©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Hydration during Generation of Coagulation Factor Xa in Aqueous Phase and on Phospholipid Membranes (*)

Maria P. McGee (§) , Hoa Teuschler

From the (1)Department of Medicine, Rheumatology Section, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27157-1058

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The energetic contribution of protein solvation-desolvation reactions to generation of coagulation activated factor X (FXa) by the extrinsic pathway protease complex was determined using the technique of osmotic stress. The initial rate of FXa generation by limited proteolysis of human FX was measured in reaction mixtures with human tissue factor (TF) and factor VIIa (FVIIa) assembled either in aqueous phase or on phospholipid membranes. Osmotic stress was induced on the surface of reacting proteins with either polyethylene glycol, or dextran of 6000 and 500,000 molecular weight, respectively. These inert polymers are sterically excluded from the the solvation shells of proteins and thus increase the water activity in the excluded spaces. The volume of water transferred either to or from the excluded spaces during formation of reaction intermediates was calculated from the ratio of change in free energy of activation with change in osmotic pressure, G*/. For aqueous phase-assembled reactions, G* values decreased with at ratios of -2.36 ± 0.38 and -2.26 ± 0.26 kcal/mol/atm for polyethylene glycol and dextran, respectively. These values correspond to 5488 ± 883 and 5255 ± 604 mol of water transferred from the reacting protein surfaces per mol of FXa generated. At a physiologic osmotic pressure of 7 atm the work of transfer corresponded to 16 kcal/mol, approximately 70% of G*. The observed osmotic effects were independent of the viscosity, temperature, and ionic strength of solutions. For reactions assembled on phospholipid membranes, G* increased with at a ratio of 0.35 ± 0.05 kcal/mol/atm, corresponding to 814 ± 116 mol of water tansferred from bulk solution to protein surfaces. At physiologic osmotic pressure the work of transfer is 2.45 kcal/mol, approximately 12% of G*. Results indicate that for factor Xa generation in aqueous phase the work of desolvation is a significant component of the free energy of activation. Results also suggest that phospholipid membranes catalyze the reaction by reducing the desolvation component of the free energy of activation.


INTRODUCTION

The generation of coagulation FXa()by limited proteolysis of FX via the extrinsic pathway is one of the key reactions of the blood coagulation cascade. The reaction is catalyzed under biologically relevant conditions, by a complex formed between tissue factor (TF), a protein co-factor and activated coagulation factor VII (FVIIa), a serine esterase. The co-factor has been identified in many extravascular tissues and is expressed on the surface of cells as a transmembrane protein (for reviews, see Rapaport (1991) and Nemerson(1988)).

There is little information about local factors that may regulate functional assembly of TFFVIIa complexes in vivo. Results from kinetics studies with purified reagents indicate that purified TF protein either in aqueous solution or reconstituted into phospholipid vesicles accelerates, by several orders of magnitude, the rate of FXa generation by FVIIa (Carson and Konigsberg, 1980). The TFFVIIa complex assembled on lipid membranes generates FXa at rates that are approximately 100-fold faster than rates of the same reaction in aqueous phase. This catalytic effect of phospholipid membranes suggests that limited exposure of reactants to solvent water may be important for efficient assembly and function of coagulation proteases.

Competition for water in biological microenvironements must favor relatively dehydrated conformations of enzymes and substrates. Further, the close apposition between proteases and their substrates required for catalysis is likely associated with changes in the volumes of water hydrating the interacting protein surfaces. Thus, it can be expected that a significant component of the free energy of activation in protein catalysis is work of hydration/dehydration required in the formation of tight fitting intermediates and attainment of chemically active configurations (Colombo et al., 1992; Rand, 1992; Timasheff, 1993; Douzou, 1994). To test this possibility in reactions catalyzed by coagulation proteases, transfer of water during generation of FXa by the extrinsic protease complex was examined using the technique of osmotic stress (OS).

The term ``osmotic stress'' refers to the strictly controlled removal of water from the surface of a macromolecular system, (Parsegian, 1986). Osmotic stress is induced experimentally on the surface of proteins using inert polymers that are excluded from the proteins' hydration shells (Knoll and Hermans, 1983). The OS technique has been used and validated extensively to measure ``hydration'' forces influencing short range intermolecular separations (Parsegian et al., 1986; Prouty et al., 1985; Rand and Parsegian, 1989; Rau and Parsegian, 1990). The technique has also been used successfully to measure the hydration volumes associated with transitions in protein conformation, transitions between lamellar and non-lamellar phase of membranes (Gawrisch et al., 1992), permeation of transmembrane channels (Zimmerberg, 1990), DNA-promoter interactions (Robinson and Sligar, 1993), and enzyme-substrate interactions (Rand et al., 1993).


MATERIALS AND METHODS

Coagulation Proteins

Pure recombinant TF was purchased from American Diagnostics. This protein was used either in aqueous solution or reconstituted into mixed phospholipid vesicles, either 75% phosphatidylcholine, 25% phosphatidylserine, or 100% phosphatidylcholine in the presence of divalent cations and deoxycholate, as described previously (Carson and Konigsberg, 1980). This procedure results in a mixture of small unilamellar and large multilamellar vesicles, (Slack et al., 1973) and molar ratio of phospholipid to TF protein of 2.5 10 to 1. Pure recombinant human FVIIa with specific activity of >2000 units/mg was a gift from Dr. Ulla Hedner of Nova Nordis (Gentofte, Denmark). Human plasma factor X with specific activity of 125 units/mg was purchased from Enzyme Laboratories Inc. (South Bend, IN). These proteins were electrophoretically homogeneous and functionally pure in chromogenic and clotting assays.

Measurement of Factor Xa Generation Rates

The rate of FX conversion into FXa was measured in reaction mixtures prepared in 50 mM Tris-HCl buffer, pH 7.3, containing 0.03-0.4 M NaCl, 5 mM CaCl, and 0.5 mg/ml bovine serum albumin. Recombinant TF protein was added at 1.5 nM for reactions assembled in aqueous phase, and lipid-reconstituted TF was added at 0.02 nM for reactions assembled in lipid phase. Factor VIIa was added at 1.7 nM for both aqueous and lipid phase reactions. This concentration of FVIIa is of the same order of magnitude as FVII concentration in human plasma and sufficient to bind all the functional TF in both types of reaction mixtures. This was demonstrated in titration experiments with fixed concentrations of all reagents, except FVIIa, which was varied from 0.2 to 3.4 nM. Reaction rates were maximal in reaction mixtures with 1.7 nM FVIIa and did not increase further with higher FVIIa concentration.

During measurements, reaction mixtures were stirred continuously and maintained at a temperature between 32 and 35 °C in a Reacti-therm heating/stirring module (Pierce). In experiments designed to examine temperature dependence of reaction rates, temperatures were varied between 17 and 37 °C using an electronically controlled thermocycler (Perkin-Elmer).

Reactions were initiated with the substrate, FX, added to final concentrations ranging from 0.01 to 3 µM. Reaction mixtures were sampled at six regular intervals of 0.25-5 min each, and samples were diluted immediately in 0.2 M EDTA solution to stop the reaction. Concentration of FXa in each sample was determined as before (McGee et al., 1992), with chromogenic substrate N-benzoyl-L-isoleucyl-L-glutamyl-L-arginine p-nitroanilide (S222L) purchased from American Diagnostica (Greenwich, CT). Initial rates of chromogenic substrate hydrolysis were followed at 405 nm using a microplate reader (V kinetic microplate reader; Molecular Devices, Palo Alto, CA). The corresponding factor Xa concentrations were calculated from standard curves calibrated with active site-titrated factor Xa, (Smith, 1973). Under the conditions of these experiments no FXa generation was detected in the absence of TF.

Reaction Rates under Conditions of Osmotic Stress

To examine the effect of osmotic stress on reaction rates, mixtures included either polyethylene glycol 6000, molecular biology grade (PEG) (Sigma) or dextran T500 (DT) (Pharmacia Biotech Inc.) at concentrations ranging from 0 to 10%. The viscosity of PEG and DT solutions was measured using a calibrated cross arm viscometer (Internal Research Glassware, Charlotte, NC). Viscosities were directly proportional to polymer concentration and much higher for DT than for PEG. Within the range of polymer concentrations used in these studies, viscosity increased by 0.959 ± 0.08 and 0.473 ± 0.07 centistokes/sec per each 1% (w/w) increase in the concentration of DT and PGE, respectively.

Observed effects of PGE in reaction rates were not significantly different in dialyzed as compared with undialyzed solutions. In contrast, DT solutions contained impurities that were inhibitory and required extensive dialysis before this polymer could be used in OS experiments. Dialysis resulting in <10% dilution of polymer was accomplished using a multichamber dialysis unit (MRA corporation, Clearwater, FL) at 10 °C with continuous stirring and back pressure with a column of water of 10 2.5 cm.

Concentrations of reactants were adjusted as molal rather than molar to avoid errors resulting from the small but significant volume occupied by the stressing polymer. At the highest concentrations of PEG used, this volume corresponded to approximately 10% of the total solution volume. Osmotic pressure exerted by each polymer concentration was calculated using published relationships between weight percent of polymer and osmotic pressure. (Parsegian et al., 1986). Osmotic pressure of DT solutions was also measured directly using a membrane osmometer (Wescor, Inc., Logan, UT). Rates of chromogenic substrate hydrolysis, Spectrozyme FVIIa (methanesulfonyl-D-cyclohexylalanyl-butyl-arginine-paranitroaniline monoacetate) (American Diagnostica, Greenwich, CT), at 5 10M, by TFFVIIa, at 1.7 nM were measured in Tris-HCl buffer, pH 7.3, 5 mM, CaCl, 0.15 M NaCl, containing 0.5 mg/ml bovine serum albumin and PEG concentrations ranging from 0 to 5% corresponding to 0-0.384 atm. Rates at 1, 2, 3, 4, and 5% PEG were 9.52 ± 0.23, 10.1 ± 0.17, 9.92 ± 0.12, 9.78 ± 0.22, and 9.78 ± 0.11 10 optical density units/min, respectively, and not significantly different from the rate (9.88 ± 0.14 10 optical density units/min) without PEG.

Calculation of Initial Rates of Factor Xa Generation

Progression curves of FX conversion to FXa were analyzed using plots of FXa concentration versus reaction time. Initial steady-state rates were calculated as the slope of straight lines fitted to experimental data points using computer routines for regression analyses (Stat-View 512, Brain Power, Inc.). Initial rates from progression curves that deviated appreciably from linearity were derived from the initial tangent to the curve calculated as the second coefficient of a second degree polynomial fitted to the observed progress curve.

Calculation of Gibbs Free Energy of Activation

The Gibbs free energy of activation, G*, of FXa generation by TFFVIIa was determined from reaction rate coefficients according to the following equation.

On-line formulae not verified for accuracy

This treatment is based on ``rate process theory'' (Glasstone et al., 1941). The observed reaction rate is proportional to the concentration of a high energy ``activated intermediate'' that is at equilibrium with reactants. The proportionality coefficient can be derived independently from general physical principles. The equilibrium constant expressed in terms of this coefficient and in terms of the observed reaction rate can then be subjected to thermodynamic reasoning as any equilibrium constant. In the resulting relationship, i.e. Equation 1, R is the gas constant (1.987 cal/mol/degree), h is Plank's constant (1.584 10 cal s), k is Boltzmann's constant (3.298 10 cal s), and the reaction rate, k, is expressed in s ([FXa]/[TF] s).

Differences in G* values, i.e. G*, were calculated from rates measured in sets of reaction mixtures containing identical preparations of protein reactants. Therefore, possible discrepancies between nominal and real concentration of either TF, FVIIa, or FX would not influence the accuracy of these differences. For determination of G* values at various osmotic pressures, the concentration of substrate was kept constant and below the apparent K of the reaction.

The volume, V, of water transferred during reactions was calculated as the differential ratio of the free energy of activation with respect to the osmotic pressure, obtained from the slope of curves constructed with data from the interval between 0.022 and 0.225 atm, accessible experimentally with both PEG and DT. Reaction rates at higher osmotic pressures were examined using PEG as the stressing polymer. The viscosity of DT in solution made preparation of reaction mixtures containing more than 10% of this polymer difficult.

Curves were generated by either linear or polynomial regression using the computer program Stat-View 512 (Brain Power, Inc.). This program calculates regression coefficients by applying the sweeping operator to the matrix of cross-product deviations. Volumes were calculated from the linear coefficients using the equivalence: 1 atm times the volume of one mol of water = 0.430 cal/mol. The effect of OS on apparent K and k was also examined by measuring initial rates at increasing concentrations of factor X in reaction mixtures with 0-5.4% PEG. The K and k were obtained as the concentration giving half-maximal reaction rate, and maximal rate divided by enzyme concentration, respectively. These values were calculated from rectangular hyperbolas fitted to data by least square minimization techniques (k, Biometallics, Inc., Princeton, NJ)


RESULTS

Effect of Osmotic Stress on the Rate of FX Hydrolysis by TFFVIIa Assembled in Aqueous Phase

Initial rates of FXa generation were measured in reaction mixtures containing fixed concentrations of TF, FVIIa, CaCl, and FX but varied concentrations of stressing polymers. Osmotic stress was induced with either PEG or DT included in reaction mixtures at concentrations ranging from 0 to 10%. The calculated increase in the osmotic pressure of reaction mixtures induced by the polymers was 0.022-1.3 atm above that of control reaction mixtures (Fig. 1). Control reaction mixtures were 290 mosm, equivalent to 7 atm, the physiologic osmolarity of plasma at 38 °C (Jeanneret et al., 1954).


Figure 1: Change in free energy of activation with osmotic stress in aqueous phase. Reaction rates were measured at increasing osmotic pressure in mixtures with 1.5 nM TF, 1.7 nM FVIIa, 5 mM CaCl, 0.15 M NaCl, 0.5 mg/ml BSA, and 80-200 nM FX in Tris buffer, pH 7.2, at 33 °C. Gibbs free energy of activation (G*) was calculated from reaction rate coefficients, k, as G* = -RT ln kh/kT (h is Planck's constant, k is Boltzmann's constant, and T is temperature, 306 K). The G* values in the ordinate are the arithmetic difference between G* values of reactions under standard control conditions and of reactions under an osmotic stress, , above control. Osmotic stress was induced with either PEG (A) or with DT (B). The slope of linear regression lines fitted to data points for the interval between = 0.022 and = 0.25 atm was 2.26 ± 0.26 and 2.36 ± 0.38 kcal/mol/atm for reactions with PEG and DT, respectively. The linear coefficient of a polynomial regression line fitted to all data points in A was 1.898 kcal/mol/atm. Data are from 12 and 5 experiments totalling 60 and 30 independent determinations of G* in A and B, respectively.



Reaction rates increased with osmotic stress induced with either PEG or DT. The change in free energy of activation with osmotic pressure calculated using linear regression for the interval between 0.022 and 0.225 atm, was -2.36 ± 0.380 and -2.26 ± 0.257 kcal/mol/atm for reactions with DT and PEG, respectively. A similar value, -1.898 kcal/mol/atm, was obtained from the linear coefficient of the curve fitted by polynomial regression to data obtained with PEG for the interval between 0.066 and 1.3 atm. The average of these values corresponds to 4597 mol of HO transferred to bulk solution from the excluded volume/mol of FXa generated by TFFVIIa. The calculated work of transfer at 7 atm is 16 kcal/mol. Apparent kinetic parameters varied with OS (). Assuming that the K value reflects the equilibrium constant of factor X-TFFVII interaction, the change of K with corresponds to the transfer of 1688 ± 400 mol of HO/mol of FXa generated.

Independence of the Effects of Osmotic Stress and Ionic Strength

Reaction rates were measured in mixtures with concentrations of NaCl ranging from 0.015 to 0.4 M, with and without osmotic stress. Osmotic stress in these experiments was induced with PEG at a fixed concentration of 3.1%, corresponding to an increase in osmotic pressure in the excluded volume of 0.15 atm.

Increasing salt concentration decreased reaction rates measured either with or without osmotic stress. The free energy of activation increased with salt concentration and to the same extent under either condition (Fig. 2). The slope of the regression lines fitted to data points were 5.9 ± 0.5 and 6.2 ± 0.5 kcal/mol/1 M increment in NaCl concentration for stressed and nonstressed reactions, respectively. These results indicate that the effects of PEG and NaCl were largely independent of one another.


Figure 2: Electrostatic double layer screening and osmotic stress. Reaction rates were measured without () and with () 3.1% PEG (0.15 atm) at increasing concentrations of NaCl. Other components of reaction mixtures were as indicated in legend to Fig. 1. The slope of regression lines were 5.9 ± 0.5 and 6.2 ± 0.5 for reactions with and without PEG, respectively.



In theory, assuming that Na and Cl ions are not excluded from the protein surfaces, increasing the concentration of NaCl results both in double layer screening of charges and in osmotic pressure increases in excluded and nonexcluded volumes. The increase in osmotic pressure is 5.1 atm/0.1 M increment in NaCl concentration and should have resulted in an unfavorable change in G* of 10.2 kcal/mol/0.1 M increment in NaCl concentration. The much smaller change observed, that is 0.6 kcal/mol/0.1 M increment in NaCl concentration, may perhaps reflect a favorable change in G* mediated by electrostatic effects. Under physiologic conditions, 0.15 M NaCl, the favorable change in G* would be of some 13.3 kcal/mol and sufficient to compensate for over 80% of dehydration work. In its simplest interpretation, this result suggests that the role of univalent salt in the reaction is to overcome electrostatic repulsion between reactants. Alternatively, NaCl may be excluded from hydration volumes different from those that exclude PEG. The electrostatic component of the reaction is currently being examined in more detail in our laboratory.

Effect of Osmotic Stress at Variable Temperature

Reaction rates were measured at temperatures ranging from 17 to 38 °C, both under standard conditions and under an osmotic stress of 0.323 atm induced with 4.95% PEG. Reaction rates increased with temperature, and for the interval 17-32 °C the slopes of Arrhenius-type plots appear linear and not significantly different for reactions with stressed and nonstressed proteins. (Fig. 3). The activation energy, E, determined from the slope was 8.24 ± 1.25 and 7.81 ± 0.95 for reactions with and without PEG, respectively. The respective values of activation enthalpy, H*, calculated from the relationship H* = E - RT were 7.63 and 7.20 kcal/mol. This result indicates that the decrease in G* observed under osmotic stress conditions is primarily due to a more favorable activation entropy rather than to a change in rate-limiting step. The value of the activation entropy at 32 °C, calculated from the free energy of activation values, G*, and the activation enthalpy, H*, according to the thermodynamic relationship G* = H* - TS*, was -40.8 and -43.96 cal/mol/degree with and without PEG, respectively. This corresponds to a favorable change in the entropy of activation of 9 entropy units/atm.


Figure 3: Temperature and Osmotic Stress. Reaction rates were measured at temperatures ranging from 17-38 °C, (290-311 K) in mixtures without () and with () 4.95% PEG ( = 0.324 atm). Other reactants were as indicated in the legend to Fig. 1. The natural log of the reaction rate coefficient is plotted against the reciprocal of the temperature in Kelvin degrees. The slope of the regression lines fitted to data points are -3.93 ± 0.63 and -4.15 ± 0.47 for reactions without and with PEG, respectively.



Effect of Osmotic Stress on the Rate of FX Hydrolysis by TFFVIIa Assembled on Phospholipid Membranes

Reaction rates were measured in mixtures prepared identically as aqueous phase FXa generation mixtures, except that recombinant TF protein was substituted by recombinant TF protein reconstituted into phospholipid vesicles. Concentration of reconstituted TF in (PC:PS) mixtures was 100- and 10-fold lower than TF protein in aqueous phase and PC reactions, respectively, in order to maintain FXa generation at a similar rate in each type of mixture.

The rate of reactions assembled on PC:PS membranes decreased with increasing osmotic stress. The free energy of activation increased with the osmotic pressure of the polymer solution. The slope of the regression line fitted to data points (Fig. 4) was 0.35 ± 0.050 kcal/mol/atm. This corresponded to 814 mol of HO transferred from bulk fluid phase to the excluded volume per mol of FXa generated. In contrast, reaction rates in mixtures with PC membranes increased with osmotic stress. For the interval between 0.063 and 0.239 atm, the free energy of activation decreased with at a ratio of -2.035 ± 0.192 kcal/mol/atm. This corresponds to 4732 ± 446 mol of HO transferred to bulk aqueous phase per mol of FXa.


Figure 4: Change of free energy of activation with osmotic stress on phospholipid membranes. Reaction rates were measured at increasing osmotic pressures induced with PEG. Reaction mixtures contained 0.02 nM TF, reconstituted into phospholipid vesicles (75% PC, 25% PS). All other reactants were as indicated in the legend to Fig. 1. The slope of the regression line fitted to data points was 0.35 ± 0.05 kcal/mol/atm, corresponding to 813.9 mol of water bound/mol of FXa generated.




DISCUSSION

Osmotic stress induced with inert polymers, either PEG or dextran, increased the rate of FXa generation by TFFVIIa when the reaction was assembled in aqueous phase. The extent of rate acceleration was independent of the chemical characteristics of the stressing polymer, of the viscosity, of the temperature, and of the ionic strength of the solutions. These results considered together with results obtained by others in completely different systems (Gawrisch et al., 1992; Rau and Parsegian, 1990; Rand et al., 1993; Robinson and Sligar, 1994) indicate that the effect observed is related to the osmotic force induced by the polymers in the excluded volumes of reacting proteins. The osmotic force that drives water out of the excluded volume is the net pressure acting on that volume. The net pressure is a direct function of the difference in water activity between excluded and nonexcluded volumes. If water is either extruded or bound during the reaction, the energetic consequences of increasing the water activity in the excluded volume is reflected in a shift in the equilibrium position between reactants and products and can be included as an additive component to Gibbs free energy of activation, G*.

Gibbs free energy of activation decreased with increasing osmotic pressure at an initial ratio of 2.2 kcal/mol/atm. This corresponds to 5116 mol of HO/mol of FXa transferred from the excluded volume during the reaction. At physiologic osmotic pressures (approximately 7 atm) the work of transfer for this volume of HO is 16 kcal/mol, very significant when compared with the free energy of activation for the reaction calculated at 21 kcal/mol. These results imply that the functional structure of the reacting proteins in the activated intermediates up to the rate-limiting step corresponded to dehydrated conformations relative to those of the reactants. Further, the large volumes of water displaced suggest that the extent of interacting surfaces is also large.

The rate of reactions assembled on phospholipid membranes was less sensitive to osmotic stress than the rate of reactions in aqueous phase. The effect of osmotic stress on membrane-assembled reactions was to decrease rather than to increase the rate. The free energy of activation increased at a ratio of 0.35 kcal/mol/atm, corresponding to 814 mol of water/mol of FXa generated. The increase in free energy of activation suggests that the activated intermediates in membrane reactions undergo net hydration relative to reactants. Alternatively, the net increase in the free energy of activation for the reaction on membranes may reflect two simultaneous but opposite effects of osmotic stress on reaction rates, mediated by hydration/dehydration reactions of lipid and protein components, that is the rate acceleration mediated by protein dehydration could be obscured by concomitant physical changes in the membrane that may interfere with either assembly of protease complex or binding of substrate. Aggregation, lipid exchange, and increases in fluidity of artificial phospholipid vesicles with PEG have been described (MacDonald, 1985; Massenburg and Lentz, 1993). However, these physical changes have been observed at PEG concentrations greater than those found to affect reaction rates in the present studies. The fact that reactions assembled on PC membranes responded to OS like aqueous phase reactions suggests that the catalytic role of membranes is mediated via interaction of factor X with the acidic membrane.

The change in slope of G*/ plots observed at > 0.25 atm in aqueous phase reactions suggests the existence of at least two different transitions reflected in the reaction rate. Protein conformation changes required for attainment of activated intermediates may involve both dehydration of apposing surfaces and stabilization by hydration of newly exposed protein regions. Since it is possible for several reaction steps with similar energy barriers to influence the reaction rate, it is also possible for hydration/dehydration volumes to vary at each one of these steps.

The practical applications and biological relevance of examining macromolecular interactions under osmotic stress has been noted before (Parsegian et al., 1986; Rand, 1992; Timasheff, 1993; Douzou, 1994). The colloidosmotic pressure (Diem, 1962; Webster, 1982), generated in plasma by solutes that cannot permeate the vascular membrane, must also generate osmotic stress on the excluded volumes of circulating coagulation factors. Similarly, in extravascular spaces, coagulation factors are subjected to osmotic stress generated by constitutive biopolymers such as diffusible matrix proteins and glycosaminoglycans. Changes in either the concentration or physical distribution of these polymers can conceivably influence hydration/dehydration reactions involved in the equilibrium between functional and nonfunctional conformations of coagulation factors. The extreme sensitivity of the factor Xa generation reaction to osmotic stress suggests the interesting possibility that changes in the rate of coagulation reactions may be regulated in vivo by osmotic pressure changes within the physiologic range of osmotic pressures measured in plasma (Diem, 1962).

The volumes of water transferred during formation of enzyme substrate complexes can provide useful data for protein structure modeling. The volumes of water displaced by binding of glucose to the active site cleft in hexokinase have been measured by OS techniques. The information has been used with space-filling molecular modeling to determine the relative contribution of surface dehydration versus protein conformation changes associated with reduction of the cleft upon substrate binding (Rand et al., 1993). The differential sensitivity to osmotic stress observed between aqueous and fluid phase reactions also has general relevance to the overall balance of the coagulation system. This balance is maintained by a series of cross-catalytic reactions including amplifying and inhibitory loops mediated by both aqueous and membrane-bound enzymes and inhibitors. Under pathological conditions, the protein and GAG composition and concentration of interstitial fluids and plasma may change considerably. For example, necrosis of atherosclerotic lesions must generate a large concentration of colloidosmotic active products derived from cellular disintegration. Subsequent changes in the osmotic and colloidosmotic equilibrium may disrupt the complex balance of the coagulation cascade. Understanding the interaction of functional coagulation proteases under osmotic stress will help in the development of predictive kinetic models for the behavior of these enzymes in biological environments of reduced water activity.

The results in this report demonstrate a significant contribution of water-protein interactions to the energy required for FXa generation by the extrinsic pathway protease. Results also provide evidence consistent with a catalytic role of phospholipid surfaces mediated by facilitation of the dehydration component of the reaction. In addition, the quantitative information on surface water obtained should provide a useful complement to x-ray crystallography and site-directed mutagenesis in the identification of the functional configuration of coagulation enzymes.

  
Table: Change in apparent kinetic parameters with osmotic stress



FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL-42812 and by American Heart Association Grant 93007890. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine, Rheumatology Section, Medical Center Blvd., Winston-Salem, NC 27157-1058.

The abbreviations used are: FXa and FVIIa, activated factor X and activated factor VII, respectively; FX and FVII, factor X and factor VII, respectively; TF, tissue factor; OS, osmotic stress; PEG, polyethylene glycol; DT, dextran T500; PC, phosphatidylcholine; PS, phosphatidylserine.


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