©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Second Kringle Domain of Prothrombin Promotes Factor Va- mediated Prothrombin Activation by Prothrombinase (*)

(Received for publication, October 14, 1994)

Karen J. Kotkow (§) Steven R. Deitcher Bruce Furie Barbara C. Furie

From the Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center and the Department of Medicine and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The incorporation of factor Xa into the prothrombinase complex, factor Xa-factor Va-phospholipid-Ca(II), results in an approximately 10^5-fold higher rate of substrate activation than that of the enzyme alone. To examine the role of the prothrombin kringle domains in the interaction with prothrombinase we have employed site-directed mutagenesis to produce prothrombin species that lack either the first kringle domain, PT/DeltaK1, or the second kringle domain, PT/DeltaK2. Previously, we have shown that these proteins are fully carboxylated and that they bind to phospholipid vesicles. In this investigation we demonstrate that cleavage at Arg-Thr and Arg-Ile peptide bonds occurs upon activation with prothrombinase to yield normal thrombin from both PT/DeltaK1 and PT/DeltaK2. In the absence of factor Va, the K(app) for the activation of PT/DeltaK1, PT/DeltaK2, or plasma-derived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent. The K(app) for the activation of PT/DeltaK2 by prothrombinase is approximately 4-5-fold higher than that obtained for plasma-derived prothrombin or PT/DeltaK1. These data demonstrate that the prothrombin kringle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction. In the absence of factor Va, equivalent k values were obtained for all of the prothrombin species when they were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold lower k value was obtained for the activation of PT/DeltaK2 by prothrombinase as compared with that obtained for plasma prothrombin or PT/DeltaK1. Collectively, these data suggest that determinants within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of substrate turnover. Indeed, in contrast to plasma-derived prothrombin, no direct binding of PT/DeltaK2 to factor Va to form the PT/DeltaK2factor Va complex could be demonstrated by 90° light scattering.


INTRODUCTION

The generation of thrombin concentrations suitable for blood clot formation on a physiologically relevant time scale requires prothrombin activation mediated by prothrombinase, a complex consisting of the enzyme factor Xa and cofactors calcium, factor Va, and membrane surfaces (for a review see Mann, 1987). Incorporation of factor Xa into the prothrombinase complex confers a catalytic rate advantage resulting in an approximately 2.8 times 10^5-fold higher rate of prothrombin activation than the rate of catalysis by the enzyme alone in solution (Nesheim et al., 1979). A body of data suggests that factor Xa associates with factor Va on the membrane surface with a 1:1 stoichiometry (Nesheim at al., 1979), independent of substrate (Nesheim et al., 1981). Both factor Va and factor Xa bind to phospholipid with high affinity. The binding of factor Va to phospholipid independently increases the affinity of factor Xa for the membrane (Krishnaswamy, 1990). The factor Va-factor Xa interaction results from lateral diffusion of the two proteins on the membrane surface (Krishnaswamy et al., 1988). The interaction of factor Va and factor Xa is stabilized at the membrane surface relative to their binding in solution (Krishnaswamy, 1990). The calcium-stabilized conformer of prothrombin initially binds with phospholipid forming a ternary complex, which then serves as the substrate for the membrane-bound enzymatic complex (Nesheim et al., 1984). From these data, the primary role of the phospholipid membrane appears to be as a vehicle for the concentration of reactants, factor Xa, factor Va, and prothrombin. Indeed, in the absence of factor Va, phospholipid lowers the apparent K of factor Xa activation of prothrombin from 131 µM to 1 µM but causes little or no change in the V(max) (Rosing et al., 1980). In contrast, the cofactor appears to function in the enhancement of the catalytic rate. In the absence of phospholipid, factor Va caused little change in the apparent K value for factor Xa activation of prothrombin but promoted a 1,000-fold increase in the V(max) value (Rosing et al., 1980; van Rijn et al., 1984). It is currently unclear how factor Va effects this dramatic increase in the catalytic rate.

We are interested in understanding the nature of the interaction between prothrombin and the prothrombinase complex. Various structural domains of prothrombin are thought to play important functional roles in binding to the different components of the prothrombinase complex, thereby regulating the generation of thrombin activity. Our previous data have suggested that membrane binding is mediated by the -carboxyglutamic acid-rich domain and/or aromatic amino acid stack domain (Kotkow et al., 1993). Indeed, we have recently demonstrated that the homologous Gla domain and aromatic amino acid stack domain of Factor IX express phospholipid binding properties (Jacobs et al., 1994). The precise role of the kringle domains of prothrombin in the activation process is unknown. However, these structural motifs are found in many other proteins involved in hemostasis and fibrinolysis and have been implicated in protein-protein interaction. For example, studies examining the mechanism of plasmin generation by tissue type plasminogen activator have determined that the fibrin specificity displayed by tissue type plasminogen activator is caused by its ability to assemble with plasminogen bound on a fibrin surface (Hoylaerts et al., 1982). Fibrin recognition and enhanced activity of tissue type plasminogen activator appear to be localized to determinants within the second kringle domain (van Zonneveld et al., 1986; Larsen et al., 1988; Urano et al., 1989). Previous studies examining prothrombin activation in solution by factor Xa-factor Va-Ca(II) using fragments of prothrombin have suggested that the second kringle domain interacts with factor Va (Esmon and Jackson, 1974; Bajaj et al., 1975). We have examined the interaction of recombinant prothrombins lacking the first kringle (PT/DeltaK1) or the second kringle (PT/DeltaK2) as substrates for the complete prothrombinase complex. We now suggest that determinants within the second kringle domain of prothrombin are important for the factor Va-dependent rate enhancement observed upon activation of prothrombin by the prothrombinase complex. Also, kinetic studies performed in the absence of factor Va indicate that the kringle domains of prothrombin do not play any direct role in the interaction of prothrombin with factor Xa.


EXPERIMENTAL PROCEDURES

Materials

Purified plasma-derived human prothrombin, human factor Xa, and bovine factor Va were obtained either from Enzyme Research Labs (South Bend, IN) or Haematolgic Technologies (Essex Junction, VT). The chromogenic substrate CBS 34.47 was from Diagnostica Stago; the chromogenic substrate S-2238 and the thrombin inhibitor I-2581 were from KabiVitrum. Phosphatidylserine and phosphatidlycholine were obtained from Avanti Polar Lipids. PT/DeltaK1 and PT/DeltaK2 were constructed, expressed, and purified as described previously (Kotkow et al., 1993).

Determination of K(m) and V(max)for activation of prothrombin species by prothrombinase

The ability of prothrombin and the recombinant prothrombin species to serve as substrates for the prothrombinase complex were evaluated as described previously for the activation of factor IX by tenase (Hertzberg et al., 1992). Factor Xa (0.01 nM), factor Va (10.0 nM), phospholipid vesicles (35 µM), and calcium chloride (2.5 mM) in TBS (^1)containing 0.1% BSA were incubated at 37 °C for 2 min. The reaction was initiated by the addition of substrate at the indicated concentrations. Aliquots of the reaction mixture were removed at 2-min intervals, and thrombin activity was assayed with the chromogenic substrate CBS.34.47 (375 µM). Kinetic constants were determined by nonlinear regression analysis using the Michaelis-Menten equation and the simple weighting function (Enzfitter; Elsevier). To establish levels of factor Va that would be saturating for determination of the K(m) and k for activation of prothrombin and recombinant species the rate of activation of these species was determined as a function of factor Va concentration. The assay was performed as described above except that 10 nM prothrombin substrate was activated with 0.10 nM factor Xa, 35 µM phospholipid vesicles, 2.5 mM calcium chloride, and the indicated concentrations of factor Va in TBS containing 0.1% BSA. Similarly, the rate of activation of prothrombin and the recombinant species as a function of phospholipid and Ca(II) concentrations was determined. Optimal concentrations of these cofactors were used in the kinetic analyses.

Determination of K(m) and V(max) for Activation of Prothrombin Species by Factor Xa-Ca(II)-Phospholipid

The ability of prothrombin and the recombinant deletion mutants to serve as substrates for factor Xa in the presence of phospholipid and calcium was evaluated as described above. Factor Xa (5 nM) in TBS containing 0.1% BSA, 35 µM phospholipid vesicles, and 2.5 mM calcium chloride was incubated at 37 °C for 2 min. The reaction was initiated by the addition of substrate at the concentrations indicated. Aliquots of the reaction mixture were removed at 0, 15, 25, and 40 min. Thrombin generation was assayed with the chromogenic substrate S-2238 (375 µM). Kinetic constants were determined by nonlinear regression analysis using the Michaelis-Menten equation and the simple weighting function (Enzfitter; Elsevier).

Determination of the Binding Constant for Interaction of Factor Va with Prothrombin Species by Light Scattering

The interaction between the prothrombin species and factor Va was measured by the relative 90° light-scattering technique using an SLM 8000 fluorescence spectrophotometer as described by Boskovic et al.(1990). Excitation and emission wavelengths were 380 nm; excitation and emission slit widths were 8 nm. Factor Va, prothrombin, and PT/DeltaK2 were dialyzed into TBS, 5 mM CaCl(2), which had been freed of particulates by filtration through a 0.22-µm filter (Millipore Corp., Bedford, MA). Protein solutions were freed of particulates by centrifugation at 12,000 times g for 10 min at 4 °C. Factor Va (2.7 µM, 100 µl) was placed in a 200-µl cuvette. Aliquots of prothrombin (52.3 µM) or PT/DeltaK2 (32.8 µM) were added to achieve the concentrations shown. Dissociation constants were calculated according to Lim et al.(1977) using Sigmaplot (Jandel).

Activation of Recombinant Prothrombin Species with Prothrombinase

Prothrombin and recombinant deletion mutants (2 µM) were activated with 0.3 nM factor Xa in the presence of 5 nM factor Va, 50 µM phospholipid (Thrombofax, Ortho), 5 mM CaCl(2), and 20 µM I-2581 in TBS at 37 °C. At the indicated times aliquots of the reaction mixture were removed, and the reaction was stopped by the addition of EDTA to a final concentration of 50 mM.

SDS-Polyacrylamide Gel Electrophoresis

Mixtures of activated prothrombin species were subjected to electrophoresis in gels consisting of a 5% polyacrylamide stacking gel and a 10% polyacrylamide resolving gel (Laemmli, 1970). All gels contained 0.1% SDS and were run under nonreducing conditions. Samples were prepared for electrophoresis by the addition of sample buffer containing 0.1% SDS and heated at 60 °C for 10 min. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 or electroblotted onto Immobilon-P membranes (Millipore Corp.) for amino-terminal sequence analysis.

NH(2)-terminal Sequence Analysis

Amino-terminal sequence was determined by automated Edman degradation using a Milligen/Biosearch Prosequencer (LeGendre and Matsudaira, 1988). Prior to sequencing, protein bands were electroblotted from SDS-polyacrylamide minigels onto polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.) in 10 mM 3-(cyclohexylamino)propanesulfonic acid (CAPS), 10% methanol (v/v), pH 11.0, at 100 mA for 1 h (LeGendre and Matsudaira, 1988).

Preparation of Phospholipid Vesicles

Large unilamellar vesicles (phosphatidylserine:phosphatidylcholine; 15:85) were prepared by extrusion. Solvent was removed from phospholipids (20 mg of phosphatidylserine, 113 mg of phosphatidylcholine) in chloroform:methanol (9:1) under a stream of nitrogen. The lipid mixture was redissolved in benzene and evaporated to dryness. The resulting powder was suspended in 4 ml of TBS and subjected to freeze-thawing 10 times before extrusion through 0.4-µm polycarbonate membranes (Szoka et al., 1978). The resulting phospholipid vesicles were stored under nitrogen at -80 °C in small aliquots. Phospholipid concentrations were determined by assay of elemental phosphorus as described (Chen et al. 1956).


RESULTS

Previously we have shown that recombinant prothrombins lacking the first kringle (PT/DeltaK1) or the second kringle (PT/DeltaK2) have the mature amino-terminal sequence of prothrombin and are fully -carboxylated (Kotkow et al., 1993). These proteins are able to assume both calcium-induced conformational transitions and to bind to phospholipid vesicles. Despite these properties, PT/DeltaK2 has only 10% of the coagulant activity of plasma-derived prothrombin, whereas PT/DeltaK1 has 50% of the coagulant activity of plasma-derived prothrombin (Kotkow et al., 1993).

SDS-Polyacrylamide Gel Electrophoresis Analysis of the Activation of Prothrombin Species by Prothrombinase

The cleavage patterns of prothrombin and the prothrombin-related species generated by the complex of factor Xa, factor Va, Ca(II), and phospholipid were compared by monitoring the appearance of the A and B chains of thrombin. Prothrombin, PT/DeltaK1, or PT/DeltaK2 was incubated with the prothrombinase complex. Aliquots of the reaction mixture were taken at various time points, and the reaction was stopped by the addition of EDTA. After SDS-polyacrylamide gel electrophoresis of the reaction products the separated protein bands were electroblotted onto polyvinylidene difluoride membranes and subjected to amino-terminal sequence analysis. Fig. 1depicts the resolved activation products after SDS-polyacrylamide gel electrophoresis and staining with Coomassie R-250. Fig. 1, A and B demonstrates that activation of both plasma-derived prothrombin and PT/DeltaK1 by prothrombinase resulted in the production of prethrombin 1 and fragment 1, as would be expected from the primary sequence of these proteins. In contrast, the presence of prethrombin 1 was not detected upon activation of PT/DeltaK2 by prothrombinase. This observation is consistent with the fact that the thrombin cleavage site at amino acids 156-157 is located in the deleted second kringle exon. (^2)Fragment 2 and truncated species of fragment 1 and fragment 1bullet2 derived from the recombinant prothrombin proteins were not retained on the gel under these conditions because of their low molecular weight. The extent of cleavage over the time course appeared to vary with each mutant and generally reflected the differences in activity observed in the coagulant activity assay. This suggests that the rate of activation, and not a defect in the resultant thrombin molecule, accounts for the lower coagulant activity displayed by these mutants.


Figure 1: SDS-PAGE analysis of the activation of plasma-derived prothrombin, PT/DeltaK1, and PT/DeltaK2 by prothrombinase. For each reaction 2 µM prothrombin substrate was activated with 0.3 nM factor Xa in the presence of 5 nM factor Va, 50 µM phospholipid, 5 mM CaCl(2) and 20 µM I-2581. A, plasmaderived prothrombin; B, PT/DeltaK1; C, PT/DeltaK2. Pre 1, prethrombin 1; F1bullet2, fragment 1bullet2; IIalphabeta, thrombin; F1, fragment 1.



Amino-terminal sequencing of the thrombins derived from limited proteolysis of PT/DeltaK1 and PT/DeltaK2 by the prothrombinase complex showed that the only observable cleavages occurred at Arg-Thr and Arg-Ile, thereby producing alpha-thrombin. These were also the cleavages observed upon activation of the plasma-derived prothrombin sample under the same conditions. These data demonstrate that the deletion of a kringle domain does not alter the specificity of the factor Xa cleavage site in the substrate.

Effect of Factor Va Concentration on the Rate of Thrombin Generation from the Prothrombin Species

The rate of thrombin generation from mutant and plasma-derived prothrombin was studied as a function of factor Va concentration (Fig. 2). In this experiment a fixed concentration of prothrombin substrate (10 nM) was activated with a fixed concentration of factor Xa (0.1 nM) and varying concentrations of factor Va. Optimal concentrations of phospholipid (35 µM) and Ca(II) (2.5 mM) were included in the reaction. In the absence of factor Va, no thrombin was detected. For plasma-derived prothrombin and the prothrombin kringle deletion mutants the initial rate of thrombin formation was dependent on the concentration of factor Va present, and at saturating concentrations of factor Va a maximal initial rate was achieved (Fig. 2). From these data 10 nM factor Va was determined to be saturating for all prothrombin species, and this factor Va concentration was used for determination of the apparent K(m) and k for all of the prothrombin species. Concentrations of 35 µM phospholipid and 2.5 mM Ca(II) were determined to yield optimal initial rates of thrombin generation from all of the prothrombin substrates. Significantly higher or lower concentrations of these cofactors yielded lower initial rates of thrombin generation for activation of all of the prothrombin species (data not shown).


Figure 2: Comparison of the rate of activation of plasma-derived prothrombin, PT/DeltaK1, and PT/DeltaK2 as a function of factor Va concentration. Prothrombin substrates (10 nM) were activated with 0.10 nM factor Xa, 35 µM phospholipid vesicles, 2.5 mM CaCl(2), and indicated concentrations of factor Va in TBS containing 0.1% BSA at 37 °C. Aliquots were removed at 2-min intervals. bullet, plasma-derived prothrombin; circle, PT/DeltaK1; times, PT/DeltaK2.



Activation of the Prothrombin Species by the Prothrombinase Complex

To define the role of the kringle domains in the interaction of prothrombin with the components of the prothrombinase complex, the rate of thrombin generation from the kringle deletion mutants was compared with that from prothrombin as a function of substrate concentration. In this experiment varying concentrations of prothrombin were activated with 0.01 nM factor Xa and saturating concentrations of factor Va (10 nM), phospholipid (35 µM), and Ca(II) (2.5 mM). The formation of thrombin was monitored as a function of time by hydrolysis of the chromogenic substrate S-2238. Under these conditions, the apparent K(m) values determined for both plasma-derived prothrombin and PT/DeltaK1 were equivalent ( Table 1and Fig. 3). The apparent K(m) value determined for PT/DeltaK2 was 4-5-fold higher than that of PT/DeltaK1 or plasma-derived prothrombin. The k value determined for PT/DeltaK2 was approximately 7-fold lower than that determined for either plasma-derived prothrombin or PT/DeltaK1. From this combined with our previous data we conclude that the first prothrombin kringle domain is not involved in phospholipid, factor Xa, or factor Va binding. These data also suggest that the second kringle domain contributes to the binding interaction and has an important influence on the rate with which the prothrombinase complex turns over substrate. This region may affect the catalytic rate by influencing the function of factor Xa, factor Va, or both.




Figure 3: Activation of plasma-derived prothrombin, PT/DeltaK1, and PT/DeltaK2 by prothrombinase. Prothrombin substrates (0.03-3.12 µM) were activated with 0.01 nM factor Xa, 10.0 nM factor Va, 35 µM phospholipid vesicles, and 2.5 mM CaCl(2) in TBS containing 0.1% BSA at 37 °C. Aliquots were removed at 2-min intervals and assayed for thrombin activity with the chromogenic substrate CBS.34.47 (375 µM). A, Plasma-derived prothrombin (bullet); B, PT/DeltaK1 (circle); C, PT/DeltaK2 (times).



Activation of the Prothrombin Species by Factor Xa-Ca(II)-Phospholipid

To further define the role of the kringle domains in substrate activation by factor Xa, PT/DeltaK1 and PT/DeltaK2 were evaluated for their ability to serve as substrates for factor Xa in the presence of phospholipid and Ca(II) but in the absence of the cofactor, factor Va. Initial rates of thrombin formation were determined under conditions employing a fixed factor Xa concentration (5 nM), optimal phospholipid and Ca(II) concentrations (35 µM and 2.5 mM, respectively), and varying substrate concentrations. Apparent K(m) and k values were calculated from the kinetic data obtained ( Fig. 4and Table 1). The apparent K(m) and k values determined for the recombinant prothrombin species were equivalent to those of plasma-derived prothrombin. From these data we conclude that the kringle domains of prothrombin are not involved in factor Xa-prothrombin interaction or phospholipid binding. These results suggest that the impaired ability of PT/DeltaK2 to serve as a substrate for the prothrombinase complex must be caused by the interaction of the second prothrombin kringle domain with factor Va. Furthermore, these results demonstrate that the alpha-thrombin derived from the prothrombin species are equivalent functionally and that deletion of the kringle domains does not disrupt the structure of the C-terminal half of the recombinant prothrombin.


Figure 4: Activation of plasma-derived prothrombin, PT/DeltaK1, and PT/DeltaK2 by factor Xa, Ca(II), and phospholipid. Prothrombin substrates (0.63-4.0 µM) were activated with 5 nM factor Xa, 35 µM phospholipid vesicles, and 2.5 mM CaCl(2) in TBS containing 0.1% BSA at 37 °C. Aliquots were removed at 0, 15, 25, and 40 min after initiation of the reaction and assayed for thrombin activity with the chromogenic substrate S-2238 (375 µM). A, plasma-derived prothrombin (bullet); B, PT/DeltaK1 (circle); C, PT/DeltaK2 (times).



Binding of Prothrombin and PT/DeltaK2 to Factor Va

To confirm that the defect in PT/DeltaK2 as a substrate for the prothrombinase complex is the result of impaired binding to factor Va we determined the relative binding affinity of plasma-derived prothrombin and PT/DeltaK2 for factor Va using the 90° light-scattering technique. Prothrombin bound to factor Va with a relative dissociation constant of 7.8 ± 1.4 µM (Fig. 5). This is in good agreement with the value of 8.8 ± 0.8 µM previously determined by this technique (Boskovic et al., 1990). In contrast, PT/DeltaK2 did not bind significantly to factor Va under the conditions employed. These results suggest that the second kringle domain binds directly to factor Va. These data support the conclusion that the impaired ability of PT/DeltaK2 to serve as a substrate for the prothrombinase complex results from the loss of the ability of PT/DeltaK2 to bind to the cofactor factor Va and the concomitant loss of the acceleration of the rate of substrate activation afforded by the cofactor.


Figure 5: Binding of plasma-derived prothrombin and PT/DeltaK2 to factor Va. The interaction of the prothrombin species with factor Va was monitored by 90° light scattering. Plasma-derived prothrombin (52.8 µM) or PT/DeltaK2 (32.8 µM) were added to a cuvette containing Factor Va (2.7 µM, 100 µl) to achieve the concentrations shown. The change in relative molecular weight is expressed as M2/M1(molecular weight of the protein complex/molecular weight of factor Va). bullet, plasma-derived prothrombin; times, PT/DeltaK2.




DISCUSSION

Our approach toward understanding the functional role of the prothrombin kringle domains in the interaction with the prothrombinase complex was to study mutant prothrombin proteins that lack either the first kringle (PT/DeltaK1) or the second kringle domain (PT/DeltaK2). Previously, we have provided evidence that posttranslational -carboxylation of these mutants is complete and that the -carboxyglutamic acid-rich domains of our purified PT/DeltaK1 and PT/DeltaK2 proteins are properly folded (Kotkow et al., 1993). In this study we have shown that activation of PT/DeltaK1 by prothrombinase results in the generation of prethrombin 1 and fragment 1, whereas these fragments are not detected upon activation of PT/DeltaK2. This pattern of activation fragments is consistent with those predicted from the primary sequence of the mutated proteins. Cleavage of both mutant zymogens by factor Xa occurred at the expected sites, resulting in the production of alpha-thrombin. Equivalent apparent K(m) and k values were obtained when the mutant and plasma-derived substrates were activated by factor Xa, Ca(II), and phospholipid. These data demonstrate that in the absence of factor Va the mutant substrates are recognized and processed as efficiently as the plasma-derived substrate, making it unlikely that the observed differences in thrombin generation were actually caused by differences in the specific activity of thrombin species derived from these mutants. These observations indicate that functional differences between the mutant prothrombin species and plasma-derived prothrombin are caused by the absence of amino acid residues on the surface of the deleted kringle domain rather than incomplete processing events or global distortion of the structure of the recombinant proteins.

The first kringle domain of prothrombin does not appear to interact with phospholipid, factor Va, or factor Xa. Kinetic analyses of PT/DeltaK1 activation by prothrombinase or factor Xa-Ca(II)-phospholipid yielded equivalent apparent K(m) and k values as plasma-derived prothrombin. Despite these results, the coagulant activity of this prothrombin species was 50% when compared with that of plasma-derived prothrombin. This inconsistency may reflect inherent differences between the two assays. The coagulant activity assays, which are performed under conditions that may not follow Michaelis-Menten kinetics, may enhance slight differences between the prothrombin species.

Data from these kinetic studies have established that the kringle domains of prothrombin do not contribute significantly to the overall binding affinity of the substrate-enzyme interaction. Equivalent apparent K(m) values were obtained when PT/DeltaK1, PT/DeltaK2, or plasma-derived prothrombin was activated by factor Xa-Ca(II)-phospholipid or when PT/DeltaK2 was activated by prothrombinase (Table 1). These apparent K(m) values were approximately 4-5-fold higher than those obtained for the activation of PT/DeltaK1 or plasma-derived prothrombin by prothrombinase (Table 1). These results indicate that the binding affinity of the PT/DeltaK2 substrate for prothrombinase is weaker than that of plasma-derived prothrombin and that this result is caused solely by the loss of binding affinity resulting from the interaction of the second kringle domain with factor Va. Indeed, we were unable to demonstrate direct binding between PT/DeltaK2 and factor Va, in contrast to the binding of prothrombin to factor Va. These results are consistent with previous studies, which have shown that the phospholipid component, not the protein cofactor, contributes significantly to the overall binding affinity of the substrate-enzyme complex (Rosing et al., 1980).

This investigation provides new insight into the mechanism by which factor Va effects an enhancement on the catalytic rate of substrate turnover when it is assembled in the prothrombinase complex (Rosing et al. 1980, Nesheim et al., 1979). Previous studies have suggested that this rate enhancement could be attributed to a direct influence on the conformation of the enzyme active site by the cofactor, factor Va. A perturbation of the active site of factor Xa has been observed upon incorporation of the enzyme into the prothrombinase complex by perturbation of the active site bound fluorophore dansyl-Glu-Gly-Arg, suggesting that factor Va induces an allosteric conformational transition in the active site of factor Xa (Krishnaswamy et al., 1988; Husten et al., 1987) Recent studies have shown that this conformational transition results in subtle changes in the accessibility of the factor Xa active site rather than in a general increase in the reactivity of the active site histidine residue (Walker and Krishnaswamy, 1992). In addition to influencing the conformation of the enzyme, other observations have suggested that the interaction of factor Va with the substrate may be critical for the enhancement of the catalytic rate. Nesheim et al.(1981) have observed that, in contrast to activation of prothrombin, the V(max) for activation of small peptide substrates by factor Xa is unaffected by the presence of factor Va. We have observed that the k value determined for PT/DeltaK2 is approximately 7-fold lower than that determined for plasma-derived prothrombin, thus implying that one aspect of the factor Va-dependent catalytic rate enhancement is dependent upon the association of the cofactor and the substrate.

Previous studies examining the ability of various prothrombin activation fragments to serve as substrates for the prothrombinase complex for the solution phase factor Xa-Ca(II)-factor Va complex have suggested that prothrombin fragment 2, comprising the second kringle domain, contains a site for factor Va interaction, which affects the factor Va-dependent catalytic rate acceleration (Esmon and Jackson, 1974; Bajaj et al., 1975). Our results confirm these data. However, using prothrombin species that bind phospholipid membranes the concentrations of reactants and the time required to monitor thrombin formation approach physiologically relevant conditions. By including the contributions of the phospholipid component to assembly of the prothrombin-prothrombinase complex, we may have also ensured that the conclusions reached as a result of these studies reflect the interactions that occur in the physiological complex.

The second kringle domain of prothrombin is not the only domain that is necessary for factor Va interaction, since we observed that the factor Va-dependent rate enhancement, approximately 200-fold for plasma-derived prothrombin, was not completely ablated in PT/DeltaK2. Indeed, our data indicate that PT/DeltaK2 displays an approximately 35-fold higher k value in the presence of saturating concentrations of factor Va than that determined for activation of the mutant by factor Xa-Ca(II)-phospholipid (Table 1). This may be because the first kringle domain is able to functionally substitute for the second kringle domain, although it is more likely that an additional site for factor Va interaction is present. This additional site may be localized to some portion of the serine protease domain. Support for these conclusions are derived from previous studies of Esmon and Jackson(1974), which have shown no effect of fragment 1 on the factor Va-dependent rate enhancement, thus indicating that the first kringle domain probably can not substitute for the second kringle domain. Also, Esmon and Jackson(1974) have shown that the rate of prethrombin 2 activation by factor Xa and Ca(II) is stimulated 5-fold by the addition of factor Va, suggesting that portions of the serine protease domain are involved in the factor Va-dependent rate enhancement. In addition, recent studies employing a chimeric factor Xa have determined that the binding of factor Va in the prothrombinase complex is mediated by the second EGF-like domain and the serine protease domain of factor Xa (Hertzberg et al., 1992) Thus, it seems likely that optimal alignment between the active site of factor Xa and the activation sites of the prothrombin serine protease domain is also mediated by factor Va.

In summary, we have determined that the first kringle domain of prothrombin is not involved in phospholipid, factor Xa, or factor Va binding. The second kringle domain of prothrombin appears to contribute only modestly to the overall binding affinity of the substrate-enzyme complex. However, our data suggest that this domain interacts directly with factor Va to effect an acceleration in the catalytic rate of thrombin formation.


FOOTNOTES

*
This work was supported by Grants HL18834 and HL42433 from the National Institutes of Health. 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.

§
This work was performed by K. J. K. in partial fulfillment of the requirements for the degree of Doctor of Philosophy from Tufts University. Present address: Children's Hospital, Boston, MA 02115.

(^1)
The abbreviations used are: TBS, Tris-buffered saline (20 mM Tris-HCl, 0.15 sodium chloride, pH 7.5); BSA, bovine serum albumin.

(^2)
Residues are numbered according to the mature human prothrombin protein sequence.


ACKNOWLEDGEMENTS

We thank Ms. Jill Scott for excellent technical assistance.


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