Cooperative Roles of Factor Va and Phosphatidylserine-containing Membranes as Cofactors in Prothrombin Activation*

Gabriel E. WeinrebDagger, Kasturi MukhopadhyayDagger, Rinku MajumderDagger, and Barry R. Lentz§

From the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260

Received for publication, August 17, 2002, and in revised form, October 17, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor Xa, enzyme; factor Va and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzIIa) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). Vmax, Km, or Vmax/Km for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzIIa and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 µM, whereas MzIIa proteolysis was saturable. First order rate constants for formation of MzIIa and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-Va-Xa complex (i.e. "channeling") also was adjusted because both the membranes and factor Va have been shown to cause channeling. kcat, Km, or kcat/Km values were reported for one lipid concentration, for which all Xa was likely incorporated into a Xa-Va complex on a PS membrane. Comparing previous results, which were obtained either with factor Va (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor Va and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor Va has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzIIa versus Pre2 pathway; and 3) PS membranes cause ~50% of the substrate to be activated via channeling at 50 µM membrane concentration, but factor Va extends the range of efficient channeling to much lower or higher membrane concentrations.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Factor Xa catalyzes the activation of human prothrombin by proteolytic cleavages at Arg273-Thr274 and at Arg322-Ile323. The polypeptide chain composed of residues 1-273 (fragment 1.2) is released as an activation peptide. Residues 274-322 comprise the A chain of alpha -thrombin, which is linked by a disulfide bond to the B chain of thrombin (residues 323-581). As two cleavages are required in the prothrombin molecule to produce thrombin, two kinetic pathways are possible in the activation of prothrombin (Fig. 1). A single cleavage at Arg273-Thr274 (reaction A) yields fragment 1.2 (F1.2)1 and an intermediate species known as prethrombin 2 (Pre2). Pre2 is composed of the A and B chains of thrombin with the crucial bond at Arg322-Ile323 intact, making Pre2 catalytically inactive. Further proteolysis at this bond (reaction B) converts prethrombin 2 to alpha -thrombin. Alternatively, a single cleavage in the prothrombin molecule at Arg322-Ile323 (reaction C) produces a species known as meizothrombin (MzIIa), a catalytically active intermediate. Further proteolysis of meizothrombin by factor Xa at the Arg273-Ile274 bond (reaction D) yields as products F1.2 and catalytically active alpha -thrombin. In two recent studies, a third possible pathway of activation, processive proteolysis of the intermediate without release from the enzyme, was demonstrated (reaction E in Fig. 1) (1, 2).

Both factor Va and phosphatidylserine (PS)-containing membranes, or a soluble form of PS, affect the rates of each of the five proteolytic events involved in prothrombin activation (Fig. 1) (1-3). In doing so, these two cofactors 1) increase the overall rate of prothrombin activation, 2) define the intermediate for activation, and 3) promote channeling. The rate-enhancing effect of both cofactors has long been recognized (4, 5), with either cofactor enhancing the first order rate constant for prothrombin activation by roughly 3 orders of magnitude but both together enhancing it by 200,000-fold (5). Until recently, it was accepted that factor Va was responsible for directing activation via the MzIIa pathway (4, 6). However, more recent results have shown that in the absence of factor Va, PS-containing membranes alone increase the preference of human factor Xa for the MzIIa pathway by more than 550-fold relative to the Pre2 pathway (2) and that this is because of the binding of individual PS molecules to specific sites on factor Xa (7). Factor Va also enhances the preference of bovine factor Xa for the MzIIa versus Pre2 pathway, although only by 25-fold (1). The cofactors also control access to pathway E. In the presence of bovine factor Va, 40% of the substrate was channeled directly to thrombin (1), whereas 50% processivity occurred in the presence of an optimal concentration of PS-containing membranes (2).

Because both cofactors influence similarly the pathway of prothrombin activation, one might ask why both factor Va and PS membranes should be needed, as apparently they are under physiological conditions, to promote efficient thrombin generation in vivo. To answer this question, we establish here the pathway of prothrombin activation by human factor Xa in the presence of saturating concentrations of factor Va and various PS-membrane concentrations. Furthermore, for one optimal membrane concentration, we estimate the second order rate constants for each of the reactions shown in Fig. 1 and compare these with the rates observed when either of the cofactors is present individually. We find that factor Va contributes most to enhancing the rate of activation and that PS dominates the choice of the MzIIa pathway. Together the cofactors promote more efficient activation than either does alone; the overall rate of thrombin formation is at least two orders of magnitude higher and occurs almost exclusively through channeling that minimizes the appearance of intermediates in the solution. In addition, a new role for factor Va has been found, namely making channeling the stable pathway of activation over the wide range of membrane concentrations that one might expect to find in vivo.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials

Ecarin from Echis carinatus snake venom and EGTA were purchased from Sigma. Bovine brain PS and dioleoyl-phosphatidylcholine (DOPC) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Outdated human plasma was obtained from the American Red Cross Center, Durham, NC. Human prothrombin, factor X, and factor Xa were obtained by published procedures (8), and MzIIa was prepared as described by Pei et al. (9). Pre2 and F1.2 were obtained as described by Wu et al. (2). Purified human factors Xa, Va, and prothrombin also sometimes were purchased from Haematologic Technologies Inc. (Essex Junction, VT). The activity of Factor Xa was assayed with the synthetic substrate S-2765 (2). Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine (DAPA) was obtained from Haematologic Technologies Inc.

Methods

Preparation of Phospholipid Vesicles-- Extruded large unilamellar vesicles (LUV) composed of 25/75 bovine brain PS and DOPC were prepared, and the phospholipid concentration was determined by the methods described in Wu et al. (2).

Fluorescence Stopped Flow Measurements-- Measurements of thrombin formation from prothrombin, MzIIa, or Pre2 (with equimolar fragment 1.2) were performed using DAPA and exploiting the large change in fluorescence intensity that accompanies the binding of this fluorophore to the products of the prothrombinase reaction. Stopped flow measurements were performed using a SLM-Aminco Milliflow® stopped flow reactor (Spectronic Instruments, Inc., Rochester, NY) attached to the SLM 48000® spectrofluorometer (Spectronic Instruments). Broadband fluorescence of DAPA was monitored at 37 °C with a 515-nm cut-off filter in the emission path and with the excitation monochromator set at 280 nm, slit = 4 nm.

Reactions were initiated by rapidly mixing an equal volume (400 µl) of the contents of the two driving syringes. Syringe A contained substrate solution and DAPA in 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5 (Buffer R); and syringe B contained pre-assembled prothrombinase (Xa, Va, and bovine brain PS/DOPC) in the same buffer. The final concentrations of factors Xa and Va in the reaction chamber were maintained as 2.5 and 30 nM, respectively, for the substrate MzIIa and prethrombin 2. The substrate/DAPA ratio was always kept at 1/5. The initial fluorescence intensity (F0) was obtained from mixing experiments with the substrate and DAPA in syringe A and the lipid only in syringe B. F0 as well as other fluorescence intensities were corrected for light scattering by subtracting the scattering intensity recorded during a push involving only the lipid and factor Xa with no DAPA (2). Prior to mixing, both syringes were allowed to incubate at 37 °C for 2-3 min. Fluorescence intensity at the completion of the reaction, after which no further changes in fluorescence intensity occurred, was considered to represent quantitative conversion of prothrombin to thrombin and was used to convert the fluorescence intensity change per unit time into units describing thrombin active site formation as a function of time. The initial rate of thrombin generation thus was determined from the initial rate of fluorescence intensity change normalized to the intensity at complete thrombin formation in accordance with Wu et al. (2).

Rapid Chemical Quench Measurements-- Quench flow methodology was used to determine the initial rate of MzIIa and IIa appearance. Rapid quench experiments were performed using the Chemical-Quench-Flow Model RQF-3 from Kintek Corp. (State College, PA). The temperature was maintained at 37 °C by a circulating water bath. Prothrombin solution in Buffer R with 0.6% polyethylene glycol, which was used to limit thrombin adsorption to surfaces, was loaded into one sample loop; also in Buffer R, preassembled prothrombinase complex, at a concentration used in the stopped flow fluorescence studies, was loaded into another sample loop. The reaction was initiated by pushing both solutions into the reaction loops and then was allowed to proceed for various times. The reaction was stopped by mixing the solutions with a quench solution (100 µl of 100 mM Na2EGTA solution, pH 7.5) from the quench syringe. The mixture then was expelled into a collection tube. Aliquots from collected samples were assayed for amidolytic activity using S-2238 in a VersaMaxTM microplate reader (Molecular Devices Corp., Sunnyvale, CA), and the concentration of the active site (IIa plus MzIIa) was determined by comparing the initial rate of S-2238 hydrolysis to a standard curve, as described by Wu et al. (2). To measure the amount of MzIIa formed, the amidolytic activity was measured after incubating the reaction mixture aliquot for 1 min in the presence of heparin (10 µg/ml) and anti-thrombin III (300 nM), thereby blocking the thrombin but not the MzIIa active site (10). The initial rate of thrombin formation was obtained by subtracting the rate of MzIIa appearance from the initial rate of total active site formation.

Analysis of Prothrombin Activation by SDS-PAGE-- These experiments were performed as described for rapid quench experiments except that aliquots of 40 µl at 0.5, 1, and 1.5 s were taken and subjected to gel electrophoresis (1.5 mm of 12% polyacrylamide) with and without reduction with 5% (v/v) 2-mercaptoethanol (11) for analysis. Protein bands were visualized with colloidal Coomassie Blue staining (12).

Kinetic Modeling-- To determine the kinetic constants for the first two proteolytic events (reactions A and C; Fig. 1), three experimentally determined time courses (thrombin formation, MzIIa appearance, and Pre2 appearance) were modeled according to a steady state kinetic scheme that assumed a parallel, sequential reaction mechanism, as described in detail by Wu et al. (2). A fraction of prothrombin consumed was considered to convert directly to thrombin without the escape of an intermediate from the membrane-enzyme-cofactor complex (2). All three sets of data were fit simultaneously by adjusting first order rate constants Ra (for reaction A; Fig. 1), Rc (reaction C), and Re (reaction E), subject to experimental constraints, namely Rb and Rd (for reactions B and D), which were fixed at the values determined independently in this paper. This global data fitting was carried out using the SigmaPlot 2000 least-squares regression package (SPSS Inc., Chicago, IL). The details of these procedures are described by Wu et al. (2).


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Fig. 1.   Schematic representation of human prothrombin activation. Prothrombin consists of a membrane-binding N-terminal domain (Fragment 1.2) and a C-terminal catalytic domain (Prethrombin 2). Two peptide bonds (Arg273-Thr274 (site (2)) and Arg322-Ile323 (site (1))) must be hydrolyzed by factor Xa to activate prothrombin. This creates two possible pathways of activation, A (cut (2)) and B (cut (1)) as well as C (cut (1)) and D (cut (2)), and also creates two possible released intermediates, Pre2 and MzIIa. A third possible pathway of activation is envisioned (E, fast consecutive cuts (1) and (2)) in which no intermediate is released, i.e. extensive channeling occurs.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Effects of Factor Va and PS/DOPC Membranes on Prothrombin Activation by Prothrombinase-- To establish the appropriate factor Va concentration for our studies, we monitored prothrombin activation using the fluorescent active site inhibitor DAPA at several concentrations of phospholipid and factor Va (see "Methods") (Fig. 2). It was not possible to saturate prothrombinase activity even at 30 nM factor Va in the presence of only 20 µM PS membranes (circles in Fig. 2). However, above this lipid concentration, prothrombinase activity always was saturated by 30 nM factor Va (Fig. 2), reflecting optimal assembly of prothrombinase at that lipid concentration. Based on this result, we fixed the factor Va concentration at 30 nM in all other experiments. We note that the saturating activity at 30 nM Va was a function of lipid concentration, with a maximum at 100-200 µM lipid, as seen in the inset in Fig. 2. This is not surprising as several laboratories have reported inhibition of prothrombinase activity by high phospholipid concentration (13-16), and we have reported a similar behavior for Xa even in the absence of factor Va (2).


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Fig. 2.   Initial rate of prothrombin activation is shown as a function of added factor Va in the presence of 25/75 PS/DOPC vesicles (LUV). Activation was initiated by stopped flow mixing of equal volumes of prothrombin (final concentration of 0.5 µM) and DAPA (final concentration of 2.5 µM) in one syringe with prothrombinase complex in another syringe. The prothrombinase was assembled using 2.5 nM factor Xa, 0-30 nM factor Va, and 20 µM (circles), 50 µM (squares), 100 µM (diamonds), 200 µM (triangles), and 2000 µM (inverted triangles) 25/75 PS/DOPC vesicles (LUV). A plot of the initial rates of prothrombin activation (Vini) obtained in the presence of 30 nM factor Va versus lipid concentration ([PL]) is shown in the inset.

Activation of Meizothrombin and of Prethrombin 2 plus Fragment 1.2 to Thrombin-- Fig. 3, A and B, shows the initial rates of activation to thrombin of MzIIa as well as Pre2 and F1.2, as obtained from the initial rates of change in DAPA fluorescence intensity. As noted previously (6, 17), activation of these intermediates to thrombin reflects the prothrombinase-catalyzed cleavage of individual peptide bonds, Arg273-Thr274 in MzIIa and Arg322-Ile323 in Pre2.


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Fig. 3.   Initial rates of IIa formation from intermediates as a function of substrate concentrations. Activation was initiated by stopped flow mixing of equal volumes of substrate and DAPA (in a ratio of 1/5) in one syringe with prothrombinase complex in another syringe. The final concentrations in the reaction chamber were 2.5 nM factor Xa and 30 nM factor Va. The substrates were MzIIa (A) and an equimolar mixture of Pre2 and F1.2 (B). The concentration of lipid was 20 µM (circles), 50 µM (squares), 200 µM (triangles), 1200 µM (open inverted triangles), and 2000 µM (open hexagons).

Because these reactions involve a single proteolytic event, it seems appropriate to apply the Michaelis-Menten model and obtain the apparent first order rate constants (Vmax/Km) as a function of membrane concentration. Plots of initial rates of MzIIa activation versus substrate concentration thus were fit to this model. The Km values determined from these fits showed no clear trend with lipid concentrations within experimental and fitting errors and had an average value of 0.9 ± 0.1 µM. The apparent first order rate constants (Vmax/Km) for proteolysis of MzIIa were nearly independent of lipid concentrations above 50 µM but did decrease slightly at 2000 µM lipid (filled triangles, Fig. 4A). A similar behavior was seen for Pre2 and F1.2 proteolysis (filled circles, Fig. 4A). For activation of Pre2 and F1.2 as well as MzIIa, a somewhat lower rate was observed at 20 µM lipid (Fig. 4A), at which only 45% of the substrate was membrane-bound and for which the rate of prothrombin activation was not saturated in Va (Fig. 2).


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Fig. 4.   First order rate constants (Ra, Rb, Rc, Rd, and Re) for the five proteolytic events of prothrombin activation as a function of phospholipid concentrations. Panel A contains experimentally determined rates (filled symbols) of second steps, whereas Panel B shows rates of initial steps obtained from modeling (open symbols). Rates of Arg273-Thr274 cuts (reactions A and D) are recorded as triangles, whereas Arg322-Ile323 cuts (reactions B and C) are shown as circles. The channeling rate, Re, is shown in squares. Panels C-E show relative rates of the three possible initial prothrombin activation steps: Pre2 formation (R<UP><SUB><IT>a</IT></SUB><SUP><IT>relative</IT></SUP></UP> triple-bond  Ra/(Ra + Rc + Re); triangles, panel C); MzIIa formation (R<UP><SUB><IT>c</IT></SUB><SUP><IT>relative</IT></SUP></UP> triple-bond  Rc/(Ra + Rc + Re); circles, panel D); and channeling (R<UP><SUB><IT>e</IT></SUB><SUP><IT>relative</IT></SUP></UP> triple-bond  Re/(Ra + Rc + Re); squares, panel E). The relative rates of channeling without factor Va also are reproduced (cross-filled symbols) from the findings of Wu et al. (2). The X symbols on the ordinates mark the relative rates in the presence of factor Va and in the absence of membrane, as reported by Boskovic et al. (1).

Determination of the Kinetic Constants of the Initial Proteolytic Steps-- To establish the pathway of activation, we do not need the rates of reactions B and D but instead those of reactions A and C. The rates of these reactions are difficult if not impossible to measure directly because of the rapid consumption of their products in the second step of prothrombin activation (Fig. 1). To overcome this problem, we have used an approach described earlier (2). In Fig. 5, the time courses for appearance of thrombin plus MzIIa (circles) and MzIIa alone (squares) are measured by the S-2238 assay at five lipid concentrations, and the time courses of thrombin plus MzIIa (open circles), MzIIa (open squares), and Pre2 (open triangles) appearance are measured by PAGE at 200 µM lipid. The fact that the rate of thrombin formation was greater than the rate of MzIIa appearance at all lipid concentrations means either that significant thrombin formation occurred through the Pre2 pathway or that a large fraction of MzIIa and/or Pre2 rapidly was processed directly to thrombin and not detected as a released intermediate. An extensive SDS-PAGE analysis was performed at one lipid concentration (200 µM) with the results matching the quench flow results and showing that no Pre2 formation was detectable (Fig. 5). Single time points confirmed that Pre2 could not be detected by SDS-PAGE at any other lipid concentration, implying that a fraction of intermediate(s) was processed directly to thrombin (reaction E in Fig. 1).


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Fig. 5.   Best fits of simulations to observed prothrombin proteolysis data at 20, 50, 200, 1200, and 2000 µM PS/DOPC LUV concentrations. Left panel, prothrombin (0.5 µM) proteolysis catalyzed by Factor Xa (2.5 nM) in the presence of 5 mM CaCl2, factor Va (30 nM), and different concentrations of PS/DOPC (25/75) ([PL]) at 37 °C. The formation of thrombin plus MzIIa (circles) and the appearance of MzIIa (squares) during prothrombin proteolysis was followed by generation of amidolytic activity toward the synthetic substrate S-2238 at all lipid concentrations and by SDS-PAGE (open symbols) at 200 µM (see "Methods"). Pre2 could not be detected by SDS-PAGE (open triangles). All experimental curves were fitted simultaneously using the SigmaPlot simulation program to obtain estimates for the rate constants Ra, Rc, and Re (channeling). Simulated curves were calculated using best fit kinetic parameters and are shown as solid lines. Right panel, thrombin formation through different pathways: total IIa (solid line), IIa formed through Pre2 pathway (dashed line), IIa formed through MzIIa pathway (small triangles), and IIa formed through channeling (small circles).

To test quantitatively for this possibility, we fit our data as described elsewhere (2) with the measured first order rate constants (Vmax/Km) Rb and Rd (Fig. 4) fixed and the values of Ra, Rc, and Re, adjusted to obtain the best possible fit to these experimental time courses. The parameter values Ra (open triangles) and Rc (open circles), resulting in the fits shown by the solid lines through the data in the left panels of Fig. 5, are plotted as a function of phospholipid concentration in Fig. 4B. The rates of Pre2 formation were set at a constant upper limit (Ra = 0.001 s-1) consistent with the fact that Pre2 was never detected.

Fig. 4B also shows, as a function of lipid concentration, the rate of direct conversion of prothrombin (Re, channeling; open squares) that was necessary to obtain the fits shown in Fig. 5. Shown in Fig. 4 are the relative rates of prothrombin activation via the following pathways: the channeling pathway (Re relative Re/(Ra + Rc + Re); open squares, panel E); the MzIIa pathway (open circles, panel D), or the Pre2 pathway (open triangles, panel C).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The purpose of this work was to ask why factor Va and PS membranes both seem to be required for efficient prothrombin activation. The results obtained here, combined with other results obtained in the presence of either of the two cofactors alone (1-3, 6), suggest the following answers: (i) the main role of the cofactor Va and the membrane is rate enhancement; (ii) the two co-factors direct prothrombin activation through the meizothrombin pathway, with PS membranes having the major role in this function; (iii) both cofactors contribute to channeling, i.e. to the creation of a fast pathway of direct conversion of prothrombin to thrombin; and (iv) factor Va allows factor Xa to function efficiently as a robust channeling enzyme over a wide range of membrane concentration. We will discuss each conclusion in order.

Rate Enhancement-- To show how PS membranes and factor Va together enhance the rate of prothrombin activation compared with the individual cofactors alone, we must obtain from our results estimates of second order rate constants. Second order rate constants are difficult to obtain because of a lack of precise knowledge about the total amount of factor Xaa in a reaction mix actually complexed with factor Va on a membrane to form the active prothrombinase complex. The difficulty in defining Xa-Va-membrane complex concentration derives from the fact that studies of prothrombin activation normally are carried out on discrete membrane vesicles so that the efficiency of association between factors Va and Xa is a function of lipid concentration (14, 15). This has been suggested to account, at least in part, for the phenomenon of lipid inhibition that we note in the inset in Fig. 2. There is no quantitative treatment of this effect that would allow prediction of the concentration of Xa-Va complex at different lipid concentrations based on first principles. Thus, we must be content to make simplifying assumptions, as in earlier studies, so that we might compare rate constants with them. Nesheim et al. (4) reported rate constants for bovine prothrombinase under conditions for which activation was maximal with respect to both lipid and Va concentrations, whereas Krishnaswamy et al. (6) based human prothrombinase concentration on an apparent assembly dissociation constant estimated from the dependence of prothrombin activation rate on factor Va concentration. Based on the results summarized in Fig. 4A, cleavage of individual bonds was optimal at 50 µM lipid. This lipid concentration is also low enough to avoid complications because of incomplete distribution of Va and Xa between discrete vesicles (7 Xa and 80 Va/vesicle). The apparent dissociation constant (1.7 ± 1 nM) obtained from the data of Fig. 2 using the approach of Krishnaswamy et al. (6) also supports the assumption that all factor Xa (at 2.5 nM) is incorporated into the Va-Xa complex at 30 nM Va and 50 µM lipid. Based on this assumption, the second order rate constants (kcat/Km) are calculated and presented in Table I for 50 µM lipid. These constants are in close agreement with the only results in the literature to which we can make a direct comparison, 2.9 × 107 and 2.3 × 107 M-1s-1 for reactions B and D, respectively (6).

                              
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Table I
Kinetic parameters (kcat/Km) for activation of prothrombin and its intermediates
The absolute values of the rate constants are expressed in M-1 s-1. The changes are given as the ratio of the absolute rate of the current row to the rate of the previous row rounded to two significant figures. PC, phosphatidylcholine.

There is no simple means by which to judge how PS membranes and factor Va increase the rate of prothrombin activation because they can affect any of five different reactions shown in Fig. 1. To begin, we focus on the cleavages of two bonds in prothrombin that lead to intermediate formation. The rates of these bond cleavages in prothrombin (reactions A and C) were very different (Table I), as both PS membranes and factor Va plus membranes inhibited Arg273-Thr274 cleavage in reaction A and promoted Arg322-Ile323 cleavage in reaction C dramatically. This shifts activation to the MzIIa intermediate almost exclusively, as we will discuss in the next section. Based on the data recorded in Table I, in the presence of PS membranes, the rate-enhancing effect of factor Va on the reaction producing MzIIa (1800) is roughly twice that of PS membranes (750). Based on results obtained with bovine proteins (1, 18, 19), in the absence of PS membranes, the rate of enhancement of this reaction due to factor Va alone can be estimated to be 14,000. From either comparison, we conclude that factor Va plays the dominant role in enhancing the rate of MzIIa formation.

We turn attention next to the activation of intermediates to thrombin. Our results show that the rate constants for proteolysis of the Arg273-Thr274 and Arg322-Ile323 bonds in MzIIa and Pre2, respectively, were quite similar in the presence of Va and PS membranes, in agreement with the conclusion reported by Krishnaswamy et al. (6) for human prothrombinase assembled in the presence of 20 µM PS/phosphatidylcholine (25/75) small unilamellar vesicles. Because the formation of Pre2 is so slow in the presence of PS membranes, we can focus attention for all practical purposes solely on MzIIa activation. PS membranes alone result in a 100-fold enhancement of the rate of human MzIIa activation (2), whereas factor Va alone results in a 940-fold enhancement of bovine MzIIa activation (18); therefore, factor Va accounts for roughly a 9-fold greater rate of MzIIa activation than do PS membranes. Comparing, at optimal membrane concentrations, the second order rate constant for reaction D for the full Xa-Va-PS membrane complex (Table I) to that reported for factor Xa bound to PS membranes (2), we see a 20-fold increase in rate because of adding factor Va to the PS membrane. Based on this comparison (9-fold versus 20- fold ± membranes), it would seem that binding to PS membranes does not alter significantly the ability of factor Va to enhance the rate of MzIIa activation, and, as with MzIIa formation, factor Va seems to be the major contributor to the cofactor-mediated rate enhancement of MzIIa consumption.

Shifting Prothrombin Activation to the Meizothrombin Pathway-- Aside from rate enhancement, the principle role of factor Va and PS-containing membranes in prothrombin activation is to direct the pathway of activation. To discuss this effect of the cofactors, we do not need absolute rates; the relative rates recorded in Fig. 4, C-E, will suffice. We note first that 96% of the initial proteolysis of human prothrombin by factor Xa leads to Pre2 formation (2). From the relative rates recorded in Fig. 4, it is clear that bovine factor Va alone reduces this fraction to roughly 31% (X in panel C) (1), whereas PS membranes reduce this fraction to roughly 4% at optimal membrane concentration (cross-filled triangles in panel C) (2). It is notable that neither cofactor alone is sufficient to completely eliminate formation of Pre2 intermediate. However, in the presence of both PS membranes and factor Va, no Pre2 could be detected (Fig. 5), and the fraction of prothrombin initially converted to Pre2 was reduced to zero at all membrane concentrations (open triangles in panel C). The fact that the fraction of prothrombin activated to MzIIa was lower for the full prothrombinase studied here (open circles in panel D) than for Xa bound to PS membranes alone (cross-filled circles in panel D) (2) simply reflects a large amount of activation via channeling (panel E), as will be discussed next. These relative rates establish that factor Va and PS membranes together direct activation exclusively via the MzIIa intermediate and/or via channeling to a greater extent than either cofactor can accomplish alone.

Effects of PS and Va Cofactors on Channeling-- Both factor Va and PS membranes have been shown to promote conversion of prothrombin to thrombin without release of the intermediate from the enzyme complex (1, 2), i.e. to promote intermediate channeling (reaction E in Fig. 1). Fig. 4E summarizes our results as well as results presented in the recent literature with respect to channeling. Here we see that bovine factor Va alone causes factor Xa initially to process roughly 39% of prothrombin via channeling (X in Fig. 4E) (1). At optimal PS membrane concentration (50 µM), 55% of prothrombin initially is processed via channeling (cross-filled squares) (2), although channeling was much less efficient at lower and higher membrane concentrations. The addition of factor Va to PS membranes had little effect on the fraction of prothrombin initially activated via the channeling pathway at 50 µM PS membranes but had a significant influence at higher and lower membrane concentrations (open squares, Fig. 4E), as will be discussed below.

Factor Va in the Presence of PS Membranes Creates a Robust Channeling Enzyme-- Even though the relative rates presented in Fig. 4 show that only about 55-68% of prothrombin initially is processed via channeling, the calculated initial time courses of thrombin generation through different pathways show that, at all lipid concentrations, the presence of factor Va and PS-containing membranes causes thrombin formation to proceed exclusively through channeling, at least at the early stage of the reaction. This finding is depicted graphically in the right-hand panels of Fig. 5 (channeling, small filled circles; Pre2 intermediate, dashed lines; and MZIIa intermediate, small filled triangles). Although these two observations at first might seem to be inconsistent, they are not because in the presence of factor Va and PS membranes, channeling occurs at a rate similar to those of meizothrombin formation and activation to thrombin (Table I). Under these circumstances, a two-step, sequential mechanism will be roughly 100 times slower than a channeling mechanism. By contrast, in the presence of PS membranes but not factor Va, MzIIa formation and channeling had comparable rate constants, but MzIIa activation occurred roughly 100 times faster (2). Thus, the presence of factor Va on a PS membrane enhances the rates of channeling and MzIIa formation substantially (~1700-fold at 50 µM lipid; Table I) but enhances the rate of MzIIa activation by only 20-fold (Table I). This converts prothrombin activation from a process that proceeds via both MzIIa and channeling in the presence of PS membranes (2) to one that proceeds exclusively via channeling when factor Va is bound to the membranes (Fig. 5).

The most remarkable effect of factor Va is that it makes factor Xa an efficient channeling enzyme over the two orders of magnitude range of lipid concentrations we considered (open squares in Fig. 4E). This could be a great advantage in vivo where the concentrations of platelet membrane vesicles (20, 21) promoting prothrombin activation could vary greatly within a wound area, even though it would be important to have stable production of thrombin under all conditions. Channeling occurs in the absence of factor Va, but it is limited to a narrow range of membrane concentrations (cross-filled squares, Fig. 4E) (2). The ability of factor Va to support channeling at higher membrane concentrations seems to be responsible for the maximal overall activation rate seen at 100-200 µM lipid in the inset in Fig. 2 compared with the minimal lipid dependence seen for individual proteolytic steps of activation (Fig. 4, A and B). It is not yet clear whether the ability of PS membranes and factor Va to support channeling at high membrane concentrations is a result of the cooperative effect of the two cofactors or the binding of PS to factor Va to enhance its channeling activity. Clarification of this issue must await a full analysis, analogous to the ones presented here and elsewhere (1, 2) of channeling in the newly described solution-assembled prothrombinase (22).

    FOOTNOTES

* This work was supported by United States Public Health Service Grant HL45916 (to B. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, CB # 7260, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260. Tel.: 919-966-5384; E-mail: uncbrl@med.unc.edu.

Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M208423200

    ABBREVIATIONS

The abbreviations used are: F1.2, fragment 1.2; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine; LUV, large unilamellar vesicles; S-2238, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride (synthetic substrate for thrombin); S-2765, N-alpha -benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline dihydrochloride (synthetic substrate for factor Xa); DOPC, dioleoyl-phosphatidylcholine; Pre1, prethrombin 1; Pre2, prethrombin 2; IIa, thrombin; MzIIa, meizothrombin; PS, phosphatidylserine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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