The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.

The seventeenth edition of Standards for Blood Banks and Transfusion Services 1 changed the transfusion expiration time for FFP from no more than 24 hours after thawing to up to 5 days when thawed plasma is used to treat coagulopathies other than factor VIII (FVIII) deficiency. Despite this, thawed plasma is frequently discarded at less than 5 days of age, most likely because of the belief that coagulation factor activity decreases during extended storage to clinically unsuitable levels. We designed a study to analyze the activity level of factors I (fibrinogen), II, V, VII, VIII, and X and in thawed plasma stored at 1 to 6°C for 5 days.Five units each of types A, B, and O FFP designated for study were thawed at 37°C, divided into five equal volumes in plastic bags, and stored at 1 to 6°C for 5 days. Daily aliquots of plasma from the divided volumes were frozen at -70°C. For each aliquot, factors II, V, VII, VIII, and X and fibrinogen were assayed by using factor-deficient commercial plasma (Dade Behring, Marburg, Germany) and an automated coagulation analyzer (Sysmex CA 6000, TOA Medical Electronics Company, Kobe, Japan). We used ABOspecific units, because FVIII is affected by ABO blood group.As expected, FVIII, being heat labile, had the greatest decline in activity, with a drop of 28 percent from the original level of activity within the first 24 hours of storage 2 (Table 1). Comparison of FVIII activity at Day 1 with that at Day 3 was significant (p values for types A, B, and O were <0.004, <0.02, and 0.001, respectively). While freeze-thaw cycles may affect factor activity levels to some extent, in this study, the freeze-thaw cycles were identical for all aliquots; hence, any decrease in FVIII would be similar. Therefore, the decrease in FVIII at 4°C during the study period represents a true decline in activity level from that after the freeze-thaw cycles. Because FFP is indicated to treat coagulopathy but not isolated FVIII deficiency, most patients requiring FFP, including liver disease patients, have clinically adequate or even high plasma levels of FVIII, which is an acute-phase reactant. 3,4 Factor V is also heat labile (37°C), but it appears to be stable at 4°C. All other clotting factors are stable for over 5 days. 5 Thawed plasma stored up to 5 days can safely be used to treat the coagulopathy of liver disease, thrombotic thrombocytopenic purpura, and disseminated intravascular coagulation and to reverse the effect of coumadin. Because all factors except fibrinogen decreased with extended storage, we elected at this time to use a more conservative expiration time of 3 days rather than 5 days. Extension to 3 days of the expiration time for thawed plasma in our institution reduced wastage from 5.1 percent with 24-hour storage at 4°C to 1.1 percent with 3-day storage, for a total estimated cost savings of $17,800 per year.

Background: The extent of preservation of clotting factors and incidence of transfusion reactions to noncommercial equine plasma is not documented.Hypothesis: Equine frozen plasma would retain its coagulation factor activity within the reference range and the incidence of transfusion reactions would be low.Animals: Ten plasma donor horses. Fifty clinically ill hospitalized horses receiving plasma were reviewed to determine the incidence of reactions.Methods: In vitro study and retrospective case review. Plasma was prepared by gravity sedimentation from whole blood refrigerated for 48 hours. The activities of factors VII through XII, antithrombin (AT), and Protein C were measured. Factor activities were compared for plasma samples obtained before blood collection (S0), after 48 hours of gravity sedimentation at 5 1C and after plasma separation (S1), and after 90 days of storage at À20 1C (S90). The medical records of 50 consecutive clinically ill horses receiving frozen plasma were reviewed to determine the incidence of transfusion reactions.Results: The combined effect of plasma harvest, gravity sedimentation, decantation, and freezing caused significant reductions in factors IX, (43% P 5 .0013), X, (33% P 5 .0001), XI, (48% P 5 .0008), AT, (10% P 5 .02), and Protein C (26% P 5 .0001). Activities for all factors analyzed, except factor X, remained within the reference ranges. Transfusion reactions were recorded for 5/50 horses.Conclusions and Clinical Relevance: Clotting factors, AT, and Protein C were well preserved. The incidence of reactions to frozen plasma was 10%.

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sullhydryl reagent 5,5'-dithiobis-(Znitrohenzoic acid) (DTNB). At 30°C or 37'C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, amhenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, amhenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate incrased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129RK231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129RK231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.

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GLUTAMATE AND POTASSIUM TRANSPORT 149 depleted of its normal potassium content, small increases occurred on incubation with the two substrates. 6. It is suggested that glucose (or lactate or pyruvate) is required as a source of energy; whilst L-glutamate serves another function in potassium transport. We wish to thank Dr W. Klyne for information on the flame photometer of Domingo & Klyne (1949) and for instruction in its use.

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