/mL and 11.4; 95% CI 8.62-14.1 μg/mL). Current dosing schemes for EACA and TEA in horses may be as much as 20× higher than necessary, potentially increasing cost of treatment and risk of adverse effects.

Population pharmacokinetics of epsilon-aminocaproic acid in infants undergoing craniofacial reconstruction surgery

Understanding the clinical pharmacology of the antifibrinolytic epsilon-aminocaproic acid (EACA) is necessary for rational drug administration in children. The aim of this study is to determine the pharmacokinetics (PKs) of EACA in infants aged 6-24 months undergoing craniofacial reconstruction surgery. Cohorts of six infants were enrolled sequentially to one of the three escalating loading dose-continuous i.v. infusion (CIVI) regimens: 25 mg kg(-1), 10 mg kg(-1) h(-1); 50 mg kg(-1), 20 mg kg(-1) h(-1); 100 mg kg(-1), 40 mg kg(-1) h(-1). Plasma EACA concentrations were determined using a validated high-performance liquid chromatography-tandem mass spectrometry assay. A population non-linear mixed effects modelling approach was used to characterize EACA PKs. Population PK parameters of EACA were estimated using a two-compartment disposition model with weight expressed as an allometric covariate and an age effect. The typical patient in this study had an age of 38.71 weeks and a weight of 8.82 kg. PK parameters for this typical patient were: pre-/postoperative plasma drug clearance of 32 ml min(-1) (3.6 ml kg(-1) min(-1)), inter-compartmental clearance of 42.4 ml min(-1) (4.8 ml min(-1) kg(-1)), central volume of distribution of 1.27 litre (0.14 litre kg(-1)), and peripheral volume of distribution of 2.53 litre (0.29 litre kg(-1)). Intra-operative clearance and central volume of distribution were 89% and 80% of the pre-/postoperative value, respectively. EACA clearance increased with weight and age. The dependence of clearance on body weight supports weight-based dosing. Based on this study, a loading dose of 100 mg kg(-1) followed by a CIVI of 40 mg kg(-1) h(-1) is appropriate to maintain target plasma EACA concentrations in children aged 6-24 months undergoing these procedures.

The Effective Concentration of Epsilon-Aminocaproic Acid for Inhibition of Fibrinolysis in Neonatal Plasma in Vitro

Pediatric patients, particularly neonates, are at high risk for bleeding complications after cardiovascular surgery because of their immature hemostatic system, small size, and the complex operations they require. Activation of intravascular fibrinolysis is one of the principle effects of cardiopulmonary bypass that causes poor postoperative hemostasis. This complication has long been recognized and treated with antifibrinolytic medications, including the lysine analog epsilon aminocaproic acid (EACA). The therapeutic plasma concentration of EACA has been scientifically determined for the adult population, but the current recommended dosage for neonates has been empirically derived from adult studies. Therefore, we investigated the appropriate concentration of EACA for neonates undergoing bypass. We conducted an in vitro study using neonatal plasma derived from the placenta/cord units from 20 term, elective cesarean deliveries. Graded concentrations of EACA were added to aliquots of the plasma pool before activating fibrinolysis with tissue-type plasminogen activator. Standard kaolin-activated thromboelastograms were then run with the primary outcome variable being estimated percent lysis. These procedures were repeated on samples of commercially available pooled adult normal plasma for comparison. We found that neonatal plasma required significantly lower concentrations of EACA to completely prevent fibrinolysis than did adult plasma (44.2 microg/mL and 47.8 microg/mL for neonatal plasma and 94.4 and 131.4 microg/mL in adult plasma for 400 and 1000 U/mL of plasminogen activator, respectively, P < 0.001). Our data establish the minimal effective concentration of EACA necessary to completely prevent fibrinolysis in neonatal blood in vitro. This concentration is significantly less than that targeted by current dosing schemes, indicating that neonates are possibly being exposed to greater levels of EACA than is clinically necessary.

ε-Aminocaproic acid inhibition of fibrinolysis in vitro: should the ‘therapeutic’ concentration be reconsidered?

The therapeutic concentration of epsilon-aminocaproic acid (EACA) has been 130 microg/ml or greater for nearly 50 years. We tested the effects on clot growth/disintegration of EACA with a plasmatic model of hyperfibrinolysis in vitro. Human plasma was exposed to 1000 U/ml tissue-type plasminogen activator containing 0, 13, 65 or 130 microg/ml EACA, with clot growth/disintegration kinetics quantified via thrombelastography. Data were analyzed with one-way analysis of variance or Kruskal-Wallis analysis of variance as appropriate. Exposure of plasma to 1000 U/ml tissue-type plasminogen activator resulted in a brief-lived clot, lasting 2 min. EACA at all concentrations tested significantly increased the rate of clot growth compared with samples with 0 microg/ml EACA. Clot strength was significantly increased by EACA in a concentration-dependent fashion. Similarly, EACA significantly prolonged the time of onset of clot lysis and decreased the rate of lysis. Samples with 130 microg/ml EACA had no sign of lysis present for 30 min. Subtherapeutic to therapeutic concentrations of EACA significantly attenuated or abolished fibrinolysis in the presence of a concentration of tissue-type plasminogen activator more than 2000-fold that encountered systemically during cardiopulmonary bypass. Further clinical investigation is warranted to determine whether smaller concentrations of EACA could provide a reduction in bleeding with a concomitant decrease in thrombotic complications.

The fibrinolytic system attenuates vascular tone: effects of tissue plasminogen activator (tPA) and aminocaproic acid on renal microcirculation.

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.

Epsilon-Aminocaproic acid plasma levels during cardiopulmonary bypass.

epsilon-Aminocaproic acid (EACA) concentrations achieved during cardiopulmonary bypass (CPB) have not been previously reported. It is unknown whether plasma concentrations reported to inhibit fibrinolysis in vitro (130 microg/mL) are achieved or whether differences in these levels relate to variability in postoperative bleeding. EACA (total intraoperative dose 270 mg/kg) was administered to 27 patients undergoing cardiac reoperation. The plasma EACA concentration was measured by using high-pressure liquid chromatography: 1) 30 min after initiation of drug administration (baseline); 2) 30 min (CPB + 30) after initiation of CPB; 3) 90 min after initiation of CPB. (CPB + 90); and 4) at cardiopulmonary bypass termination (end CPB). Plasma EACA concentrations (microg/mL, min - max, mean +/- SD) were 276-998, 593 +/- 154 at baseline; 147-527, 302 +/- 95 at CPB + 30; 112-500, 314 +/- 100 at CPB + 90; and 84-537, 317 +/- 100 at end CPB. Twenty-four-hour postoperative thoracic drainage and allogeneic red blood cell transfusions were not associated with plasma levels at any time. Although plasma EACA concentrations greater than 130 microg/mL were consistently achieved, we observed a marked variability (more than sixfold) in plasma concentrations and bleeding outcomes despite the use of a weight-based dosing regimen. This variability in drug levels appears to have little relevance to bleeding outcomes, possibly since mean plasma levels exceeded 130 microg/mL during CPB, and nearly all patients (26 of 27) achieved that target level.

Role of tryptophan-63 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability, folding, and ligand binding properties.

Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2tPA), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties of this isolated protein domain. The variants were expressed in Pichia pastoris cells. The binding constants of epsilon-aminocaproic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrations of the alterations in intrinsic fluorescence of the variant kringles with the ligands. As compared to wild-type r-K2tPA, increases in the Kd (dissociation) values of approximately 15-fold and 20-200-fold were found for the W63F and W63Y mutants, respectively, toward these three ligands. Neither the W63H nor the W63S variant interacted with these same ligands. Differential scanning calorimetric analyses were also performed on each of the peptides to determine whether the alterations affected the conformational stability of wtr-K2tPA. The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (Tm) values that were 12-20 degrees C lower than that of wtr-K2tPA. Addition of EACA resulted in increases (approximately 12 degrees C) in the Tm values of r-[W63F]-K2tPA and r-[W63Y]K2tPA, a result showing that EACA stabilized the native conformations adopted by these kringle domains. As expected from its greatly diminished binding to r-[W63H]K2tPA and r-[W63S]-K2tPA, high concentrations of EACA had little effect on the Tm of thermal denaturation of these latter mutants. 1H-NMR analysis of the two aromatic mutant kringles was employed to assess their overall comparative folding properties. The high upfield chemical shifts (-0.98 ppm) of the CH3(delta') protons of L47, a major signal of proper kringle folding, were slightly lowered to -0.83 to -0.86 ppm in the cases of all of the mutants. This is due to alterations in the W25-L47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distance relationships of these two amino acid side chains. Assignments of nearly all of the protons of the aromatic residues in the W63F and W63Y mutants were accomplished, and few additional differences from their wild-type counterpart were noted. Reactivities of the mutants against four different monoclonal antibodies directed to wtr-K2tPA revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W63. We conclude that W63 possesses an important direct role in the ligand binding properties of r-K2tPA. This residue also contributes significantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.

Contributions of Individual Kringle Domains toward Maintenance of the Chloride-Induced Tight Conformation of Human Glutamic Acid-1 Plasminogen

The roles of each of the three omega-amino acid-binding kringles (K) of Glu1-Pg, viz., [K1Pg], [K4Pg], and [K5Pg], in engendering the Cl(-)-induced alteration to its tight (T) conformation and in effecting the epsilon-aminocaproic acid (EACA)-mediated change to the relaxed (R) protein conformation have been investigated by mutagenesis strategies wherein the omega-amino acid ligand-binding energies in the individual kringles in recombinant (r)-Glu1-Pg were greatly reduced. This was accomplished in the most conservative manner possible by altering a critical Asp residue in each relevant kringle to Asn. The particular mutations chosen were r-[D139N]Glu1-Pg, r-[D413N]Glu1-Pg, and r-[D518N]Glu1-Pg, in which a conserved Asp residue at a homologous sequence position in each of the three kringle domains is eliminated. These changes also lead to a great reduction of the EACA-binding strength of [K1Pg], [K4Pg], and [K5Pg], respectively. The s0(20,w) of wild-type (wt) r-Glu1-Pg in the presence of levels of Cl(-)-sufficient to fully occupy its binding sites on this protein was 5.9 S, a value reduced to 4.9 S as a result of addition of saturating concentrations of EACA to the Cl-/Glu1-Pg complex. Neither Cl- nor EACA substantially altered the s0(20,w) value of 5.2 S for r-[D139N]Glu1-Pg (4.8 S) or r-[D413N]Glu1-Pg (4.5 S). On the other hand, the s0(20,w) value of 5.2 S for r-[D518N]Glu1-Pg at saturating levels of Cl- is slightly reduced to 4.8 S upon addition of binding maximal concentrations of EACA.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation.

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of in vitro fibrinogenolysis during laboratory monitoring of thrombolytic therapy with streptokinase or APSAC

In the present study, we systematically investigated aprotinin, epsilon-aminocaproic acid (EACA) and tranexamic acid as inhibitors of fibrinogen breakdown and of the generation of fibrinogen degradation products (FgDP). The experimental setting very closely imitated the conditions in practice when collecting blood from patients receiving thrombolytic therapy with streptokinase or APSAC. The minimal concentration of aprotinin required to completely inhibit fibrinogen breakdown and FgDP generation was 200 KIU/ml blood. This was sufficient even at the highest concentrations of streptokinase and APSAC expected to occur in patients (300 U/ml and 46 nM, respectively). However, 200 KIU/ml aprotinin heavily interfered in the determinations of plasminogen and alpha 2-antiplasmin. Relatively low concentrations of EACA (200 mM) and tranexamic acid (35 mM) were sufficient to prevent FgDP generation, but they interfered in the Clauss assay of fibrinogen. A non-interfering concentration of EACA (7 mM) allowed the inhibition of lower concentrations of APSAC (20 nM) and streptokinase. We conclude that at least 200 KIU aprotinin per ml blood is necessary to effectively inhibit in vitro fibrinogenolysis under circumstances likely to be met in clinical practice during thrombolytic therapy.

Analysis of the stabilizing effect of epsilon-aminocaproic acid by electrophoretic techniques and immunoblotting

The effect of epsilon-aminocaproic acid (EACA) on the degradation of aqueous pollen extracts was studied by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. The extracts were stored at 4 °C for 7 days and at 37 °C for 1, 4, and 7 days. Addition of 0.1 mol/L of EACA before storage partly protected the extract from degradation. The protective effect of EACA could be demonstrated most clearly by immunoblotting, suggesting that more epitopes were preserved in an antigenic configuration. The stabilizing effect increased with higher EACA concentrations.

Control of human plasminogen activation.

The activation of Glu1-plasminogen (Glu-Pg) by streptokinase (SK), urokinase (UK) and tissue plasminogen activator (tPA) is under rigorous control by molecules such as epsilon-aminocaproic acid (EACA), fibrinogen (Fg), fibrin (Fn) and, as we have recently discovered, anions. This presentation will focus on the biochemical mechanisms that are involved in these processes. In the case of activation by SK, a species of activator complex, composed of Glu-Pg and SK, can be identified that is inhibited by anions, such as Cl-, and stimulated by Fg and Fn. This species rapidly decays to another activator complex, also consisting of Glu-Pg and SK, that is much less sensitive to control by these effector molecules. The most stable activator complex, containing equimolar SK and plasmin, is not affected to a great extent by anions, Fg or Fn. In the overall activation of Glu-Pg by SK, Cl- behaves as a mixed inhibitor, with a Ki of 6.4-9.2 mM, and Fg functions as a mixed activator, displaying a Ka of 110-240 nM. These results show that activation of Glu-Pg by SK in physiological samples would be considerably inhibited by Cl- in the absence of Fg. The activation of Glu-Pg by both high- and low-molecular weight UK is also inhibited by Cl-, but is stimulated by EACA. The inhibition by Cl- does not occur in the presence of concentrations of EACA that saturate its weak binding sites on Glu-Pg, and the stimulation by EACA is maximally exhibited in the presence of Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Autologous fibrin tissue adhesive biodegration and systemic effects

A series of experiments was conducted to investigate the rate of Autologous Fibrin Tissue Adhesive (AFTA) degradation by the fibrinolysis inhibitor, epsilon amino caproic acid (EACA). The duration of AFTA clots in vitro, subcutaneous, and in the middle ear was prolonged for a time interval that was proportional to the concentration of EACA in Component II of the adhesive. No toxic reactions were observed in the middle or inner ear. Systemic pathology (thrombosis or emboli) could not be related to the presence of EACA applied in the middle ear or directly into the blood stream at concentrations (mg/kg body weight) up to 1,500 times that expected to occur during surgery on humans.

High does epsilon-aminocaproic acid prolongs the bleeding time and increases rebleeding and intraoperative hemorrhage in patients with subarachnoid hemorrhage

epsilon-Aminocaproic acid (EACA) has been used to prevent rebleeding in patients with intracranial aneurysms because it crosses the blood-brain barrier and is an inhibitor of fibrinolysis. Recommended doses have ranged from 24 to 48 g/day. We now describe an inhibitory effect on platelet function at the higher dose range. In vitro, a dose-dependent inhibition of adenosine diphosphate- and collagen-induced platelet aggregation was observed with concentrations of EACA beginning at 7.6 mM. In vivo, prolongation of the template bleeding time was observed in all eight patients receiving 48 g/day (greater than 20 minutes in four), in all five on 36 g/day (greater than 20 minutes in three), and in none of seven on smaller doses. More importantly, rebleeding and excessive intraoperative bleeding (requiring more than 1 litre of blood replacement) occurred predominantly in patients receiving the larger doses of EACA. Within 48 hours of the discontinuation of EACA, the bleeding times returned to normal values in all but one patient. We conclude that EACA exerts a dose-dependent inhibitory effect on platelet function and that patients receiving doses in excess of 24 g/day may be at risk of serious bleeding. Patients receiving EACA should be monitored with serial bleeding time tests.

Partial purification and characterization of keratokinase, the fibrinolytic activator of the cornea

Keratokinase (KK) is a fibrinolytic enzyme released by in vitro cultures of cornea. KK converts plasminogen into plasmin producing on the molecule of plasminogen the same pattern of limited proteolysis as urinary activator, urokinase (UK) does. The mol. wt. of KK is around 55 000 daltons, i.e. in the same range of that of UK. Like UK, but unlike tissue activator (TA, derived from vascular endothelium) KK shows a biphasic activity response to increasing concentrations of epsilon aminocaproic acid (EACA) and is scarcely bound to fibrin during clotting. KK may play a role in a number of pathophysiological states of the cornea such as corneal swelling, inflammation and collagen metabolism.

Isolation of normal human IgG3. Identical molecular weight for normal and monoclonal gamma-3 chains

Staphylococcal protein A has been shown to bind all IgG subclasses except IgG3 (Kronvall & Williams, 1969). This differential binding provides a method for the purification of normal IgG3, since chromatography of normal IgG on insolubilized protein A (e.g. protein A-Sepharose) should result in the elution of unbound IgG3 with neutral buffer. whereas IgGl. IgG2 and IgG4 molecules would be retained and would require a low pH for elution. This procedure was first used by Hjelm (1975) but his estimate of 56.000 for the mol. wt of normal gamma-3 chains was significantly lower than the previously reported mol. w’t of 59.50&60,950 for gamma-3 chains released by monoclonal IgG3 proteins (Saluk & Clem, 1971 ; Virella & Parkhouse, 1972). This discrepancy led us to attempt to repeat the purification of normal IgG3 and to determine the mol. wt of normal gamma-3 chains. Normal IgG was separated from the freshly collected serum of a normal donor by precipitation with ammonium sulfate at 33”)” saturation followed by ion-exchange chromatography in DE-52 (Whatman) using 0.01 A4 sodium phosphate buffer, pH 6.5. for the elution of IgG. This is a standard procedure in our laboratory, which has been found to yield IgG free of contamination with other proteins or immunoglobulins. The purified IgG was chromatographed in a protein A-Sepharose (Pharmacia) column measuring 0.9 x Scm. with a bed volume of 4.3 ml. Unbound protein was washed with 0.1 M sodium phosphate buffer, pH 7.0. to which 0.2 M epsilon-aminocaproic acid was added to protect IgG3 from proteolytic degradation (Virella & Yeh. 1977). Bound protein was eluted with 1 A4 acetic acid containing the same concentration of epsilon-aminocaproic acid. Fractions containing unbound and bound protein were pooled separately and concentrated by negative pressure ultrafiltration, with simultaneous dialysis against phosphate-buffered saline. The concentrated and neutralized pools were then characterized for their IgG subclass content by double immunodiffusion (Ouchterlony, 1962) in 17, (w/v) Noble agar (Difco) in 0.3 M potassium phosphate buffer. pH 8.0, using sheep antisera to IgG subclasses produced in the Department of Experimental Pathology, University of Birmingham, U.K. and absorbed in our laboratory (except for anti-IgGl, which was provided in absorbed form). The molecular weights of the heavy chains released by the bound and unbound fractions of normal human IgG

The effect of epsilon-aminocaproic acid on biochemical changes in the development of the cellular slime mould Dictyostelium discoideum.

epsilon-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain AX2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of N-acetylglucosaminidase, alpha-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells.

Differentiation between Plasminogen Activators by Means of Epsilon- Aminocaproic Acid

SummaryThe differentiation between plasminogen activators from tissue and urine by means of the patterns of inhibition produced by epsilon-aminocaproic acid (EACA) in fibrin plate assays was investigated. Different inhibition patterns reflected genuine differences between two types of activators. The method allows the differentiation between two types of activators in weakly fibrinolytic and impure activator solutions.A biphasic pattern of inhibition by EACA was observed with human urine as well as with the purified urokinase preparations. The degree of enhancement of fibrinolysis observed in the millimolar concentration range of EACA fluctuated with the fibrin substrate and was, in part, related to its plasminogen content. Increasing concentrations of EACA produced a uniformly increasing inhibition of preparations of tissue activator obtained in various degrees of purity from porcine ovary and heart. Plasmin was uniformly inhibited by EACA. Inhibition of plasmin required about 100 times higher concentrations of EACA than inhibition of tissue activator-induced fibrinolysis. EACA produced a slightly biphasic pattern of inhibition with plasminogen activator from human uterine tissue.

Caseinolytic and Fibrinolytic Activities of Human Plasma in Starch Block Electrophoresis

SummaryHuman plasma was subjected to starch electrophoresis with veronal buffer at pH 8.6, μ = 0.05. The veronal buffer eluates of each of the 40 sections into which the block was cut were used for the study of caseinolytic and fibrinolytic activity after activation with streptokinase or urokinase. A high fibrinolytic activity was induced by streptokinase in the γ-globulin fraction. A high caseinolytic activity was induced by urokinase in the β-globulin fraction and by streptokinase in the α1-globulin fraction. Caseinolytic activity in the α1-globulin fraction was not inhibited by a concentration of epsilon-aminocaproic acid (2 × 10-3 M) which gave a marked inhibition of the fibrinolytic activity elicitable in the β- and γ-globulin fractions. Inhibition of the caseinolytic activity in the α1-globulin fraction was effected by dicarbobenzoxy-lysine (2 × 10-3 M) which had only a slight inhibitory effect on the fibrinolytic activity of the β- and γ-globulin fractions.