The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin*

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479–Leu480–Gln481–Val482. Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu480 and Gln481 as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIaS478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIaSLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIaΔSLQ (where rIIaΔSLQ is recombinant human α-thrombin with amino acids Ser478/Leu480/Gln481 deleted). These data provide new evidence demonstrating that amino acid sequence Leu480–Gln481: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg320; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.

In the presence of a procoagulant membrane surface and divalent metal ions, factor Va (fVa) 3 binds factor Xa (fXa) to form prothrombinase. Prothrombinase is the two-subunit enzymatic complex where the non-enzymatic regulatory subunit (fVa) controls the rate and directs cleavage of prothrombin (FII) by the catalytic subunit (fXa) at two spatially distinct sites resulting in timely ␣-thrombin (IIa) formation at the place of vascular injury (1)(2)(3). Cleavage at Arg 271 and Arg 320 of FII is required to form the active serine protease IIa. The essential IIa molecule bears strong homology with other serine protease enzymes, such as activated protein C (APC), chymotrypsin, and fXa. Several different numberings of IIa residues appear in the literature based on either the chymotrypsin numbering (4) or IIa numbering (5,6) or the entire FII sequence (7). The latter nomenclature is used herein with the appropriate chymotrypsin numbering in parentheses when required for comparison with the existing data in the literature.
Historically, it has been shown that in the absence of fVa, initial cleavage at Arg 271 of FII results in the generation of the inactive intermediate prethrombin-2 and fragment 1⅐2. Further cleavage of prethrombin-2 at Arg 320 generates IIa (prethrombin-2 pathway) (8 -21). Concurrent with the appearance of excess fVa during clotting and in the presence of a procoagulant surface, the order of cleavages is reversed, and initial cleavage at Arg 320 generates a transient enzymatically active intermediate, meizothrombin, that has much higher catalytic efficiency than IIa toward chromogenic substrates usually employed to assess IIa activity (18,(22)(23)(24). Meizothrombin is next cleaved at Arg 271 resulting in the generation of IIa and fragment 1⅐2 (meizothrombin pathway). Although efficient cleavage at each site requires the presence of phospholipids, initial cleavage at Arg 320 is entirely fVa-dependent.
In the absence of fVa, the two activation cleavage sites are not readily available, and FII is activated at a slow non-physiological rate by membrane-bound fXa alone. Interactions between fXa and FII are known to exist in the presence and absence of fVa; however, the enhanced activity of fXa within prothrombinase toward both activating cleavage sites is controlled solely by the membrane-bound non-enzymatic cofactor (3,16,17,25,26).
Consequently, the innate process of coagulation rests on specific molecular interactions involved in the fVa-dependent activation of FII by prothrombinase. In relation to fXa alone, the relative rate of IIa formation by prothrombinase is increased by 300,000-fold because of the increase in the rates of both FII cleavages. This increase is mainly associated with a large (3,000fold) increase in the k cat value of fXa within prothrombinase with a 100-fold decrease in the K m value of the enzyme (16). This substantial increase in enzymatic activity resulting in rapid and physiologically relevant IIa generation at the place of vascular injury is credited through precise and unique interactions of the cofactor with specific amino acids affiliated with both membrane-bound fXa and membrane-bound FII as recently demonstrated (27). Accordingly, the introduction of the nonenzymatic cofactor into prothrombinase equips the organism's coagulation artillery necessary for the explosive arrest of vasculature bleeding.
The necessary fVa-dependent activation of FII by prothrombinase is a widely studied mechanism of coagulation but is still poorly understood. Numerous fVa-binding sites are acknowledged to exist on FII. Earlier investigations have shown the existence of binding sites on FII for fVa in each of the kringle domains (39 -41) and within the Gla domain (42). Furthermore, significant protein-protein interactions between the acidic COOH-terminal region of fVa and a region rich in basic amino acids of FII have been inferred and characterized indirectly by employing molecular techniques involving specific hirudin-like ligands and the anion-binding (pro)exosite I (ABE I) of FII derivatives, as well as directly using a specific acidic peptide derived from the COOH-terminal region of the fVa heavy chain and recombinant fVa molecules (43)(44)(45)(46)(47)(48)(49). Site-directed mutagenesis of the basic residues in the proenzyme generated a recombinant FII molecule impaired in its ability to be activated by fully assembled prothrombinase (50). Although a crystal structure and a model of fVa have been available for some time now (51,52), the crucial interaction of the acidic hirudin-like COOH-terminal portion of the heavy chain of the cofactor with FII required for efficient IIa formation was initially ignored because it was missing from the crystal structure of the cofactor (51). This interaction was further discounted without providing any solid evidence (53) despite initial findings by Guinto and Esmon (54) and more recent original find-ings from our laboratory (47)(48)(49). A very recent model of prothrombinase using as a template the crystal structure of prothrombinase from the snake venom of Pseudonaja textilis verified and established the critical role of the acidic COOHterminal region of fVa heavy chain for optimal rates of FII cleavage at two spatially distinct sites by prothrombinase resulting in timely IIa formation at the place of vascular injury (27,55).
Additional studies with several recombinant prethrombin-1 molecules, where seven critical basic amino acids within (pro) exosite I were individually changed to glutamic acid, confirmed the interaction of (pro)exosite I with fVa acidic regions (50). Notably, the data revealed that although mutated prethrombin-1 is a poor substrate for prothrombinase, the same molecule was activated by membrane-bound fXa alone with similar rates as wild type prethrombin-1. Supplementary to these studies, Yegneswaran et al. (56), utilizing synthetic peptide derived from a highly conserved region of FII, postulated the existence of an fVa-dependent binding exosite for fXa within the sequence 473-487 of FII (chymotrypsin numbering 149D-163 (4)) that is in close spatial arrangement to (pro)exosite I. The same authors have also identified an fVa-independent site for fXa on prothrombin (amino acids 557-571) (57).
This study was initiated to identify and investigate the identity and role of the minimum required amino acid stretch within sequence 473-487 of FII that is conserved in a wide range of mammalian species and regulates peptide bond specificity and FII activation by prothrombinase in an fVa-dependent manner. Our findings identify for the first time two specific amino acids within FII that have a dual role. They are required for efficient fVa-dependent tethering of fXa needed for timely FII cleavage at Arg 320 and IIa formation, while also serving an important role in directing efficient cleavage of IIa's physiological substrates. The latter is a prerequisite for expression of optimal physiological IIa activity.

Experimental Procedures
Materials-Phenylmethylsulfonyl fluoride (PMSF), o-phenylenediamine dihydrochloride, Hepes, Trizma (Tris base), and Coomassie Blue R-250 were purchased from Sigma. fV-deficient plasma was purchased from Research Protein Inc. (Essex Junction, VT). Secondary anti-mouse, anti-sheep, and antiequine IgG coupled to peroxidase were from Southern Biotechnology Associates, Inc. (Birmingham, AL). L-␣-Phosphatidylserine (PS) and L-␣-phosphatidylcholine (PC) were from Avanti Polar Lipids (Alabaster, AL). Chemiluminescent reagent ECL Plus, heparin-Sepharose, and Mono Q 5/50 columns were from GE Healthcare. Normal reference plasma and chromogenic substrate Spectrozyme-TH were from American Diagnostica Inc. (Greenwich, CT). S-2238 was from AnaSpec (Fremont, CA); recombiPlasTin used in the clotting assays was purchased from Instrumentation Laboratory Co. (Lexington, MA). The reversible fluorescent IIa inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA), human plasma-derived protein C, human plasma-derived IIa, human plasma-derived FII, and FII-deficient plasma were purchased from Hematologic Technologies Inc. (Essex Junction, VT). The purified human plasma-derived protein C preparation used contained both heavy chain isoforms that are activated to APC with similar rates as described earlier (58,59). Human fXa was purchased from Enzyme Research Laboratories (South Bend, IN). The plasmid pZEM229R-lite encoding human recombinant prothrombin (rFII) was a generous gift from Dr. Kathleen Berkner (Cleveland Clinic Foundation, Cleveland, OH). QuikChange II XL site-directed mutagenesis kit was obtained from Agilent Technologies Genomics (Santa Clara, CA). All molecular biology and tissue culture reagents, specific primers, and medium were obtained from Gibco, Invitrogen, or as indicated. Monoclonal antibodies to fV (␣HFV HC 17 and ␣HFV LC 9), monoclonal antibody ␣HFV1 coupled to Sepharose used to purify plasma and recombinant fV molecules, and a polyclonal antibody to FII used for immunoblotting experiments during rFII production were provided by Dr. Kenneth G. Mann (Department of Biochemistry, University of Vermont, Burlington). Plasma factor V (fV plasma ) and plasma fVa (fVa plasma ) were purified as described previously (60 -62).
Expression of Wild Type and Mutant rFII Molecules in Mammalian Cells-rFII expression in baby hamster kidney (BHK-21) cells has been described previously in detail (63). Briefly, BHK-21 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10%), and a streptomycin/penicillin (1%) mixture. Isolated plasmids (4 -6 g) for wild type and mutant rFII molecules were transfected into the BHK-21 cells using a lipid-based transfection reagent, Lipofectamine (Invitrogen), according to the manufacturer's instructions. Following an incubation period of 48 h, DMEM was supplemented with fetal bovine serum (10%), streptomycin/penicillin (1%) mixture, and methotrexate (1 M) and added to the cells. After 3 weeks of treatment with the selection medium, colonies were isolated, grown, and screened for levels of rFII expression by Western blot analysis using a monoclonal antibody and compared with plasmaderived FII as a standard (1 g/ml). Identification of the highest secreting rFII clone was further used in large scale protein expression with serum-free Opti-MEM supplemented with ZnCl 2 (50 M), vitamin K 1 (10 g/ml), and penicillin/strepto-mycin/Fungizone (1% v/v) mixture, and the medium were collected every 2 days for 2-3 weeks. Following collection, the media were stored at Ϫ80°C until the desired amount (usually 4 liters) was obtained and used for purification.
Purification of rFII Molecules-Purification of rFII was performed through a well established protocol previously described in detail (63). Briefly, collected media were thawed, filtered (0.45 m), and loaded on a tandem column setup of Amberlite XAD 2 and Q-Sepharose. Following the complete addition of medium to the two columns, the Q-Sepharose column was separated and washed with TBS (0.02 M Tris, 150 mM NaCl, pH 7.4). The bound material containing rFII on the Q-Sepharose was eluted with 0.02 M Tris, 0.5 M NaCl, pH 7.4. The material was treated with barium citrate, and the isolated pellet was dissolved in a minimum volume of EDTA (0.5 M, pH 7.4). The dissolved pellet was dialyzed twice in fresh TBS (two times, 4 liters) and filtered (0.45 m) prior to being loaded onto a General Electric FPLC instrument, equipped with a strong anionic exchanger Mono Q 5/50 column. The column was equilibrated in TBS, and a stepwise gradient of calcium (0 -50 mM) in TBS was used to isolate fully ␥-carboxylated rFII. Tubes containing the rFII molecules were concentrated using an appropriate Millipore Centricon (Billerica, MA), and aliquots were frozen at Ϫ80°C to avoid repeated freeze-thaw cycles. Following purification and before any experiment, all rFII molecules were characterized as extensively described below.
The level of ␥-carboxylation of all rFII molecules was determined at the Protein Chemistry Facility, Texas A&M University, by alkaline hydrolysis followed by amino acid analysis as described (64,65). All purified molecules were found to be properly carboxylated (Table 1). To verify that rFII WT and rFII ⌬473-487 are processed at the appropriate cleavage sites when incubated with prothrombinase or fXa alone and produced the expected fragments, the recombinant proteins were incubated with PCPS vesicles and fXa in the presence and absence of fVa. Following gel electrophoresis, fragments were identified following NH 2 -terminal sequencing from PVDF membranes (see below). All fragments derived from the recombinant FII molecules have the expected NH 2 -terminal sequence following cleavage by either prothrombinase or membranebound fXa alone (data not shown).
The fact that the rFII ⌬473-487 molecule contains a 15-amino acid deletion was verified by cDNA sequencing. However, in view of the surprising and unexpected data presented herein, it was important to confirm the existence of the deletion in the purified recombinant protein. This was accomplished by mass spectrometry. Briefly, following activation of rFII ⌬473-487 and FII plasma by prothrombinase, aliquots were analyzed in triplicate under reducing conditions on an SDS-12% PAGE. Following staining/destaining, the B-chain of ⌱⌱a was excised from the gel, and the proteins were reduced and alkylated with iodoacetamide. Digestion (in gel) was accomplished with porcine trypsin. Analysis of the resulting peptides was performed with an ␣-cyanohydroxycinnamic acid (matrix) Kratos Axima CFR MALDI-TOF mass spectrometer (reflector mode; 25,000 accelerating voltage) in the Protein Chemistry Laboratory, Texas A & M University, under the direction of Dr. Larry Dangott. The data obtained were compared with the peptide map following digestion of the B chain of IIa obtained from ExPASy/Swiss-Prot and verified the existence of the deletion in rFII ⌬473-487 . Similar experimental work performed with some other mutant molecules demonstrated that the rFII molecules described herein are fully carboxylated, can be appropriately processed by prothrombinase and fXa alone, and do indeed contain the expected deletion/mutations.
Gel Electrophoresis, Western Blotting, and Amino Acid Sequence from PVDF Membranes-SDS-PAGE was performed according to the method of Laemmli (66), using 9.5% gels following reduction with 2% ␤-mercaptoethanol. Screening for high levels of rFII-secreting clones was performed by Western blotting using PVDF according to a modified method initially described by Towbin et al. (67). Successfully transferred proteins were visualized by chemiluminescence using ECL Plus reagents following incubation with a polyclonal antibody specific to prethrombin-1. In some experiments, proteins were transferred to PVDF membranes and stained with Coomassie Blue, and NH 2 -terminal sequencing analysis was performed at the Biomolecular Resource Facility at the University of Texas Medical Branch (Galveston TX) as described previously in detail by our laboratory (68).
Studies of the Pathway for FII Activation by Gel Electrophoresis-The investigation of the activation rates of plasmaderived and of all rFII molecules, cleavage and activation by fXa alone or prothrombinase was performed according to a protocol previously described by our laboratory using plasma-derived FII or rFII (47,49,69). The calculation of the rates of all FII molecules consumption by fXa alone or by prothrombinase were performed as described previously with the software Prizm (GraphPad) (47,49,69).
Kinetic Titrations of Prothrombinase-To investigate the kinetic constants (K m and k cat ) of prothrombinase, assays with a set amount of plasma-derived fVa and fXa (as described in the legend to the figures) were executed as described by our laboratory in many instances (47,49,69,70). The initial rate of IIa generation was analyzed with the software Prizm (GraphPad) according to the Michaelis-Menten equation, and all final numbers reported are derived directly from the graphs. Each experiment used to report final numbers was run at least in duplicate, and the goodness of fit (R 2 ) for every model tested is provided under the "Results." The change in transition-state stabilization free energy, which measures the effect of the mutations in rFII, was calculated for the double and triple mutants as extensively detailed in the literature and previously reported by our laboratory (71)(72)(73)(74)(75)(76)(77).
Recombinant Thrombin Activity-rFII molecules were converted to rIIa by 1 nM prothrombinase. Full conversion of rFII to rIIa under these conditions was verified by gel electrophoresis. The chromogenic substrate S-2238 was used to assess rIIa activity by employing serial dilutions of the enzyme in Tris-NaCl buffer in the presence of 0.1% PEG 8000. The final concentrations of S-2238 used in the reactions were 0.94, 1.87, 3.75, 7.50, 15, and 60 M. The reaction was started by the addition of 4 nM rIIa. The data were obtained at 1 min using a SpectraMax M2 plate reader (Molecular Devices). The optical density was automatically adjusted for a 1-cm pathlength, and the V max was calculated from the optical density using the established extinc-tion coefficient of S-2238 at room temperature (78) following plotting of the data to the Michaelis-Menten equation using the software Prizm (GraphPad).
Activation of fV and fVIII by rIIa-rFII molecules were converted to rIIa by 1 nM prothrombinase. Full conversion of rFII to rIIa under the conditions described was assessed by gel electrophoresis. The resulting wild type and mutant rIIa were assessed for their ability to cleave and activate the cofactors over time, by SDS-PAGE. Reaction mixtures containing either 500 nM plasma-derived human fV or recombinant human fVIII were diluted in Tris-NaCl buffer in the presence of Ca 2ϩ . The final concentration of rIIa in the mixtures was 4 nM.
Activation of Protein C by Plasma-derived IIa or rIIa-rFII molecules described herein were converted to IIa by 1 nM prothrombinase. Full conversion of rFII to rIIa under the conditions described was assessed by gel electrophoresis. The resulting IIa molecules were assessed for their ability to cleave and activate protein C in the presence of thrombomodulin and PCPS vesicles according to a procedure previously described (79) in Tris-buffered saline with Ca 2ϩ . Protein C activation was verified by SDS-PAGE under reducing conditions. The final concentration of IIa in all mixtures was 8 nM. Gels were stained with Coomassie Brilliant Blue.
FII Clotting Assay-To assess the function of all FII molecules in whole plasma, a clotting assay using FII-deficient plasma was employed. The clotting assay was performed as described previously (80), and the time needed for formation of a fibrin clot was monitored at 37°C using a Diagnostica Stago STart 4 hemostasis analyzer as described previously (80). The analyzer was set up to automatically measure the time to clot up to 120 s.
Structural Analysis-To evaluate the structural features of the Ser 478 , Leu 480 , and Gln 481 residues, crystal structures of FII and IIa were superimposed and compared. The three human FII crystal structures that have been reported, show similar conformations for the residues of interest and neighboring regions; the highest resolution of these structures was chosen for detailed analysis (38). From the many human IIa crystal structures that are available, several representative examples in different bound states were compared and found to have similar conformations for the region containing the residues of interest. A high resolution structure of unbound IIa was chosen as the representative structure for detailed analysis (81). The program COOT was used to inspect structural features and determine distances (82). AREAIMOL (83)(84)(85) was used to calculate the solvent-accessible surface areas for specific residues, and molecular figures were prepared with the PyMOL Molecular Graphics System, version 1.5.0.4 (Schrödinger, LLC).
Prothrombin Time-The ability of FII and all rFII molecules to be activated under physiological conditions and to promote fibrin clot formation was first assessed using prothrombin times (PTs) (Fig. 2). The results shown in Table 1 demonstrate that although FII plasma , rFII WT , and rFII S478A had comparable clotting times of 12.7, 12.2, and 11.7 s, respectively, rFII L480A (where rIIa L480A is recombinant human ␣-thrombin with the mutation L480A) exhibited a minimal but significant prolonged PT of ϳ30 s (Fig. 2). Surprisingly, although rFII SL3AA (where rFII SL3AA is recombinant human prothrombin with the mutation S478A/L480A) and rFII SQ3AA had slow but compa-FIGURE 1. Schematic of FII. FII is converted to IIa through two fXa-catalyzed cleavages at Arg 271 and at Arg 320 resulting in IIa formation. The red rectangle denotes the fVa-independent site for fXa on FII (57), and the yellow rectangle represents the fVa-dependent site for fXa (56,95) studied herein. The light blue rectangle denotes the amino acids composing (pro)exosite I (50). All mutants created, stably transfected, purified to homogeneity, and used in the study are shown together with their assigned name used throughout this work.
rable PTs of ϳ30 s, the triple mutant rFII SLQ3AAA was severely ineffective in fibrin clot formation (PT ϳ116 s), whereas rFII ⌬SLQ (where rFII ⌬SLQ is recombinant human prothrombin with amino acids Ser 478 /Leu 480 /Gln 481 deleted) had a PT around 140 s (data not shown). In contrast, rFII ⌬473-487 , rFII ⌬N10 , rFII ⌬C10 , and rFII ⌬S5V were unable to induce clotting under the conditions described. These functional data demonstrate that either rFII SLQ3AAA or rFII ⌬SLQ cannot get activated to rIIa in a timely fashion, or that rIIa SLQ3AAA and/or rIIa ⌬SLQ formed are catalytically impaired because of the mutations, or both. Because previous data have shown that the S478A transition in IIa is of no consequence for its chromogenic and proteolytic activity (5,6), overall these results demonstrate for the first time that both Leu 480 and Gln 481 have a profound effect on IIa generation and/or IIa activity during fibrin clot formation or both.
Activation of rFII Molecules-To ascertain the effect of region 473-487 of FII on its ability to be activated by membrane-bound fXa alone, in the absence of fVa, we assessed the pattern of activation by gel electrophoresis over a 2-h time period (Fig. 3). Fig. 3A shows a control experiment and demonstrates that FII plasma activation by membrane-bound fXa alone proceeds following initial cleavage at Arg 271 , through the intermediate prethrombin-2 with very slow gradual appearance of the B-chain of IIa because of inefficient rate of cleavage at Arg 320 . Surprisingly, with the removal of amino acids 473-487 from prothrombin (Fig. 3B), there is acceleration of rFII ⌬473-487 consumption through initial cleavage at Arg 271 that is evident by the prompt appearance of prethrombin-2. Additional examinations of the intensity of the B-chain of thrombin reveal a substantially delayed cleavage at Arg 320 of rFII ⌬473-487 compared with rFII WT resulting in insignificant IIa generation. Scanning densitometry of the gels shown in Fig. 3, A and B, showed that the rate of rFII ⌬473-487 consumption by membrane-bound fXa is ϳ4-fold increased compared with the rate of cleavage of rFII plasma under similar experimental conditions (Table 1). These data suggest that amino acid sequence 473-487 of prothrombin provides a potential obstruction for efficient initial cleavage of prothrombin at Arg 271 by membrane-bound fXa alone in the absence of fVa.
To further improve our understanding of the fundamental role of amino acid region 473-487 for FII activation by prothrombinase, we studied the pattern of FII activation by fully assembled prothrombinase with gel electrophoresis over a 2-h time period. A control experiment (Fig. 3C) demonstrates that under the conditions used the activation of FII plasma proceeds efficiently following initial cleavage at Arg 320 , through the enzymatically active intermediate meizothrombin, as confirmed by the appearance of fragment 1⅐2-A. Rapid cleavage of this fragment at Arg 271 leads to the formation of IIa. In contrast, activation of rFII ⌬473-487 under similar experimental conditions is significantly delayed through the same pathway as verified by the late appearance of the B-chain of IIa (Fig. 3D). Scanning densitometry of the gels shown in Fig. 3, C and D, showed that rFII ⌬473-487 is consumed with a rate that is ϳ27-fold slower compared with the rate of FII plasma consumption or ϳ23-fold slower compared with the rate of rFII WT consumption under the experimental conditions used (Table 1). These data suggest that under conditions of saturating amounts of fVa with respect to fXa, amino acid sequence 473-487 of FII plays a preeminent role because it is required for fast and efficient initial cleavage of FII at Arg 320 by prothrombinase.
To further investigate the effect of the deletions and point mutations on rFII cleavage and activation by membrane-bound fXa alone, we studied rFII activation by gel electrophoresis of all mutants detailed in Fig. 1. Fig. 4A shows a control experiment and demonstrates that rFII WT activation by membrane-bound fXa proceeds typically following initial cleavage at Arg 271 , as its plasma counterpart through the intermediate prethrombin-2 with very slow and minimal appearance of the B chain of IIa because of a nonproductive rate of cleavage at Arg 320 . With the removal of amino acids 478 -482 from rFII ⌬S5V (Fig. 4B), there is acceleration of rFII ⌬478 -482 consumption by fXa alone through initial cleavage at Arg 271 that is evident by the rapid appearance of prethrombin-2. The fact that only trace amounts of B-chain of IIa are apparent under the conditions employed suggests a substantially deferred rate of cleavage at Arg 320 of the deletion mutant compared with cleavage of rFII WT resulting in insignificant amounts of IIa generation. Scanning densitometry of similar gels shown in Fig. 4, A and B, showed that the rate of consumption of all rFII molecules by membrane-bound fXa alone is ϳ2.3-8-fold increased compared with the rate of cleavage of rFII WT or FII PLASMA (Fig. 4C and Table 1). However, although with rFII S478A , rFII L480A , rFII SL3AA , and rFII SQ3AA minimal amounts of the B-chain of IIa are formed (data not shown), when studying rFII ⌬N10 , rFII ⌬C10 , rFII ⌬S5V , rFII SLQ3AAA , and rFII ⌬SLQ activation, there is accumulation of prethrombin-2 with no significant amounts of B-chain of IIa generated suggesting impaired cleavage of prethrombin-2 at Arg 320 by membrane-bound fXa (Fig. 4, B and D). These data confirm our findings with rFII ⌬473-487 (Fig. 3) and reveal that the dipeptide Leu 480 -Gln 481 within the 15-amino acid stretch 473-487 of FII appears to be responsible for the sim- ilar effects observed with rFII ⌬N10 , rFII ⌬C10 , rFII ⌬S5V , rFII SLQ3AAA , and rFII ⌬SLQ when studying rFII molecular activation by membrane-bound fXa alone in the absence of fVa (Fig. 4D and Table 1).
To improve our understanding of the essential role of amino acids Leu 480 and Gln 481 for FII activation, we studied the pattern of all rFII molecules activation shown in Fig. 1 by fully assembled prothrombinase (i.e. in the presence of an excess of a Clotting times were determined using FII-deficient plasma as described under "Experimental Procedures" in quadruplicate. b The rates of rFII consumption were obtained following scanning densitometry of gels studying rFII activation. Some of the gels used are shown in Figs. 3-5. The final rate of rFII consumption in the presence of membrane-bound fXa or prothrombinase was calculated using the apparent first-order rate constant, k (s Ϫ1 ), obtained directly from the graph following plotting of the data as described under "Experimental Procedures." c The numbers in parentheses represent the goodness of fit (R 2 ) to the equation representing first-order exponential decay using the software Prizm from where the firstorder rate constant was obtained. d No clotting time could be detected following a 120-s incubation time period. Aliquots were withdrawn at various time intervals and treated as described (47,69). M represents the lane with molecular weight markers (from top to bottom): 98,000, 64,000, 50,000, and 36,000, respectively. Lanes 1-19 show samples from the reaction mixture before (0 min) the addition of fXa and 20, 40, 60, 80, 100, 120, 150, 180, 210, and 240 s and 5, 6, 10, 20, 30, 60, 90, and 120 min, respectively, after the addition of fXa. Following scanning densitometry as described under "Experimental Procedures," the data representing FII consumption as a function of time (s) were plotted using non-linear regression analysis according to the equation representing a first-order exponential decay and the rates of FII consumption using the apparent first-order rate constant, k (s Ϫ1 ), obtained directly from the fitted data, were calculated as described (47) and are reported in Table 1. FII-derived fragments are identified to the right of A-D as follows: FII, prothrombin (amino acid residues 1-579); P1, prethrombin-1 (amino acid residues 156 -579); F1⅐2-A, fragment 1⅐2-A chain (amino acid residues 1-320); F1⅐2, fragment 1⅐2 (amino acid residues 1-271); P2, prethrombin-2 (amino acid residues 272-579); B, B-chain of IIa (amino acid residues 321-579); F1, fragment 1 (amino acid residues 1-155). JANUARY 22, 2016 • VOLUME 291 • NUMBER 4

JOURNAL OF BIOLOGICAL CHEMISTRY 1571
fVa) by gel electrophoresis over a 2-h time period (Fig. 5). A control experiment (Fig. 5A) demonstrates that under the conditions used, rFII WT proceeds as its plasma counterpart following initial cleavage at Arg 320 , through the enzymatically active intermediate meizothrombin, as confirmed by the appearance of fragment 1⅐2-A. Rapid cleavage of this fragment at Arg 271 leads to the formation of rIIa. Similar results were found when using rFII S478A (Fig. 5B) demonstrating that the S478A transition alone is of no consequence for timely FII activation by prothrombinase. In contrast, activation of rFII ⌬S5V and rFII SLQ3AAA under similar experimental conditions was significantly delayed through the same pathway as verified by the lingering of fragment 1⅐2-A at the late time points and the late appearance of the B-chain of rIIa (Fig. 5, C and D). Similar results were obtained with rFII ⌬SLQ (Table 1). A systematic analysis of the activation of all rFII mutant molecules by pro-thrombinase using similar experimental procedures, followed by scanning densitometry of the gels and calculation of the rate of rFII consumption, revealed the existence of two groups as follows: a group of molecules represented by FII plasma , rFII WT , and rFII S478A (also containing rFII L480A , rFII SL3AA , and rFII SQ3AA ) that are efficiently activated by prothrombinase; and a second group of proteins represented by rFII ⌬S5V and rFII SLQ3AAA (including rFII ⌬N10 , rFII ⌬C10 , and rFII ⌬SLQ ) that are activated by fully assembled prothrombinase with a rate that is ϳ13-18-fold slower than the rate observed with rFII WT (Fig. 6A, inset, and Table 1). The data suggest that under conditions of saturating amounts of fVa with respect to fXa, the dipeptide Leu 480 -Gln 481 of prothrombin plays a leading role during FII activation because it is required for fast and efficient initial cleavage at Arg 320 by prothrombinase (Fig. 6B, the deficient step is represented by the red arrow). , and 120 min respectively, after the addition of fXa. C, the two gels shown in A and B together with similar gels obtained with all rFII studied were scanned, and rFII consumption was recorded as described under "Experimental Procedures." Following scanning densitometry and normalization to the initial FII concentration, the data representing rFII consumption as a function of time (seconds) were plotted using non-linear regression analysis according to the equation representing a first-order exponential decay using the software Prizm (GraphPad, San Diego). Prothrombinase was assembled with rFII WT (filled circles; R 2 0.98), rFII ⌬C10 (filled squares; R 2 0.98), rFII ⌬N10 (filled triangles; R 2 0.99), rFII ⌬S5V (filled inverse triangles; R 2 0.99), rFII S478A (filled diamonds; R 2 0.99), rFII L480A (open squares; R 2 0.99), rFII SQ3AA (open circles; R 2 0.98), rFII SL3AA (open triangles; R 2 0.99), and rFII SLQ3AAA (open inverse triangles; R 2 0.99). The rates of rFII consumption shown in C, using the apparent first-order rate constant, k (s Ϫ1 ) obtained directly from the fitted data, were calculated as reported (47), and the data are shown in Table 1. D, schematic representation of fragments derived following FII activation by membrane-bound fXa alone. The red arrow indicates impaired cleavage at Arg 320 in rFII ⌬C10 (filled squares), rFII ⌬N10 (filled triangles), rFII ⌬S5V (filled inverse triangles), and rFII SLQ3AAA (open inverse triangles) resulting in prethrombin-2 accumulation. FII-derived fragments are identified to the right of each panel, according to the description provided in the legend of Fig. 3.

Kinetic Analyses of the Activation of rFII Molecules-To
understand the effect of the S478A/L480A/Q481A substitutions on the activity of prothrombinase in activating the rFII molecules, we first examined the rates of rIIa formation from all rFII molecules under similar experimental conditions. Historically, this method was designed to identify any deficiency in fVa or fXa as part of prothrombinase in cleaving and activating FII and is measured indirectly by using IIa generation as a reporting probe with a chromogenic substrate. The comprehensive kinetic data for several mutants are shown in Fig. 7 with the kinetic constants derived directly from the fitted data reported in Table 2. The combined findings demonstrate that whereas the single and double alanine substitutions rFII mutants are activated by prothrombinase similarly, providing kinetic constants comparable with the wild type or plasma FII molecules, kinetic analyses of prothrombinase activation of rFII SLQ3AAA demonstrate a modest 2.7-fold decrease in the k cat and a large 21-fold increase in the K m value of the reaction. Similar experiments studying rFII ⌬SLQ activation by fully assembled prothrombinase revealed a 29-fold increase in K m with a concomitant 16-fold decrease in the k cat values of the reaction. A direct comparison between the data obtained with rFII SLQ3AAA with the data obtained with rFII ⌬SLQ strongly suggest an important contribution of the backbone structure of the peptide bond between these three amino acids for efficient rFII activation by prothrombinase.
To quantify the interaction between the two sets of double mutations (S478A/L480A and S478A/Q481A) and to confirm their apparent synergistic detrimental effect on prothrombinase function for activation of rFII SLQ3AAA , we have further calculated the difference in free energy of the transition state analog (⌬⌬G int ) for the triple mutant as described previously by our laboratory (75,76). The large positive value of ⌬⌬G int (ϩ2.4 kcal/mol) for the combination of the mutations at Leu 480 and Gln 481 together with the sizable 55-fold decrease in the secondorder rate constant of prothrombinase for rFII SLQ3AAA activation signify that there is a deficiency in recognition between prothrombinase and rFII SLQ3AAA . These findings solidify our previous conclusion that these substitutions are detrimental to the activation of rFII bearing the triple amino acid substitution by fully assembled prothrombinase. However, it is important to note that it is also possible that rIIa SLQ3AAA may also be deficient in its own catalytic activity as observed with rIIa ⌬SLQ , and the effect observed with rFII SLQ3AAA activation may be likewise due to the deficiency of rIIa in cleaving the chromogenic substrate. Thus, although we cannot yet assign the poor performance of prothrombinase in cleaving rFII SLQ3AAA solely to a deficiency in recognizing the mutated substrate, and because the S478A transition is of no consequence for either rFII S478A activation or rIIa S478A activity, the overall data presented thus far suggest that amino acid sequence Leu 480 -Gln 481 may have a dual effect in properly directing prothrombinase rec- ognition of FII, as well as providing the resulting enzyme with the appropriate surface required for proper substrate tethering and cleavage. However, it is also possible that these two amino acids are allosterically involved in both prothrombinase interactions with FII as well as the expression of the enzymatic activity of IIa.
Analyses of the Activity of rIIa Molecules-Although many investigations have identified the specific amino acid residues from FII/IIa participating in either prothrombinase recognition or IIa activity toward its physiological substrates, respectively, few studies have shown that identical residues are involved in both FII recognition by prothrombinase and IIa activity. To understand the effect of the deletions/mutations on IIa activity, we further assessed the amidolytic and biological activity of selected rIIa molecules generated herein toward the chromogenic substrate S-2238 and toward thrombin's natural substrates, fV, fVIII, and protein C.
To understand the effect of the mutations on the amidolytic activity of IIa, we determined the kinetic constants for the hydrolysis of S-2238 by the rIIa molecules under steady state conditions. The data shown in Table 3 reveal the following: 1) rIIa WT produced under the conditions described by our laboratory has similar activity as previously found with other rIIa WT preparations, and 2) rIIa S478A has similar catalytic efficiency (k cat /K m ) as rIIa WT , as demonstrated previously (6,86). In addition, we also found that whereas rIIa SLQ3AAA was devoid of activity toward S-2238, rIIa SQ3AA (where rIIa SQ3AA is recombinant human ␣thrombin with the mutation S478A/Q481A) has similar amidolytic activity as rIIa WT , whereas rIIa L480A and rIIa SL3AA (where rIIa SL3AA is recombinant human ␣-thrombin with the mutation S478A/L480A) are the most deficient in S-2238 hydrolysis among the single and double alanine mutants when compared with rIIa WT or rIIa S478A ( Table 3). The combined data clearly demonstrate that amino acid Leu 480 plays an important role during the expression of IIa amidolytic activity and that the integrity of amino acid sequence Leu 480 -Gln 481 is required for optimal expression of this activity.
The data shown in Figs. 8 and 9 demonstrate that whereas rIIa WT and rIIa S478A cleave and activate fV and fVIII with similar rates (Figs. 8, A and C, and 9, A and C), rIIa ⌬C10 (rIIa ⌬C10 is recombinant human prothrombin missing amino acids SVLQVVNLPI), and rIIa ⌬S5V are totally deficient in cleaving both cofactor molecules over a 3-h incubation period (Figs. 8, B  and H, and 9, B and H). These data are in complete agreement FIGURE 6. Analyses of the rates of activation of rFII by prothrombinase. A, gels shown in Fig. 5, together with similar gels obtained with all rFII studied, were scanned, and rFII consumption was recorded as described under "Experimental Procedures." Following scanning densitometry, the numbers were normalized to the initial concentration of rFII studied, and the data representing rFII consumption as a function of time (seconds) were plotted using nonlinear regression analysis according to the equation representing a first-order exponential decay using the software Prizm (GraphPad, San Diego). rFII WT (filled circles; R 2 0.98), rFII ⌬C10 (filled squares; R 2 0.99), rFII ⌬N10 (filled triangles; R 2 0.94), rFII ⌬S5V (filled inverse triangles; R 2 0.99), rFII S478A  The inset shows the progress of the reaction during the first 180 s. The rates of rFII consumption using the apparent first-order rate constant k (s Ϫ1 ), obtained directly from the fitted data, were calculated as reported (47) and shown in Table 1. B, schematic representation of fragments derived following rFII activation by prothrombinase in the presence of excess fVa with respect to fXa. The red arrow indicates impaired cleavage (at Arg 320 ) in rFII ⌬C10 (filled squares), rFII ⌬N10 (filled triangles), rFII ⌬S5V (filled inverse triangles), and rFII SLQ3AAA (open inverse triangles).   Table 2 were extracted directly from the fitted data shown herein.
with our findings shown in Table 1, explaining the fact that rFII ⌬C10 and rFII ⌬S5V are devoid of clotting activity, and further attest to the crucial dual role of the dipeptide Leu 480 -Gln 481 during coagulation. Further analyses of the single or double mutants reveal a slight differentiation in cleavage and activation of the two cofactors by the various rIIa molecules. Although rIIa L480A and rIIa SL3AA appear devoid of activity toward fV (Fig. 8, D and E), both molecules slowly cleave fVIII at the Arg 372 -and Arg 1689 -activating cleavage sites (Fig. 9, D and E) (87)(88)(89)(90). Similarly, although rIIa SLQ3AAA has no apparent activity toward fV (Fig. 8G) over a 3-h time period, the mutant enzyme cleaves fVIII slowly at the non-activating Arg 740 cleavage site (Fig.  9G). Finally, although rIIa SQ3AA cleaves fV efficiently at Arg 709 to produce the heavy chain of fV and an M r 220,000 intermediate (Fig. 8F), the enzyme is also efficient in cleaving fVIII at the Arg 372 -and Arg 1689 -activating cleavage sites (Fig. 9F). These two cofactors have strategic functions within the amplified coagulation response to vascular damage and must be activated to perform accordingly within their respective enzymatic complexes. The combined data explain the impaired procoagulant activity of rFII SLQ3AAA (Fig. 2), which is deficient in producing large amounts of rIIa in a timely fashion (Fig. 5D). However, even when rIIa SLQ3AAA is generated, the recombinant enzyme is deficient in activating the pro-cofactors.
We next assessed the capability of the rIIa molecules in the presence of thrombomodulin to activate protein C and produce APC. Fig. 10 shows the results of such an experiment and demonstrates that although rIIa ⌬S5V (rIIa ⌬S5V is recombinant human ␣-thrombin with region SVLQV deleted) cannot cleave and activate protein C, rIIa SLQ3AAA has small but significant activity generating minute amounts of APC (Fig. 10, lanes 8 and 9), which in turn can cleave fV at Arg 506 /Arg 306 and produce the characteristic M r 30,000 fragment (data not shown) (68,91). All other rIIa mutant molecules tested,forAPCgeneration,havesimilaractivitiesasrIIa WT orplasmaderived IIa under the condition described (Fig. 10).
These data demonstrate a differential requirement of IIa for cleavage and activation of both the pro-cofactor molecules and protein C and attest to the sensitive requirements of fV for cleavage and activation by IIa. Overall these results demonstrate that amino acids Leu 480 and Gln 481 within the serine protease domain of FII serve a dual purpose, and thus both are required for efficient cleavage at Arg 320 by prothrombinase and may be involved in the presentation of an obligatory exosite for timely fV, fVIII, and protein C activation by IIa.  Fig. 7). Kinetic constants were derived directly from the fitted data.  The K m value for S-2238 is listed for wild-type rIIa and selected rIIa mutants and determined as described under "Experimental Procedures" according to the Michaelis-Menten equation using the software Prizm. Kinetic constants shown were derived directly from the fitted data. b k cat ϭ V max /[enzyme]; the V max was calculated as described under "Experimental Procedures," and the enzyme concentrations of rIIa was 4 nM for all experiments shown. c R 2 is the goodness of fit of the data points to the Michaelis-Menten equation using the software Prizm. d NP is no plot; no data could be plotted to the Michaelis-Menten equation using the software Prizm.

Discussion
Our data demonstrate that amino acid region 473-487 of FII is required for timely activation of FII through the meizothrombin pathway. Although prior work using synthetic peptides suggested that this region of the cofactor may contain an fVa-dependent fXa-binding site for FII, the data presented herein with recombinant FII molecules provide for the first time a mechanistic interpretation of these findings and identify the crucial amino acids from this sequence responsible for the effect observed (56).
To elucidate the number and identity of the required amino acids within amino acid sequence 473-487 of FII, we constructed, expressed, purified to homogeneity, and studied several rFII molecules with deletions and point mutations within this important regulatory region. We first investigated the effects of the 15-amino acid deletion with rFII ⌬473-487 , followed by experiments with rFII molecules containing overlapping deletions within this segment (rFII ⌬N10 and rFII ⌬C10 and rFII ⌬S5V and rFII ⌬SLQ ). Several rFII molecules bearing single mutations (rFII S478A and rFII L480A ), double mutations (rFII SL3AA and rFII SQ3AA ), and a triple mutation (rFII SLQ3AAA ) were subsequently made. Membrane-bound fXa cleaves FII sequentially at Arg 271 followed by Arg 320 , forming small amounts of IIa. Under these conditions, the activation of the deletion mutants rFII ⌬473-487 , rFII ⌬N10 , rFII ⌬C10 , rFII ⌬S5V , the triple alanine mutant, and the deletion mutant (rFII SLQ3AAA and rFII ⌬SLQ ) resulted in a modest increase of the rate of activation. In addition, activation of these six rFII molecules by fXa alone resulted in accumulation of prethrombin-2, with very little IIa formed. In contrast, activation of all these rFII mutants by fully assembled prothrombinase is significantly delayed. The combined data suggest that amino acids Leu 480 and Gln 481 within region 473-487 of FII either represent or are responsible for the presentation of an fVa-dependent site for fXa on FII, which is essential for optimal rate of cleavage at Arg 320 , which in turn is required for timely IIa formation at the place of vascular injury.
The autolysis loop of APC bears strong homology with the FII sequence 473-487 (chymotrypsin numbering 149D-163) (92). Replacement of several basic amino acids from this homologous region in APC by site-directed mutagenesis to alanine demonstrated the ability of this exosite to interact with its sub- strate fVa and to differentiate between the Arg 506 and Arg 306 cleavage sites (93). Yegneswaran et al. (56) using synthetic peptides provided initial evidence that sequence 473-487 of FII is able to disrupt prothrombinase assembly only in an fVa-dependent manner. Chen et al. (50) identified a sequence within proexosite I of prothrombin containing basic residues Arg 35 , Lys 36 , Arg 67 , Lys 70 , Arg 73 , Arg 75 , and Arg 77 (chymotrypsin numbering), which is in close spatial proximity to region 473-487 of FII. These investigations revealed that following replacement of all basic residues from pro-exosite I with Glu, there was a significant effect on fXa within prothrombinase when compared with fXa alone in cleaving and activating FII, suggesting that these amino acids are specific fVa-dependent recognition sites for fXa on FII. Further kinetic studies by Chen et al. (50) using the hirudin COOH-terminal peptide (hirugen) showed that the peptide inhibited wild type prethrombin-1 activation by prothrombinase, whereas hirugen had no inhibitory effect on the activation of the mutated zymogen lacking the basic residues in pro-exosite I by fXa alone. The combined studies of Chen et al. (50) and Yegneswaran et al. (56) suggest the requirement of both sites for optimum productive interaction of prothrombinase with FII and timely IIa formation.
Research with discontinuous assays using a chromogenic substrate for IIa revealed that when fVa is incorporated into the prothrombinase complex, the resulting K m value of the reaction was decreased by 100-fold (corresponding to a 100-fold increase in affinity of prothrombinase for FII as compared with the affinity of fXa alone for the substrate), whereas the catalytic efficiency (k cat ) of fXa was increased by 3,000-fold resulting in a 300,000-fold overall increase in the activity of prothrombinase (second-order rate constant) for FII compared with the activity of fXa alone toward FII (16). The significant increase in affinity of prothrombinase for its substrate is attributed to tighter binding of the enzymatic complex to FII because of its localization on the membrane surface by fVa. The longstanding hypothesis that fVa "localizes and positions" FII in an optimum position for efficient catalysis by fXa consistent with the classical role of a cofactor for catalysis was recently confirmed by computational studies with prothrombinase by Shim et al. (27). These studies demonstrated that the acidic COOH-terminal portion of the FIGURE 9. Activation of recombinant fVIII by rIIa. rfVIII (500 nM) was incubated with rIIa (4 nM) as described under "Experimental Procedures." At selected time intervals, aliquots of the mixtures were removed, mixed with 2% SDS, heated for 5 min at 90°C, and analyzed on a 4 -12% SDS-PAGE followed by staining with Coomassie Blue. Lane 1 in all panels depicts aliquots of the mixture withdrawn from the reaction before the addition of rIIa. Lanes 2-8 represent aliquots of the reaction mixture withdrawn at 10, 20, 30, 45, 60, 120, and 180 min. The positions of all rfVIII fragments are indicated on the right. Fragments from rfVIII are identified as previously demonstrated (80). The rIIa molecule used each time is indicated under each panel.

Function of Sequence Leu 480 -Gln 481 of Prothrombin
heavy chain of fVa that is contiguous to the A2 domain of fVa is essential in its ability to interact and snare the serine protease domain of FII. In that manner, this acidic amino acid sequence reposition the Arg 320 cleavage site at an optimum position for timely cleavage by fXa and FII activation at the site of vascular injury as earlier suggested (54) and more recently experimentally demonstrated by our laboratory with synthetic peptides and recombinant fVa molecules mutated at these specific sites (45,47,48).
We show that following removal of the amino acid sequence 473-487 from FII, prothrombinase loses the ability to efficiently form IIa because of impaired fVa-dependent cleavage of FII by fXa at Arg 320 . One easy explanation of these results was that elimination of such a huge portion of the molecule results in significant structural changes of the molecule that in turn have deleterious effects on FII molecular conformation resulting in deficient prothrombinase activity. Despite the fact that rFII ⌬473-487 was activated following the same pathways as rFII WT in the presence or absence of fVa, albeit with different rates, and in the absence of a crystal structure of rFII ⌬473-487 , there was still doubt about the structural integrity and function of a molecule bearing such a large deletion. Experiments using more modest overlapping deletions (with rFII ⌬N10 , rFII ⌬C10 , and rFII ⌬S5V ) as well as with a triple alanine mutant (rFII SLQ3AAA ) and a triple deletion mutant (rFII ⌬SLQ ) demonstrated that these molecules are also hindered in their fVa-dependent cleavage at Arg 320 to a similar level as rFII ⌬473-487 (Table 1 and Fig. 6). These data provide original evidence demonstrating that the minimal sequence of FII required for the 3,000-fold increase in the catalytic efficiency of prothrombinase, as defined ϳ36 years ago (16) for efficient cleavage of FII by prothrombinase at Arg 320 , is carried at least partially by amino acid sequence Leu 480 -Gln 481 of FII. The findings presented herein silence the notion that the effect seen with rFII ⌬473-487 may be due to a structural change of the mutant molecule rather than to specific amino acid(s) missing from rFII ⌬473-487 .
The kinetic findings presented herein revealed comparable K m and k cat constants for prothrombinase when rFII molecules bearing the single and double alanine mutations were used as substrate. However, when rFII SLQ3AAA was the substrate for prothrombinase in the same discontinuous assay, there was a significant 21-fold increase in the K m value and a modest 2.7fold decrease in the k cat of the enzyme. Similar results were obtained with rFII ⌬SLQ . Furthermore, rFII SLQ3AAA and rFII ⌬SLQ were also found to be substantially deficient in clot formation in an assay using FII-deficient plasma, whereas rIIa SLQ3AAA was also deficient in S-2238 hydrolysis. rIIa SLQ3AAA was also impaired in cleaving fV, fVIII, and to a lesser extent protein C. These data dovetail nicely with results obtained with rIIa ⌬S5V and rIIa ⌬473-487 . We can thus hypothesize that the substantial increase in the K m of prothrombinase toward rFII SLQ3AAA is due to a deficiency in prothrombinase in recognizing the mutant molecule because of the lack of Leu 480 -Gln 481 , whereas the decrease in enzymatic activity of the resulting rIIa SLQ3AAA molecule is also the result of the absence of these two important amino acid side chains. Additional data with rFII ⌬SLQ provide further evidence of the crucial role of amino acids Leu 480 -Gln 481 and the peptide bond backbone between these two amino acids because, when these residues are completely eliminated, the K m value of the reaction increases by 32-fold, and the k cat value decreases by 16-fold (Table 2). Keeping in mind that the S478A substitution is of no consequence on both rFII activation and rIIa function, these results provide strong evidence in favor of the dual role of amino acids Leu 480 and Gln 481 . Moreover, these amino acids are required by prothrombinase to efficiently promote cleavage of FII at Arg 320 and are also required by IIa for optimum amidolytic activity as well as to proficiently cleave and activate fV, fVIII, and protein C. Finally, the possibility that elimination of these two residues from rFII results in an allosteric transition of the amino acids around/within the active site of rIIa, thus modifying the critical distances between the specific residues of the catalytic triad resulting in impaired catalysis, cannot be eliminated.
A comparison of crystal structures of FII, meizothrombin, IIa, prethrombin-1, and prethrombin-2 was carried out to identify structural differences in/near the Gly 473 -Ile 487 segment comprising the fVa-dependent fXa-binding site. These residues adopt similar conformations in all of the crystal structures, with the NH 2 -terminal residues Gly 473 -Gln 476 being quite solventaccessible or flexible, and residues Pro 477 -Ile 487 being variable in their degree of solvent exposure. Residue Ile 487 is significantly more exposed in prothrombin (accessible surface area of Ͼ30 Å 2 compared with Յ10 Å 2 in meizothrombin and thrombin), as well as the adjacent Pro 486 (accessible surface area of ϳ15-30 Å 2 reducing to Ͻ10 Å 2 in meizothrombin and thrombin). The amount of solvent exposure of Ile 487 and Pro 486 appears to be heavily influenced by the flanking loops encompassing residues Ala 446 -Tyr 454 and Lys 511 -Ser 525 , which adopt different conformations upon FII activation (Fig. 11). Recently, Pozzi et al. (94) used the crystal structure of Gla-domainless FII with active site S525A to demonstrate that fVa has recognition sites in close proximity to Arg 320 (Arg 15 chymotrypsin number- FIGURE 10. Activation of protein C by rIIa. Plasma-derived protein C (80 nM) was incubated with rIIa (8 nM), thrombomodulin, and PCPS as described under "Experimental Procedures." Following a 3-h incubation period, each individual solution was dried with a vacuum concentrator, resuspended in Tris buffer, mixed with 2% SDS and 2% ␤-mercaptoethanol, heated for 5 min at 90°C, and analyzed on a 5-15% SDS-PAGE followed by staining with Coomassie Blue. Lane 1, protein C alone no IIa; lane 2, protein C alone, no IIa incubated with buffer for 3 h; lane 3, protein C and rIIa ⌬S5V ; lane 4, protein C and plasma-derived IIa; lane 5, protein C and rIIa WT ; lane 6, protein C and rIIa S478A ; lane 7, protein C and rIIa L480A ; lane 8, protein C and rIIa SL3AA ; lane 9, protein C and rIIa SQ3AA ; and lane 10, protein C and rIIa SLQ3AAA . Positions of protein C and APC heavy and light chain fragments are indicated at the right (a/b heavy chains, and c light chain). The two heavy chains of protein C in plasma (a and b) have been identified earlier, differ by one glycosylation site, and have been extensively studied (58,59).

Function of Sequence Leu 480 -Gln 481 of Prothrombin
ing). These sites create a strong electrostatic potential due to a number of basic residues described by Chen et al. (50). Through analysis of this published crystal structure, we have located this basic region to be in the vicinity of the Leu 480 -Gln 481 amino acid sequence of FII that we found to be required for efficient initial cleavage at Arg 320 by prothrombinase. It is noteworthy that a very recent study by Pozzi et al. (38) demonstrated a crucial role for linker 2 for the rate of activation of FII by prothrombinase and suggested that this region may be involved in the interaction of FII with the cofactor. These data are in complete accord with data showing that fragment 1, more precisely the kringle 2 region, is involved in the interaction of fVa as part of prothrombinase with FII (39,41). Finally, a close comparison of crystal structures of FII and IIa revealed that residues Ser 478 , Leu 480 , and Gln 481 adopt similar conformations in both structures. The Ser 478 side chain is exposed on the surface of both molecules, whereas the Leu 480 side chain is surrounded by other residues and is not accessible to solvent. Gln 481 is partially solvent-exposed in both FII and IIa. The Ser 478 /Leu 480 /Gln 481 residues are near ABE-I (Fig. 11), but Ͼ15 Å from the catalytic Ser 525 residue, and even more distant from ABE-II.
In conclusion, in this study we provide evidence for the dual effect of amino acids Leu 480 and Gln 481 of FII. Future mutagenesis studies within the amino acids uncovered herein, paired with selected mutations within pro-exosite-I and/or pro-exosite-II of FII, should be able to elucidate the intermolecular communications within FII, required for both optimal fVa-dependent activation of FII and subsequent IIa catalytic activity toward its numerous physiological substrates. Finally, our results provide evidence for the production of large quantities of rFII ⌬S5V , rFII SLQ3AAA , or rFII ⌬SLQ that could be used as therapeutic agents because these molecules would compete with the natural substrate in vivo, when infused in individuals with prothrombotic tendencies.
Author Contributions-J. R. W. designed, performed, and analyzed most experiments and participated in the writing of the paper; J. H. designed, performed, and analyzed some of the experiments; V. C. Y. designed and produced the structural pictures of prothrombin and thrombin shown in Fig. 11; M. K. conceived and coordinated the study and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.