Choline inhibition of prothrombin activation

THROMBOSIS RESEARCH 62; 635648,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

CHOLINE INHIBITION

OF PROTHROMBIN

ACTIVATION

Richard D. Leach*, Sally A. DeWind, Charles W. Slattery and E. Clifford Herrmann Department of Biochemistry, School of Medicine, Loma Linda University, Loma Linda, California, U.S.A. 92350 (Received 451990;

accepted in revised form 12.3.1991 by Editor G.V.F. Seaman)

ABSTRACT A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amldolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely x.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki values (mM): 22+3 and 48k7 without factor Va, 25+4 and 41+4 with factor Va.

INTRODUCTION Expression of procoagulant activity by clotting factors commences when circulating proenzymes undergo distinct proteolytic events of the blood coagulation cascade. After this proteolytlc activation process, the catalytic activity of the PLdependent proteases 1s markedly enhanced when these enzymes become components of heteromolecular complexes (l-7). The two sequential cascade complexes Key Words: anticoagulant; choline; chromogenic peptide substrate; enzyme inhibitor; factor Va; prothrombinase Abbreviations: A400, absorbance at 400 nm; ACT, activated coagulation time; LB, Lineweaver-Burk; pNA, paranitroaniline; PC, phosphatidylcholine; PL, phospholipid; PS, phosphatidylserine; s, second; S-2238, Phe-Pipecolyl-Arg-p-nitroanilide *Present address: Department of Family Medicine, Riverside General Hospital University Medical Center, Riverside, CA 92503 $Author to whom correspondence should be addressed 635

636

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

containing serine roteases IXa (EC 3.421.22) and Xa (EC 3.4.21.6);known as intrinsic Xase or tenase (7,8s and prothrombinase (9) respectively; are similarly constituted of calcium ions, a negatively charged PL surface as in platelet membrane, and a nonenzymatrc second protein playing the role of cofactor (7). Factor Va is the second protein of the prothrombinase complex (2). Ultimate of the PL-dependent enzyme complexes in the cascade, and currently the most studied as well as best understood, rothrombinase (S-12) catalyzes the conversion of prothrombin to thrombin (E E 3.4215). Thrombin accumulation leading to patho enic thrombosis (13) is precursory to infarction, both myocardial and cerebral (14,lf ). Secondary to the thrombotic event one postischemic outcome is reperfusion inlury, the rapid irreversible tissue damage resulting from the production of reduced oxygen species (16-21). Problems in clinical medicine stemming from the multifarious thrombus formation process have elicited a spectrum of therapeutic and preventive modalities. On one hand, in treating acute infarction, the logic of administerin a thrombolytic agent, recombinant human tissue-type plasminogen activator 22,23), together with recombinant human P superoxide dismutase (1924), is to restore blood flow and to minimize the extent of reperfusion injury simultaneously. In contrast, anticoagulant approaches (25-28) focus on thromboprophylaxis. In this study we evaluated the anticoagulant role of choline, a small physiological quaternary amine molecule. Nath and Vaishwanar had re orted that choline incubation with human plasma prolonged the Stypven time (29.P Experiments with an in vitro system containing brain extract, fibrinogen, and prothrombin, led Norrid and Cooper to conclude that choline inhibits prothrombin conversion to thrombin (30). Following the lead of these studies we initiated (31-35) a systematic investigation to characterize the quantitative inhibitory effects of choline on blood coagulation. In order to perform these studies we used purified prothrombinase components in a system similar to that described by Mann (2,8,9). A computerinterfaced assay for prothrombin activation was developed, utilizing the same principle described by Kosow to measure activation of factor X (36) and plasminogen (37). We obtained kinetic data indicating that choline exerts a mixed-type of inhibition on the factor Xa catalyzed activation of prothrombin in the presence of Ca2+ and PL, whether factor Va was present or absent. Physiological protein anticoagulants have been classified into two groups (3S,39) on the basis of inhibitory activity toward rotease coagulation factors (39,40) or nonenzymatic protein cofactors ~$%e42~ Although choline is a nonprotein small molecule, it is an endobiotic which activation, de&ease’s V max and increases K,,, of factor Xa catalyzed prothrombin similar to vascular anticoagulant protein classed in the second group above (39). MATERIALS AND METHODS Reagents. Vacutainer tubes No. 6522) containing purified siliceous earth were from Becton, Dickinson and &ompany, Rutherford, NJ. Sodium barbital was purchased from Biochemical Laboratories, Redondo Beach, CA. The chromogenic peptide substrate S-2238 was obtained from KabiVitrum, Stockholm, Sweden. Egg yolk PC was from Calbiochem-Behring Diagnostics, La Jolla, CA. Bovine brain PS, polyethylene glycol 20,000 (PEG), three times recrystallized choline chloride, Russell’s viper venom (RVV), bovine factors IIa and Xa (RVV-Xa, bovine factor X activated by RVV), were from Sigma, St. Louis, MO. Factor V-deficient and factor II-deficient plasmas were from American Dade, Miami, FL. Thromboplastin was purchased from Dade Diagnostics, Aguada, PR. Polyst rene cuvettes with a 10 mm optical path were from VWR Scientific, Los Angeles, e A. All other reagents were of the highest grade commercially available. Protein Preparation. Human factor II was isolated as a by-product of factor IX purification according to the method of Bajaj et al. (43). The activity levels of factor

Vol. 62, No. 6

CHOLINE INHIBITS PROTHROMBINASE

637

II preparations were estimated by a factor IIdeficient plasma clotting assay with RVV as the activator. As described by Nesheim et al. (44), bovine factor V was isolated and its activity determined, following re-activation with thrombin, by correction of the prolonged prothrombin time o P factor V-deficient plasma. The definition we used of an activity unit (U) was that amount of factor Ha, Va, or Xa, were stored at cor$ained in one ml of normal human plasma. Protein preparations -20 C until used. PL Vesicle Preparation. The chloroform-methanol solvent of purchased PC and PS solutions was blown off under an Ar stream at room tern erature to constant weight. In a glass tube ap ropriate amounts of redissolved P 8 and PS were mixed in a three to one ratio (w/w 5 and solvent was a ain removed. Ice cold Verona1 buffer (733 mM sodium barbital-Cl at pH 735) was a d ded to the PL-coated tube at 1 ml per mg PL. Phospholipid vesicles (2) werg prepared by microtip sonication of the aqueous PL mixture for 9 minutes at 4 C with an output setting of 35 and a 50% pulsed duty cycle. Vesicles prepared in this manner, with sonicator model W-225R from Heat Systems-Ultrasonics, Inc, Plainview, NY, were used routinely in our experiments and simply referred to as PL. Incubation cuvettes with 300 or 500 ~1 assay Reaction Mixture Preparation. mixtures contained 7.l3 mM sodium barbital-Cl (pH 735), 2.5 mM CaC12, 100 pM S2238, and choline chloride at 0,15,30, or 45 mM. Appropriate amounts of NaCl were used to constitute a constant ionic strength with respect to low molecular weight solutes, 310 mOsm/L, within the range of normal human plasma (45,46). The amounts of non-protein macromolecular corn onents, expressed on a per ml basis, were 1 mg PEG and 0.1 mg PL (PC-PS, 31, w Pw). The quantities of factor Xa, and also of factor Va in experiments with the complete prothrombinase system, were constant in all the reaction mixtures from which an experimental data set was obtained. A data set consisted of each reaction mixture composition in duplicate and Fig. 1A portrays one. By adjustment within the concentration ranges appearing in the legend of Fig. 1, variability in activity among different preparations of factors Va and Xa was accommodated within the instrumental capabilities of the data acquisition system for monitoring the reaction rate. The concentrations of factor II were also varied, as described in the same legend. Enzyme mixtures were prepared in cuvettes on ice by combining CaC12, PEG, and factor Xa. Factor Va was an additional component in experiments with the complete prothrombinase system. Ten minutes later, choline chloride (when used), Verona1 buffer, NaCl, PEG, and PL were also pipetted into the cuvettes in that order. To diminish dissolved gas in each enzyme mixture, its cuvette was agitated under reduced pressure with a Bransonic 220 Ultrasonic Cleaner, from Branson Cleaning Equipment Company, Shelton, CT. This degassing prior to the assay period prevented bubble formation on cuvette walls that would have interfered with spectrophotoOmetric readings. The enzyme mixture was subsequently equilibrated at 37 C for 20 min. This incubation also provided conditions for activation of factor V (when present) by factor Xa (7,47). Substrate mixtures containing factor II, PEG, CaC12, NaCl, Verona1 buffer, and S-2238 were equilibrated at 37 C for 10 min. Both enzyme and substrate mixture temperature equilibration was scheduled to end synchronously! and thrombin production was initiated by addition of substrate mixture containmg prothrombin to the enzyme mixture. The cuvette contents were mixed before placement in the spectrophotometer. Optimizing Assay Conditions For Kinetic Experiments. The conditions of our in vitro assay system were carefully selected on the basis of preliminary experiments within a framework to simulate physiologic conditions in the reaction mixtures and to yield consistently reproducible results. Optimizing the addition order of reagents and coagulation factors to compose the reaction mixtures as well as determining an appropriate range of prothrombin concentrations were also on the basis of preliminary kinetic experiments as were adjustments in amounts of factors Xa and Va to achieve approximate activity standardization among experiments while utilizing different lots or preparations of the protein factors.

638

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

Rosing et. al. (48) and Lindhout et. al. (49) reported that when Ca2+ concentration was varied between 0 and 5 mM an optimum rate of prothrombin activation was observed at 3 mM. Because of its proximity to this reported optimum for in vitro prothrombin activation, we chose 25 mM, the mid-range Ca2+ concentration of normal human plasma. Rationale For Assay Of Prothrombin Activation. The assay method for prothrombin activation by factor Xa, without and with factor Va, was patterned after kinetic determinations for the activation of factor X (36) and plasminogen (37) developed by Kosow and coworkers. In our system the primary reaction components for kinetic experiments were factors Xa and II, Ca2+, and PL, in aqueous Verona1 buffer; also Factor Va in experiments with the complete prothrombinase complex. Under initial velocity conditions as factor Xa catalyzed the conversion of zymogen factor II into catalytically active thrombin at a constant rate on a PL surface, designated the primary reaction according to coupled enzyme assay terminology (50), the secondary indicator reaction simultaneously released pNA at a constant acceleration rate by thrombin-catalyzed hydrolysis of the saturating thrombin-specific chromogenic peptide substrate S-2238. Accumulation of pNA was monitored at 400nm. According to the mathematical rationale described by Kosow (37), the velocity of thrombin formation in our system was proportional to the slope, a/2, in the equation: A400 = v,t + at212

where linear acceleration is represented by a. Since the spontaneous hydrolytic rate of S-2238 (v,) was undetectable over the time these reaction mixtures were incubated, the v,t term of the acceleration equation became zero. Therefore, observed A400 values versus s2 (second) yielded a linearized acceleration plot (37) and its numerical a/2 slope value was defined arbitrarily as the quantitative velocity (v = A4&s2) of prothrombin activation in this study. Data Acquisition. Thrombin production was initiated at 20 s intervals in four incubation cuvettes. After starting the fourth assay reaction an automated data acquisition system1 was activated to serially monitor reaction progress in the assay cuvettes. An automatic cuvette positioner repetitively cycled the cuvettes in the Gilford spectrophotometer with a 7 s dwell time at each position. Constant temperatyre in the cuvette holding chamber was maintained by circulating liquid from a 37 C bath. Time based spectrophotometric data, A400 signals (proportional to accumulating and analyzed by a pNA) along with elapsed incubation time, were acquired Cromemco personal computer interfaced to the photometer and positioner. Each of the four assay cuvettes underwent one 7 s period of continuous spectrophotometric monitoring beginning at 28 s intervals, and this cycle was repeated for a maximum incubation duration of 10 min. One coordinate pair data point (x,y) per 7 s period The y was obtained by the data acquisition system m the following manner. coordinate, average A4m value from the 17 digitized analog output signals of the spectrophotometer during the 7 s period, was calculated and stored along with the x coordinate, s uare of elapsed reaction time midway through the 7 s dwell period (~2). Then for eat ?I of the four simultaneously incubated cuvettes, the software selected a data point set within a common time frame of assay reaction duration. A ty ical window spanned an elapsed assay time falling between approximately 90 and (!00 s, from initiation of individual reactions. Rationale For Data Analysis. Thrombin generated at a constant rate by factor Xa in the primary reaction for the time period of each assay, would produce an increase in PNA absorbance congruent with the acceleration function only so long as the indicator reaction was saturated with respect to S-2238. Data obtained from individual secondary indicator reactions were statistically analyzed for adherence of

‘For a description of the automated data acquisition Section of this issue of Thrombosis Research.

system see the Software

Survey

Vol. 62, No. 6

CHOLINE INHIBITS PROTHROMBINASE

639

temporally increasing A400 to the acceleration function. With coordinate pairs of sz and A400 values, the acceleration function (37) was transformed into linearized format thus permitting linear regression analysis. Consequently a resultant high correlation coefficient would imply closeness of fit of the indicator reaction progress to the acceleration function, and in turn, linearity of thrombin production in the primary reaction thus validating the a/2 slope values as a quantitative measurement proportional to the velocity of prothrombin activation. RESULTS Inhibition Of Whole Blood Coagulation By Choline. The anti-coagulant role of choline (29) was confirmed by determining its effect on the ACT of whole blood (51) obtained from volunteers in our laboratory. Using siliceous earth in vacutainers, the ACT of normal whole blood was 116_+10s. When choline was included at final concentrations of 30 mM and 45 mM, the ACT was increased by 17% to 134+3 s and by 23% to 142+12 s, respectively. Statistical Evaluation Of Prothrombin Activation Assays. To verify linearity of thrombin generation by the primary reaction in each assay cuvette, linear regression analysis via the software was applied to the set of 17 (usually) coordinate pairs, sr From the 40 correlation and Aqm values, obtained from the secondary reaction. coefficient values corresponding to points of the data set portrayed by Fig. lA, a

l/Factor II (ml/U)

l/Factor II (ml/U)

Fig. 1 LB plot demonstration of mixed-type inhibition by choline of prothrombin activation, without (panel A) and with (panel B) factor Va. Panel A shows data from one experimental set in which each incubation mixture contained 33 U factor Xa/ml. Factor II concentration was varied, 0.10, 0.20, 0.50, 0.75 or 1.0 U/ml, at fixed choline chloride concentrations. Panel B consists of composite data from 9 experimental sets with factor Va. Velocity data from the replicate experiments were normalized mathematically, based on the apparent Vmax value obtained with zero choline in each experimental set, to compensate for variations in activity among factors Va and Xa and PL preparations. The concentration levels of factor II were the lower four of those stated for panel A. Although constant within an experimental set, the concentrations of the other two protein factors ranged from 35 x lo-4 to 5.5 x lo-4 U factor Xa/ml and from 0.7 x D4 to 1.1 x lw U factor Va/ml in the 9 replicate experiments.

640

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

mean of 0.9962+.0005 (SE) was calculated. Similarly when factor Va was present, a mean correlation coefficient of 0.996+.006 (SD) was obtained from 307 assays. Most of those data points (n=288) are represented in Fig. 1B. Thus results from our experimental assays, in the absence and presence of factorva, did verify that the secondary reaction progress data consistently fit the acceleration function within high confidence limits and therefore the calculated numerical a/2 slope values provided valid quantitative estimates proportional to the velocity of prothrombin activation. Inhibition Of Prothrombin Activation By Choline. In control experiments, to evaluate potential interactions among various reaction mixture components, hydrolysis of the chromogenic substrate S-2238 was undetectable in the presence of either factors II or Xa alone, and choline did not inhibit catalytic hydrolysis of S2238 by factor IIa. Linearity, portrayed in the double reciprocal plots of Fig. 1, demonstrates Michaelis-Menten kinetics of factor Xa catalyzing thrombin formation in the absence (panel A) or presence (panel B) of factor Va. Linear regression analysis is consistent with visual inspection. For example, correlation coefficients of the 36 individual lines from 9 replicate experiments represented as a composite in Fig. 1B gave a mean value of 0.97k.02 (SD). To determine the best line positioning with a common intersection point, the sets of data represented in Fig. 1 were sublected to multiple regression forcing a number of intersecting lines through a single point (52). The observed intersection in the second quadrant is characteristic of general non-competitive (mixed) inhibition, exerted in this case by choline on prothrombin Vmax and an increase in activation, where there is both a decrease in apparent apparent K, (53). Since mixed inhibition was indicated by the LB plot in Fig. lA, slopes and ordinate intercepts therefrom were replotted versus choline concentration in Figs. 2A and 3A to determine inhibition constants (54) of choline for factor Xa (KJ and for the factor Xa - factor II complex (Ki). Characteristic of mixed-type inhibition,

Choline (mM)

Choline (mM)

Fig. 2 Graphical determination of choline inhibition constants, apparent Ki values, for factor Xa without (panel A) and with ( anel B) factor Va. Slopes of lines from the respective panels of Fig. 1 were plotte B versus choline concentration. Data in panel A were from the single experimental set described in Fig. 1A. Error bars delineate 95% confidence limits in Panel B which is composed of normalized data from the 9 replicate experiments of Fig. 1B.

Vol. 62, No. 6

CHOLINE INHIBITS PROTHROMBINASE

641

the apparent Ki (2253 mM choline) and Ki (4S+7 mM choline), mean values from 9 replicate experimental sets, were significantly different, p K: apparent Km value in the presence of inhibitor, we observed an increase from 0.13 U factor II per ml in the absence of inhibitor, to 0.26 U factor II per ml in the presence of 30 mM choline. Kinetic data from the total prothrombinase system produced an intersection point in the second quadrant of a Dixon plot (Fig. 4). The apparent Ki determined from this plot was 2754 mM choline (SE), in agreement with analysis by replot of LB slo es shown in Fig. 2B. With mixed inhibition data, linearity of the Dixon plot in a icated that the factor Xa - factor II - choline complex was nonproductive. These observations indicated that choline is a mixed-type inhibitor of prothrombin activation, whether factor Va is present or absent. When absent, the same inhibition pattern and similar apparent inhibition constants were also obtained n=3) and 5.0 (Ki=18+2, n=2) mM Caz+ concentrations, and with a one at 1.5 (Ki=27+4, to three PC-PS ratio (Ki=27klO, n=5), the reciprocal ratio of that routinely employed. Thus, under the varied conditions of our experiments, choline inhibition appeared to and PL composition, suggesting be independent of factor Va, Ca2+ concentration, interaction between choline and factors Xa and/or II.

Choline (mM)

Choline (mM)

Fig. 3 Graphical determination of choline inhibition constants, apparent K’values, for the factor Xa - factor II complex without (panel A) and with (panel B) factor Va. Ordinate intercepts from respective panels of Fig. 1 were plotted versus choline concentration. Data in panel A were from the single experimental set described in Fig. 1A. Error bars delineate 95% confidence limits in panel B which is composed of normalized data from the 9 replicate experiments of Fig. 1B.

642

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

Fig. 4 Dixon lot demonstration of a non-pro Buctive complex of factor Xa - factor II - choline. Raw data are those of Fig. 1B.

Choline (mM)

DISCUSSION The incubation components of the PL-dependent primary reaction were chosen intentionally to mimic in vivo conditions for activity of factor Xa toward factor II. Simultaneously the indicator reaction provided the advantage of a kinetic assay by spectrophotometric monitoring at 400nm. As a means of quality control, data from each assay mixture were statistically evaluated for congruence of the indicator reaction to the characteristic acceleration function. We observed that choline is a mixed-type inhibitor of prothrombin activation. (1,55,56, The dissociable factor Xa - factor Va - PL complex, prothrombinase catalyzes the activation of prothrombin by a well established mechanism (48,55,56.1 The negatively charged PL surface associates with y-carboxyglutamic acid residues of factor Xa via CaZ+ bridges, while factor Va binds to the PL by hydrophobic and/or electrostatic interactions. Prothrombin interacts with all three components at the active site of prothrombinase. Our data indicate that choline inhibition of prothrombin activation seems unaffected by the presence or absence of factor Va. As a mixed-type inhibitor, both decreasing the turnover number of factor Xa and increasing its apparent Km for factor II, choline through its positively charged quaternary amine group may be envisioned to interact with negatively charged moieties of factors II, Xa, and/or the PL surface thereby interfering with the membrane-bound aspect of prothrombin activation. Along that line of reasoning the increased apparent Km could be explained by competition between choline and factor II-Caz+ binding to the PL surface on one hand, and the decreased apparent Vmax may result from competition with factor Xa-Ca2+ binding to the PL surface on the other. However, a conformational change of enzyme alone can alter both Km and Vmax. Thus additional experiments, such as to determine whether choline inhibition is competitive with PL, will be necessary to rule out or gain support for these or alternative explanations. The kinetic data with choline are consistent with the mixed inhibition pattern of group B protein vascular anticoagulant (39). Binding studies may be useful to make further comparisons. Characterization of choline interactions with prothrombinase and earlier PLdependent components of the clotting cascade will require additional studies. Investigating processes which alter the activity of clotting enzymes in vivo may provide further rationale applicable to solving clinical thrombotic problems. Several quaternary amines are known to either inhibit or stimulate the action of Roberts et. al. (57) explored the effect of choline and various many enzymes.

Vol. 62, No. 6

CHOLINE INHIBITS PROTHROMBINASE

643

rimary to quaternary amines on the enz matic activit of bovine factor Xa, and human and bovine thrombin. They o i:served that t Bese compounds inhibited a modified one-stage prothrombin test and also the clotting of fibrin0 en by thrombin. We did not observe choline inhibition of thrombin activity with S-1 238 as substrate (ie. not fibrinogen). They observed inhibition to increase re ularly as the size of the alk 1 group increased, with the quaternary amines causing t a e most inhibition. amines tested. E holine is an endobiotic and thus unique among the quaternary Human blood choline concentrations of O.OOSmMto 024 mM have been reported (5& 60). Many studies have been done in which manipulation of serum choline has been looked at in terms of its effect on neurological brain transmitters. Few, if any, have looked at its effect on coagulation. There have been reports of elevated serum choline levels in response to dietary intake of lecithin (6063) . Even though the inhibition constants we observed are two to three orders of magnitude greater than the reported serum choline levels achieved by dietary manipulation, choline may play a role in thromboprophylaxis for reasons discussed below. The single stage prothrombin activation assayed in our studies is only one of the sequential steps comprising the coagulation cascade. Similarity in structural composition among PL-dependent complexes, for example between intrinsic Xase and prothrombinase, provides a conceptual basis for functional similarity (964). Then by analogy it may be speculated that the inhibition effected by choline on prothrombinase may occur in a similar manner at earlier reaction steps, PLdependent, in the cascade thus further diminishing the potential for clot formation in viva If choline were inhibitory at more than one site in the cascade sequence, amplified inhibition may be anticipated. The inhibitor constant values we obtained are based on the assumption of homogeneous choline concentration within the confines of the assay system, but choline properties would seem to su gest a necessity for further study and consideration. One consideration woul d center around the potential tendency for choline partitioning from the aqueous phase of the assay system to the PL micelles. Whether sonication to remove dissolved gas from assay mixtures would facilitate artitioning or alter functional characteristics of the PL in the assay system has not g een investigated yet, but the procedure was applied uniformly to each assay mixture. Another focus of interest would be the glass binding propensity of choline, assuming similarity to i4[C]-choline (65), which would seem to imply that the inhibitor constant values we obtained may be somewhat overestimated because the inside surface of glass containers may have partially depleted the concentration of choline during preparation and dilution of stock solutions. Similarly, the amount of choline available for the inhibition of clotting in the ACT may also have been decreased by physical contact between reaction components and the internal surface of the glass tube, but the very high specific surface area of the suspended siliceous earth might be expected to have a much greater capacity for sequestering choline thus having resulted in a more substantial decrease in its concentration. On the other hand any contact of sample with glass during determination of plasma choline levels, described in the literature, may have resulted in underestimation of choline concentrations. If binding were. to occur between choline and negatively charged groups exposed at sites that nutlate thrombosis, then elevated levels of circulating choline within the vasculature may also play a role in diminishing the tendency for clot formation at even earlier steps of the cascade. These considerations may have merit in evaluating the potency of choline as an inhibitor of blood coagulation. Kosow demonstrated that S-2238 inhibited the roteolytic turnover of factor II by factor Xa with a Ki of 70 ~.LM(66). Therefore, in t Ke reaction mixtures of our studies, the observed velocities were less than half the values that would have been observed in the absence of this chromogenic peptide substrate. Nevertheless, with appropriate consideration of the S-2238 inhibition type reported (66), the conclusions we have drawn remain valid. The Physicians’ Health Study (27) reported the efficacy of buffered aspirin in preventing heart attack. Aspirin inhibits clotting by a well understood platelet

10th

644

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

recess

(67). Our study would seem to indicate that blood choline, which may elevated either by means of dietary intake (60,62,63) or possibly by parenteral administration., would be inhibitory of the plasma clotting cascade via the PL-de endent prothrombmase step. Thus it may be proposed that a combination of two t romboprophylactic agents affecting both protein and cellular hemostatic responses (9) may be administered as a single compound, choline acetylsalicylate. The cationic choline moiety of this salt may be dually beneficial via its inhibitory regulation of the rotein clotting cascade and its neutralization of aspirin, the latter a benefit to in Bividuals for whom buffering is advocated to lessen adverse gastrointestinal affects. With appropriate software modification, the data acquisition system could be applied to the assay of other primary reactions cou led with chromogenic secondary reactions. Stored data point sets can be rea Bily retrieved and subjected to alternative analyses simply by changing calculation parameters for the generation of various analytical kinetic patterns.

&ecome

R

In summary: A spectrophotometer-computer interface and software were developed to kinetically characterize choline inhibition of prothrombin activation. Quantitative estimates of prothrombin activation rate in the primary reaction were validated by the observed high correlation coefficients demonstrating precise adherence of the indicator reaction to the acceleration function. In this assay system choline was a mixed-type inhibitor of the PL-dependent prothrombin activation process. On the basis of our experiments, choline could be assigned functionally to the group of anticoagulants which interfere with the processes involving accessory components of procoagulant complexes (38,39). Compositional resemblance among the PL-dependent complexes suggests that choline may also exert an inhibitory affect on analogous procoagulant reactions, tenase for example. Thus elevation of blood choline levels in vivo may effect amplified inhibition in the clotting cascade. Whether choline possesses anticoagulant potential, capable of contributing to the induction and maintenance of an antithrombotic state in individuals at high risk of myocardial and cerebral infarction due to thrombogenesis, has yet to be determined. Nevertheless, for individuals with a thrombogenic tendency, it may be proposed that levels above the normal range of blood choline, elevated by enteral (60) or parenteral delivery, administered as a PLmoiety or as the salt of acetylsalicylic acid, may play a role in shifting the delicate balance of hemostasis toward thromboprophylaxis via anticoagulation. REFERENCES 1. NELSESTUEN, dependent proteins

G.L., KISIEL,W. with membranes.

and DI SCIPIO, R.G. Interaction Biochemistry

of vitamin

K

27,2134-2138,1978.

NESHEIM, M.E., TASWELL, J.B. and MANN, K.G. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J. Biol. Chem. 254, 10952-

2.

10962,1979. 3. JACKSON, 765811,198O.

C.M. and NEMERSON,

Y. Blood coagulation.

Heterogeneous 4. HEMKER, H.C. and ZWAAL, R.F.A. generation of thrombin. Trends Biochem. Sci. 7,37&381,1982.

Anna Rev. Biochem. 49,

biocatalysis

in the

5, VAN DIEIJEN, G, VAN RIJN, J.L.M.L., GOVERS-RIEMSLAG, J.W.P, HEMKER, H. C. and ROSING, J. Assembly of the intrinsic factor X activating complex-interactions between factor IXa, factor VIIIa and phospholipid. Thromb. Haemost. 53,396X)0,1985.

Vol. 62, No. 6

CHOLINE INHIBITS PROTHROMBINASE

6. FURIE, B. and FURIE, 505518,1988.

B.C. The molecular

645

basis of blood coagulation,

Cell 53,

7. MANN, K.G., JENNY, R.J. and KRISHNASWAMY, S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Annu. Rev. B&hem. 57, 915956,1988. 8. TRACY, P.B. and MANN, K.G. A model for assembly of coagulation factor complexes on cell surfaces: prothrombin activation on platelets. In: Biochemistry of Platelets. D.R. Phillips and M.A. Shuman (Eds.) Orlando-London: Academic Press, 1986, pp. 295-318. 9. MANN, K.G. The assembly Biochem. Sci. 12,229-233,1987.

of blood clotting

complexes

10. SHUMAN, M.A. and GREENBERG, C.S. Platelet formation. In: Biochemistry of Platelets. D.R. Phillips Orlando-London: Academic Press, 1986, pp. 319-346.

of thrombus re ulation an f M.A. Shuman (Eds.)

Quantitative features of the structure, 11. NESHEIM, M.E. modulation of prothrombinase. Surv. Synth. Path. Res. 3,219-232,1984. Membrane and 12. ZWAAL, R.F.A. B&him. Biophys. Acta. 515,163205,1978. 13. ZWAAL, R.F.A. Preface. Raton, FL: CRC Press, 1989.

lipid

Trends

on membranes.

involvement

In: Coagulation and Lipids.

function

in blood

and

coagulation.

R.F.A. Zwaal,

(Ed.) Boca

14. HARLAN, J.M. and HARKER, L.A. Current Concepts: Thrombosis and Coronary Artery Disease. Kalamazoo, MI: The Upjohn Company, 1983. 15. BLAKESLEE, D. Stroke. In: SpeciaZ Research Report. D. Blakeslee, CA: Research Institute of Scripps Clinic, May, 1985.

(Ed.) La Jolla,

16. BLAKESLEE, D. Inflammation. In: SpeciaZ Research Report. D. Blakeslee, La Jolla, CA: Research Institute of Scripps Clinic, January/February, 1988. 17. McCORD, J.M. Oxygen-derived EngZ. J. Med. 312,159-163,1985. 18. FRIDOVICH, I. Biophys. 247,1-11,1986.

Biological

19. FRIDOVICH, I. Superoxide J. BioZ. Chem. 264,7761-7764,1989.

free radicals effects

in postischemic

of the superoxide

dismutases.

An adaptation

radical.

(Ed.)

tissue injury.

N.

Arch. Biochem.

to a paramagnetic

20. GUNTHER, M. and CAUGHEY, W-S. Myoglobin is a potential reduced oxygen species in ischemic myocardium. FASEB J. 2, A487,1988.

source

gas. of

21. ZWEIER, J.L. and KUPPUSAMY, P. Electron paramagnetic resonance measurements of free radicals in the intact beating heart: A technique for detection and characterization of free radicals in whole biological tissues. Proc. NatZ. Acad. Sci. USA 85,5703-5707,1988. 22. GARABEDIAN, J.A., THORNTON,

H.D., GOLD, H.K., LEINBACH, R-C., YASUDA, T, JOHNS, D. and COLLEN, D. Laboratory monitoring of hemostasis

646

CHOLINE INHIBITS PROTHROMBINASE

during thrombolytic therapy with activator. Thromb. Res. 50,121-133,1988.

recombinant

human

Vol. 62, No. 6

tissue-type

plasminogen

23. FU, K.P., LEE,

S, HUM, W.T., KALYAN, N., RAPPAPORT, R, HETZEL, N. and HUNG? P.P. Disposition of a novel recombinant tissue plasminogen activator, A2-89 TPA, m mice. Thromb. Res. 50,33-41,1988.

24. BECK, Y, BARTFELD, D., YAVIN, Z, LEVANON, A, GORECKI, M. and HARTMAN, J.R. Efficient production of active human manganese superoxide dismutase in Escherichia coli. BiolTechnology 6,930-935,1988. 25. BELLINGER, D.A., NICHOLS, T.C., READ, M.S, REDDICK, R.L., LAMB, M.A, BRINKHOUS, K.M., EVATT, B.L. and GRIGGS, T.R. Prevention of occlusive coronary artery thrombosis by a murine monoclonal antibody to porcine von Willebrand factor. Proc. Natl. Acad. Sci. USA 84,8100-8104,1987. 26. VITOUX, J.F., AIACH, M, RONCATO, M. and FIESSINGER, J.N. Should thromboprophylactic dosage of low molecular weight heparin be adapted to patient’s weight? Thromb. Haemost. 59,120,1988. 27. STEERING COMMITTEE OF THE PHYSICIANS HEALTH STUDY RESEARCH GROUP. Final report on the aspirin component of the ongoing physicians’ health study. N. Engl. J. Med. 321,129-135,1989. 28. ROGERS,

P.C., SILVA, M.P., CARTER, J.E. and WADSWORTH, and response to therapy in a newborn due to protein

vein thrombosis

L.D. Renal C deficiency.

Eur. J. Pediatr. 149,124-125,1989. 29. NATH,

aceto-acetate

MC. and VAISHWANAR, I. In vitro effect of hydrolysed glucose cycloon coagulation of blood in health and metabolic disorders. Ann.

Biochem. Exp. Med. 23,187-192,1%3.

Inhibition of the conversion 30. NORRID, H.G. and COOPER, W.A. prothrombin to thrombin by choline and acetylcholine. Tex. J. Sci. 20,89-91,1968. 31. HERRMANN, E.C., DEWIND, S.A. and SLATTERY, of factor Xa activity. Thromb. Haemost. 54,204,1985. 32. LEACH, R.D., SLATTERY, C.W. and HERRMANN, prothrombinase complex activity. Clin. Res. 33,48A, 1985. 33. VASSANTACHART, P.B., NYANZI, F.A, HERRMANN, EC. Choline inhibition of thrombin membrane phospholipids. Clin Res. 31,40A, 1983. 34. HARRIS,

inhibits 1981.

intrinsic

W.B., SLATTERY, blood coagulation

35. AWAD, I.A. and SLATTERY, role of phospholipids in prothrombm

C.W. Choline EC.

D.P.

Kinetic

inhibition

inhibition

of

SLATTERY, C.W. and production is enhanced by

C.W. and HERRMANN, E.C. Blood choline in response to dietary lecithin. Clin. Res. 29,4lA, C.W. Simple method yields new insights activation. Clin. Res. 26,35A, 1978.

36. KOSOW, D.P., FURIE, B. and FORASTIERI, H. Activation properties of the reaction. Thromb. Res. 4,219-227,1974. 37. KOSOW, streptokinase.

Choline

of

mechanism

of the activation

Biochemistry 14,4459-4465,1975.

of factor

of human

into

X: kinetic

plasminogen

by

CHOLINE INHIBITS PROTHROMBINASE

Vol. 62, No. 6

647

38. ESMON, C.T. The roles of protein C and thrombomodulin blood coagulation. J. Biol. Chem. 264,47434746,1989.

in the regulation

of

39. REUTELINGSPERGER, C.P.M., KOP, J.M.M, HORNSTRA, G. and HEMKER, H.C. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation. Eur. J. B&hem. 173,171-178,1988. 40. TRAVIS, J. and SALVESEN, Rev. Biochem. 52,655709,1983.

G.S. Human

plasma

proteinase

inhibitors.

Annu.

41 BOFFA, M-C, VERHEIJ, H.M. and DE HAAS, G.H. Procoagulant phospholipid hydrolysis, penetration ability and anticoagulant activity of phospholipases A2. Thromb. Haemost. 47,299,1982. 42. THIAGARAJAN, P, SHAPIRO, S.S. and DE MARCO, L. Monoclonal immunoglobulin MA coagulation inhibitor with phospholipid specificity. J. Clin. Invest. 66,397-405,1980. 43. BAJAJ, purification 412,198l.

S-P, RAPAPORT, S.I. and PRODANOS, C. A simplified procedure for of human prothrombin, factor IX and factor X. Prep. Biochem. II, 397-

44. NESHEIM, M-E, KATZMANN, Methods Enzymol. 80,249-274,198l.

J-A, TRACY,

P.B. and MANN,

K.G. Factor

V.

45. ANNINO, J.S. Blood pH and electrolyte relationships. In: Clinical Chemistry: Principles And Procedures. Boston: Little, Brown, 1964, pp. 122-132. 46. KAPLAN, L.A. and PESCE, A.J. Physiology And Pathophysiology Of Body Water And Electrolytes. In: Clinical Chemistry; Theory, Analysis And Correlation. St. Louis: C.V. Mosby, 1984, pp. 363-386. 47. FOSTER, W.B., NESHEIM, M.E. and MANN, K-G. activation of factor V. J. Biol. Chem. 258,13970-13977,1983.

The

factor

Xa-catalyzed

48. ROSING, J., TANS, G, GOVERS-RIEMSLAG, J.W.P., ZWAAL, R.F.A. and HEMKER, H.C. The role of phos holipids and factor Va in the prothrombinase complex. J. Biol. Chem. 255,274-283,1 880. 49. LINDHOUT, T, GOVERS-RIEMSLAG, J.W.P., VAN DE WAART, HEMKER, H.C. and ROSING, J. Factor Va-factor Xa interaction. Effects phospholipid vesicles of varying composition. Biochemistry. 2Z,5494-5502,1982.

P, of

50. BERGMEYER, H.U. Determination of optimum conditions for the auxiliary and indicator reactions in coupled enzyme reactions. In: Principles Of Enzymatic Analysis. H.U. Bergmeyer and K. Gawehn (Eds.) Weinheim-New York: Verlag Chemie, 1978, pp. 69-75. 51. HATTERSLEY, 150-154,1966. 52. DEWIND,

P.G.

T.J. Personal

Activated

coagulation

time of whole

blood.

JAMA. 196,

Communication.

53. SEGEL, I.H. Rapid equilibrium Kinetics. New York: Wiley-Interscience,

partial and mixed-type 1975, pp. 161-226.

inhibition.

In: Enzyme

648

CHOLINE INHIBITS PROTHROMBINASE

54. DIXON,

M. and WEBB, E.C. Enzyme inhibition New York: Academic Press, 1979, pp. 345-352.

55. NELSESTUEN, G.L. Interactions calcium ions and phospholipid membranes.

Vol. 62. No. 6

and activation.

In: Enzymes.

of vitamin Kdependent proteins Fed. Proc. 37,2621-2625,1978.

with

56. VAN RIJN, J.L.M.L., GOVERS-RIEMSLAG, J.W.P., ZWAAL, R.F.A. and ROSING, J. Kinetic studies of prothrombin activation: Effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry. 23, 4557-4564,1984. 57. ROBERTS, P.S., OTTENBRITE, R.M, FLEMING, P.B. and WIGAND, effects of amines on the esterase activities of thrombin and thrombokinase several clotting tests. Thromb. Diath. Haemorrh. 31,309-318,1974. 58. OSER, B.L. Blood analysis. McGraw-Hill, 1965, p. 979. 59. ZEISEL, S.H. Dietary Annu Rev. Nutr. 1,95-121,198l.

In: Hawk’s Physiological

choline:

biochemistry,

physiology,

60. WURTMAN, R.J., HIRSCH, M.J. and GROWDON, raises serum-free-choline levels. Lancet. 2,68-69,1977.

Chemistry. New

York:

and pharmacology.

J.H. Lecithin

61 WOOD, J.L. and ALLISON, R.G. Effects of consumption of choline on neurological and cardiovascular systems. Fed. Proc. 41,3015-3021,1982. 62. MAGIL, egg lecithins 170,198l.

J. The and on

consumption and lecithin

S.G., ZEISEL, S.H. and WURTMAN, R.J. Effects of ingesting soy or on serum choline, brain choline and brain acetylcholine. J. Nutr. 8,166-

63. JOPE, R.S., DOMINO, E.F., MATHEWS, B.N., SITARAM, N., JENDEN., D.J. and ORTEZ, A. Free and bound choline blood levels after phosphatidylcholme. CIin. Pharmacol. Ther. 31,483-487,1982. 64. GILBERT, G.E., FURIE, B.C. and FURIE, B. Binding phospholipid vesicles. J. Biol. Chem. 265,815-822,1990. 65. Radiochemical Specifications Nuclear, Boston, MA. 66. ORTHNER, CL., MORRIS, coagulation factor Xa by thrombin

Sheet on Choline

Chloride.

of human

factor

DuPont-New

Inhibition of S. and KOSOW, D.P. substrates. Thromb. Res. 23,533-539,198L

J.R. Arachidonic 67. MONCADA, S. and VANE, interactions between platelets and blood-vessel walls. 1979.

VIII to England human

acid metabolites and the N. En@. J. Med. 300, 1142-1147,

ACKNOWLEDGEMENTS Acknowledgement is gratefully extended to A. Emsiriratana, S. Slattery, Bruce Vassantachart, L. Wann, and J. Westengard for experimental assistance; G. Maeda and R. Tatum for providing expertise to develop the interface between spectrophotometer and computer; J.R. Farley and P. Yahiku for discussions. This investigation was supported in part by a grant to CWS and ECH, URAC# 4164.