©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Development of a Novel Recombinant Serpin with Potential Antithrombotic Properties (*)

Paul C. R. Hopkins (§) , Damian C. Crowther (¶) , Robin W. Carrell , Stuart R. Stone

From the (1) Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

Recombinant -antitrypsin with a P arginine residue (Arg--antitrypsin) is a rapid inhibitor of both thrombin and activated protein C (APC). A series of mutants were made in an attempt to increase the specificity of this serpin for thrombin over APC. Initially, P and P` residues of Arg--antitrypsin were replaced in single and double mutations by the corresponding residues in antithrombin and C1 inhibitor which are very poor inhibitors of APC. No improvement in selectivity was achieved by these mutations. In fact, all P/P` substitutions led to a decrease in selectivity for thrombin over APC. For example, replacement of the P proline of Arg--antitrypsin by glycine decreased the association rate constant (k) with thrombin by 37-fold while the k value with APC was reduced by only 16-fold. Co-operative effects were observed with the double P and P` substitutions; the mutational effects were not additive. The decrease in the k for thrombin caused by the mutation of the P proline to alanine or glycine was 3-fold greater when threonine was present in the P` position instead of the normal serine. In contrast to the disappointing results with the P/P` mutations, replacement of the P to P` residues of -antitrypsin by those of antithrombin led to a dramatic increase in selectivity. Although this substitution only affected the k value with thrombin by 10-fold, a 12,500-fold decrease in this value with APC was observed. Substitution of proline for the P glycine of this chimeric serpin increased the k values with thrombin and APC by 7- and 90-fold, respectively. The effect of the P substitution was again found to depend on the sequence surrounding the residue; the change in the k for APC caused by the P Pro Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of the k values of the chimeric serpin with a P proline in light of the likely rates of inhibition of thrombin and APC during antithrombotic therapy with heparin suggested that this serpin may have kinetic parameters suitable for an antithrombotic agent.


INTRODUCTION

Thrombin plays a pivotal role in both the platelet activation and fibrin generation inherent to thrombosis. In the blood, thrombin is produced through the action of the prothrombinase complex (Factors Xa, Va, Ca, and phospholipid). Thrombin further stimulates its own production by activating factors V, VIII, and platelets (1) . These procoagulant activities of thrombin are blocked by the binding of thrombin to the endothelial cell surface protein thrombomodulin. In complex with thrombomodulin, thrombin becomes a more efficient activator of protein C, a protease with anticoagulant activity. In addition, the thrombomodulin-thrombin complex is no longer able to cleave fibrinogen and factor V and cannot activate platelets. Furthermore, activated protein C (APC)() turns off the coagulation cascade by inactivating factors Va and VIIIa which are essential cofactors for the formation of thrombin (2) . Thus, hemeostasis depends on a balance between the activity of thrombin, which promotes clot formation, and of APC, which prevents excessive clotting

Heparin is the current mainstay of antithrombotic therapy. The antithrombotic effect of heparin is primarily due to its acceleration of the inhibition of thrombin (and factor Xa) by antithrombin (3) . One of the principal drawbacks of the therapeutic use of heparin is the requirement for intensive laboratory monitoring of its anticoagulant effect because of pronounced individual variation in its anticoagulant response. Moreover, heparin can be inactivated by platelet factor 4 and heparinase (4) , both of which are released by activated platelets, and fibrin monomers have been found to protect thrombin from inactivation by heparin-antithrombin (5) . Heparin will also be ineffective in patients with antithrombin deficiency. Many of the limitations of heparin are not observed with the direct thrombin inhibitors currently in clinical trials. These inhibitors (most notably hirudin and hirulog) do not require cofactors for their activity and are not subject to inactivation by platelet proteins. Moreover, the anticoagulant responses of these compounds are reproducible and, therefore, intensive laboratory monitoring of anticoagulant effects is not required (6, 7) . However, one potential disadvantage that hirudin and hirulog share with heparin is their short plasma half-life (<1 h (8, 9) . Intravenous infusion or repeated subcutaneous injections would be required for a persistent anticoagulant effect. The necessity for such procedures could be obviated by increasing the plasma half-life of a direct thrombin inhibitor. Engineered plasma serine protease inhibitors (serpins) appear to be good candidates for such a thrombin inhibitor as their plasma half-life can be as long as several days (10, 11) . The engineered serpin would have to be a rapid inhibitor of thrombin and not require cofactors such as heparin. In this respect, the Pittsburgh variant of -antitrypsin is a good starting point for the development of a recombinant serpin with antithrombotic properties. In this variant, the P methionine of -antitrypsin has been replaced with an arginine, and the resulting molecule (PRS-AT) is a moderately rapid inhibitor of thrombin. This inhibitor has been tested in a baboon model of septic shock (12) . Activation of coagulation is the main cause of death associated with septic shock. Arg--antitrypsin was, however, unable to prevent the activation of coagulation. In addition to inactivating thrombin, Arg--antitrypsin was an effective inhibitor of APC (13) with the result that APC anticoagulant pathway was inhibited, and persistent activation of the coagulation cascade occurred. Thus, although rapid activation of thrombin is a requirement for an antithrombotic serpin, specificity is also important; in particular, inhibition of APC should be avoided. This is vital in the treatment of septic shock where APC appears to have a protective role (14) . Consequently, the aim of the current investigation was to engineer PRS-AT to increase its specificity for thrombin over APC while maintaining its fast inhibition rate with thrombin.

The primary determinant of the specificity of a particular serpin is the sequence of the reactive site loop which binds to the active site cleft of the protease. The most important residue within this sequence is the P residue which interacts with the S pocket of the protease. The P residue is, however, not the sole residue in the reactive site loop which contributes to specificity; many serpins have a P arginine residue yet have quite different inhibitory profiles. It has been shown that mutating the P` residue of antithrombin can dramatically affect its inhibition of thrombin, while having a relatively minor effect on the inhibition of factor Xa (15) . Mutagenesis of the P residue of a number of different serpins has shown that it also can contribute to serpin specificity (16, 17, 18, 19) . Antithrombin and C1 inhibitor are very poor inhibitors of APC and differ from PRS-AT in the P and/or P` positions. The P-P-P` sequences of antithrombin and C1 inhibitor are Gly-Arg-Ser and Ala-Arg-Thr, respectively. The significance of the P alanine/glycine and the P` threonine in regulating inhibitory specificity toward APC was tested by making a series of single and double mutations in these positions. The results indicated that the inhibitory specificity of antithrombin and C1 inhibitor was not due solely to the P-P-P` sequence. The poor inhibition of APC by antithrombin and C1 inhibitor could have been due to other sequences within the reactive site loop or regions in other parts of the molecule. For antithrombin, it was found that the poor interaction with APC was due mainly to sequences within the reactive site loop. Substitution of the reactive site loop of antithrombin (P-P`) into -antitrypsin resulted in a chimeric serpin whose association rate constant with APC was reduced 10-fold compared with PRS-AT.


EXPERIMENTAL PROCEDURES

Materials

The chromogenic substrate S-2266 (D-Val-Leu-Arg-p-nitroanilide) was purchased from Kabi-Pharmacia (Mölndal, Sweden). Oligonucleotides were synthesized by the Department of Biochemistry, University of Cambridge. Protease-free bovine serum albumin and p-nitrophenyl p`-guanidinobenzoate were purchased from Sigma (Poole, United Kingdom). Thrombin was prepared as described previously (20) . APC was a gift of Dr. J. Stenflo and Dr. A. hlin (Malmö, Sweden). All other chemicals were of the highest grade commercially available.

Production of Mutants of -Antitrypsin

-Antitrypsin mutants were produced as recombinant protein in Escherichia coli using the vector pN15. This vector was derived from the vector pTermat (21) by deletion of the 15 5` codons of the -antitrypsin sequence; as described previously (22) expression levels of -antitrypsin were significantly higher with the 15 5` codons deleted than with the intact N terminus. Reactive loop mutants were made by PCR exploiting the AvaI restriction site that lies at a position corresponding to the codons for residues P` and P`. Mutagenic oligonucleotides contained the desired mutations as well as the AvaI site, and these were used in a PCR reaction with an oligonucleotide complementary to an upstream region. The PCR product could be cut with BstXI and AvaI and cloned into the expression vector cut with the same two restriction enzymes. The entire cloned region was sequenced to ensure the presence of the desired mutation and the absence of PCR-induced error. Proteins were expressed and purified as described previously (21) .

Determination of Association Rate Constants

All serpins were found to be slow tight-binding inhibitors of thrombin and APC, indicating that there was no significant problem associated with modification of the single cysteine, which is reported to convert -antitrypsin to a reversible competitive inhibitor (23). All assays were performed at 37 °C in 30 mM sodium phosphate buffer, pH 7.4, containing 0.16 M NaCl, 0.1% polyethyleneglycol (M 4000), 0.2 mg/ml protease-free bovine serum albumin, and 200-400 µM S-2266. Slow-binding kinetics were performed using a Hewlett-Packard diode-array spectrophotometer. The production of p-nitroanilide was measured between 400 and 410 nm. For the analysis of APC inhibition, the plastic cuvettes were coated with bovine serum albumin and polyethyleneglycol by incubating overnight with a solution of 10 mg/ml bovine serum albumin and 0.1% polyethyleneglycol (M 8000). After washing in distilled water, the cuvettes were dried at 37 °C before use. Each slow-binding inhibition experiment consisted of six to seven assays with one in the absence of serpin and five to six others with increasing serpin concentrations. After initiation of the reactions by the addition of either thrombin or APC, the release of p-nitroaniline was monitored. Data points where substrate utilization was in excess of 10% of total substrate concentration were excluded from the analyses. The mechanism of inhibition was well described by where E, S, P, and I represent enzyme, substrate, product and serpin respectively; Kand k are the Michaelis and catalytic rate constants for the enzyme-substrate reaction, and k and k are the association and dissociation rate constants for the enzyme-inhibitor complex. Values of Kfor S-2266 with thrombin and APC were 262 ± 14 and 654 ± 62 µM, respectively (13) . Progress curve data were analyzed as in Hermans and Stone (13) to yield estimates for k. For each enzyme-inhibitor combination, at least two slow-binding experiments were performed, and the k values given represent the weighted mean of the determinations. The estimates obtained for Kwere 1 nM or less in all cases.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

For the reaction of LS-AT with APC, the k value was too low to be estimated by slow-binding kinetics; the k values for this mutant were therefore determined as described by Beatty et al.(24) .

Determination of the Active Concentrations of Proteases and Serpins

Thrombin and APC were titrated with p-nitrophenyl p`-guanidinobenzoate (25) . Serpins were titrated against thrombin in microtiter plates by incubating increasing amounts of serpin in 50 µl of buffer containing 50 nM thrombin for 3-4 h at 37 °C. The microtiter plate was then allowed to reach room temperature prior to adding 150 µl of reaction buffer containing 300 µM S-2266, and the hydrolysis of the substrate was monitored at 405 nm using a Thermomax microtiter plate reader. Regression analysis of the substrate conversion rate against volume of inhibitor added (13) was used to calculate the concentration of inhibitor. All recombinant serpins displayed roughly the same specificity activity of over 70%.

RESULTS AND DISCUSSION

Mutation of P and P` Residues in PRS-AT

In order to function as an antithrombotic agent, a serpin should inhibit thrombin rapidly. Rapid inhibition of thrombin will not only hinder the formation of fibrin and thrombin-dependent platelet aggregation, but it will also prevent the feedback activation of the coagulation cascade by thrombin (1). At the same time, an antithrombotic agent should not rapidly inhibit APC, since the activity of APC is required to shut down the coagulation cascade (2) . We have used PRS-AT as a starting point for engineering a serpin with a suitable kinetic profile for an antithrombotic agent. PRS-AT rapidly inactivates thrombin (50-100 times more rapidly than antithrombin), but it also reacts rapidly with APC (). Thus, if the rapid inactivation of APC could be dissociated from that of thrombin, PRS-AT would a exhibit a suitable kinetic profile for use as an antithrombotic. In a previous study, it was found that antithrombin and C1 inhibitor inactivated APC very slowly (13) . An earlier study with peptidyl chloromethane inhibitors had shown that compounds with a P glycine failed to inhibit APC (26) and, thus, it was suggested that the P glycine of antithrombin may be responsible for its slow reaction with APC (13) . Examination of the reactive site loop sequence of C1 inhibitor did not reveal any obvious reasons for its poor inhibition of APC apart from the fact that the usual P` serine was replaced by a threonine in this serpin (13) . Another serpin which displayed a reasonable selectivity for thrombin over APC was protease nexin 1. The ratio of the association rate constants (k) for this serpin with thrombin and APC was 290 compared with 7 for PRS-AT (). Like C1 inhibitor, protease nexin 1 contains an alanine in the P position. Thus, on the basis of these previous studies, five mutations were made in PRS-AT to examine the possibility of increasing the selectivity of this serpin: the P proline was mutated to glycine or alanine, and the P` serine was replaced by threonine. In addition, double mutations of the P and P` residues were made. These mutations reproduced the P-P-P` sequences of antithrombin (Gly-Arg-Ser), protease nexin 1 (Ala-Arg-Ser), and C1 inhibitor (Ala-Arg-Thr) in -antitrypsin. The effect of these mutations on the k values with thrombin and APC are given in .

The results in indicate that the mutations did not improve the selectivity of PRS-AT. The replacement of the P proline of PRS-AT by alanine resulted in decreases in the k values with thrombin and APC of 7- and 4-fold, respectively. The k values determined for ARS-AT with thrombin and APC agree with those previously reported (16, 17, 27). The effects of the substitution of the P residue by glycine were larger; 37- and 16-fold decreases in the k values were observed with thrombin and APC, respectively. Thus, for both substitutions of the P proline, the decrease in the k value with thrombin was about twice as large as that observed with APC with the result that the selectivity of the mutants was decreased (). The replacement of the P` serine of PRS-AT by threonine did not markedly affect the k values with either protease (). These results indicated that the poor inhibition of APC by antithrombin and C1 inhibitor cannot be attributed solely to the P and P` residues in these serpins.

The effects of the replacement of the P proline on the inhibition of thrombin can be rationalized on the basis of the structure of the covalent complex between thrombin and D-Phe-Pro-ArgCH(28, 29) . In this structure, the P proline of the inhibitor is bound in a hydrophobic pocket formed by the side chains of TyrAla, TrpAsp, His, Trp, and Leu. All the contacts made by the P proline would be lost upon replacement of the residue by glycine or proline, and the loss of these contacts would be more than sufficient to explain the observed decreases in k due to the P Pro Ala/Gly mutations. Since the tertiary structure of APC has not been determined, the results with this protease cannot be interpreted reliably in terms of tertiary structure. However, comparison of the sequences of APC and thrombin indicates that several of the residues in thrombin that interact with the P proline are not present in APC. TyrAla and TrpAsp are found in an insertion that is not present in APC, and Leu in thrombin is replaced by a threonine in APC. These changes in APC would result in an S pocket less suited for a proline and, thus, a smaller decrease in k with the P Pro Ala/Gly substitutions may be expected.

The effects of the P Pro Ala/Gly mutations on the k values with thrombin and APC illustrate the difficulties in predicting the effects of mutations in the reactive site loop of serpins from results obtained with small peptide substrates and inhibitors. Studies with peptide substrates and inhibitors have demonstrated that both thrombin and APC displayed a marked preference for proline at P over glycine (26, 30, 31) . The effects of the mutations on the k values of PRS-AT with thrombin and APC are, however, smaller than those that would be expected from these studies. The average k/Kvalue for thrombin with a series of seven p-nitroanilide substrates with a P proline was 130-fold higher than the corresponding average value with six substrates containing a P glycine (30). A similar difference in k/Kvalues was also observed for p-nitroanilide substrates with APC; the average k/Kfor substrates containing a P proline was 300-fold higher than those containing a glycine in this position (26) . Thus, although the results from studies with synthetic peptide substrates and inhibitor predicted the observed decrease in the k value upon the replacement of the P proline, they could not accurately predict the magnitude of the change. The magnitude of the effect of a P substitution is clearly modulated by the context of the individual serpin reactive site loop. Serpins interact with proteases over a more extended area than peptide substrates and inhibitors. In particular, p-nitroanilide substrates and chloromethane inhibitors only bind to the S/S-S sites and do not interact with the S` sites. As discussed below, interactions with the P` residue can modulate those made by the P amino acid and Madison et al.(32) have demonstrated that contacts with P` residues of plasminogen activator inhibitor I are important in determining the specificity of this serpin.

The effects of the double mutations demonstrated cooperative interactions between the P and P` residues in the inhibition of thrombin; the effects of the mutations were not additive. Non-additivity in mutational effects is conveniently interpreted in terms of double mutant cycles (33) as shown in Fig. 1A for the effect of the P Pro Ala and P` Ser Thr mutations on the k for thrombin. With threonine in the P` position, the effect of replacing the P proline by alanine (or glycine) was about 3-fold larger. The P Pro Ala mutation caused a 22-fold decrease in k with threonine in P` compared with the 7-fold decrease with serine in this position (Fig. 1A). If the mutational effects were additive, the P Pro Ala mutation would cause the same decrease in k irrespective of the P` residue. It follows from the symmetry of the cycle shown in Fig. 1A that the P` Ser Thr mutation was approximately 3-fold more detrimental with alanine as the P residue. The P Pro Ala and P` Ser Thr mutations are coupled; the magnitude of the mutational effect in one postion depends on the nature of the amino acid in the second position. A similar degree of coupling was observed for the P Pro Gly and P` Ser Thr mutations; the decreases in k with thrombin caused by the P Pro Gly substitution were 96- and 37-fold when threonine and serine, respectively, were the P` residues. Similar, but smaller, cooperativity effects between P and P` substitutions were also observed with APC; the effect of the P Pro Ala/Gly substitutions were about twice as large with threonine in the P` positions. These results indicate that the interactions made by the P residue within the reactive site loop of serpins are influenced by the nature of the P` residue. The presence of a threonine in the P` position of PRS-AT has made it more difficult for thrombin to accommodate the P Pro Ala/Gly substitutions.


Figure 1: Non-additivity of mutational effects in the reactive site loops of serpins. Additivity of mutational effects can be analyzed by constructing double mutant cycles as described by Horovitz (33, 45). The effects of the P Pro Ala and P` Ser Thr substitutions on the k with thrombin are shown in A; changes in k for APC caused by the -antitrypsin antithrombin loop swap (PRS LS) and the P Pro Gly mutation are shown in B. If the effects of the mutations are independent of each other, the magnitude of change in k should be equal on parallel sides; i.e. the effect of a mutation should be independent of the residues in the other positions. Cycle A shows that the effects of P Pro Ala and P` Ser Thr substitutions are not independent of each other; the P Pro Ala replacement causes a 3-fold greater decrease in k when threonine is the P` residue (similarly, the effect of the P` Ser Thr substitution is three times larger with glycine in the P position). Cycle B shows the cooperative effect between the P glycine and the rest of the antithrombin reactive loop sequence; the decrease in the k with APC caused by the P Pro Ala mutation is about 6-fold greater in the antithrombin loop.



Replacement of the Reactive Site Loop of PRS-AT

The results of the above experiments indicate that the slow inhibition of APC by antithrombin and C1 inhibitor cannot be attributed solely to the P-P-P` sequence within the reactive site loop. Additional sequences in these serpins must be responsible for their poor inhibition of APC. These sequences could reside within the reactive site loop; alternatively, the structural motifs responsible for the poor inhibition could be found elsewhere in the molecules. In order to test these two alternative explanations for antithrombin, a chimeric serpin was constructed in which the reactive site loop of PRS-AT was replaced by that of antithrombin. The results obtained with this serpin (LS-AT) indicated that most of the structural information that causes the poor inhibition of APC is found within the reactive site loop. The k value for LS-AT with thrombin was 10-fold lower than that observed with PRS-AT and slightly higher than the value observed for antithrombin (). The effect of the substitution on the inhibition of APC was dramatic. The k value for APC was reduced by 12,500-fold. This value is about 40-fold higher than antithrombin in the absence of heparin and only 3-fold higher than antithrombin in the presence of heparin. The improvement in selectivity of LS-AT over PRS-AT was striking. The ratio of the k values for thrombin and APC increased from 7 for PRS-AT to 8200 for LS-AT; the value of this ratio was only 10-fold lower than that observed with antithrombin ().

Despite the improved selectivity of LS-AT, the k value of this serpin with thrombin is still somewhat lower than would be required for an antithrombotic agent (see below). It was only slightly higher than that observed for antithrombin in the absence of heparin. In an attempt to improve the k value of LS-AT with thrombin, the P glycine was replaced by a proline (LS-Pro-AT). From the results obtained with PRS-AT, the P Gly Pro would be expected to yield increases in the k values for thrombin and APC of about 40- and 15-fold, respectively. In the context of the antithrombin reactive site loop, however, the relative increases in the k were opposite to those expected from the PRS-AT substitutions. The increase for thrombin was only 7-fold while a 90-fold increase was seen with APC (). However, LS-Pro-AT still remained reasonably selective; the k value with thrombin was similar to that observed with PRS-AT (3.1 10M s) while the corresponding value with APC (4.9 10M s) was 140-fold lower than that of PRS-AT. The increase in selectivity of LS-Pro-AT over PRS-AT was 90-fold. It was observed above that the effect of the P Pro Ala/Gly substitution was influenced by the nature of the P` residue. The results observed with the P Gly Pro substitution in LS-AT indicated that residues within the reactive site loop other than the P` residue were able to affect the magnitude of the change observed upon the mutation of the P residue. Fig. 1B shows the double mutant cycle for the inhibition of APC by PRS-AT, GRS-AT, LS-Pro-AT, and LS-AT. The decrease in k caused by the P Pro Gly substitution was six times larger (90- versus 16-fold) in the antithrombin reactive site loop compared with the -antitrypsin loop. Evidently, the interactions made by a particular residue in the reactive site loop are influenced by the nature of other residues in the loop, and cooperativity effects in the binding of residues to the active site of proteases play a role in determining serpin specificity.

The cooperativity between serpin reactive site loop residues observed here is in notable contrast to the Kazal inhibitor ovomucoid third domain. Over 100 variants of the ovomucoid third domain, many of which can be arranged into double mutant cycles, have been examined; the effect of the mutations was shown to be additive, the contribution of a particular residue being independent of the other residues in the inhibitor (34, 35, 36, 37) .

Suitability of PRS-AT Variants for Use as Antithrombotic Agents

For use as an antithrombotic agent, a serpin should inhibit thrombin rapidly and APC slowly. Some indication of required rates of inhibition can be obtained by examining the rates of inhibition of these two proteases by their natural inhibitors. The rate constant (k) for the inactivation of a protease will be given by k = k[I] where [I] is the concentration of the serpin. The half-life (t) for the inactivation will be t = ln(2)/k = ln(2)/(k[I]). The major inhibitor of thrombin in plasma is antithrombin which is present at a concentration of 2.3 µM and has a k value of about 10M s in the absence of heparin (38) . Thus, the rate constant (k) for the inactivation of thrombin by antithrombin in the absence of heparin is about 0.03 s. However, since the rate of inhibition of thrombin during thrombus formation is too slow, an antithrombotic agent should exhibit a higher k value. Heparin is the current mainstay of antithrombotic therapy, and the rate of inhibition thrombin by the heparin-antithrombin complex represents a target rate for other antithrombotics. The plasma level of heparin during effective antithrombotic therapy is usually 0.2-0.4 units/ml which corresponds to a concentration of about 60-120 nM. Using values for the kinetic constants of the inhibition of thrombin by heparin-antithrombin (38) , it can be calculated that at this concentration of heparin, the k value for the inactivation of thrombin will be about 1-2 s. From this value of k, it is possible to calculate concentrations of the recombinant serpins that would be necessary to achieve an equivalent antithrombotic effect to that achieved usually during heparin therapy. Assuming that a k value of 1 s is sufficient, the plasma concentrations of serpins required will be 1/k. Values calculated on this basis ranged from 2 to 3 µM for PRS-AT, PRT-AT, and LS-Pro-AT to 185 µM for GRT-AT. For the best inhibitors of thrombin (PRS-AT, PRT-AT, LS-Pro-AT), the concentrations required (2-3 µM) are about equal to the plasma concentration of antithrombin and represent about 0.1 g/liter.

In plasma, the activity of APC is controlled by the serpin protein C inhibitor (PCI). Antithrombotics should not augment the rate of inhibition of this protease. The k value for PCI with APC is 8 10M s(13) , and the plasma concentration of the inhibitor is 0.1 µM (39) which yields k value for the formation of the inhibited complex of 8 10 s. In the presence of heparin, the k for PCI with APC is moderately increased, and in the presence of therapeutic levels of heparin (0.2-0.4 units/ml) the k value will be about 2 10 s(13) . An inhibitor will not significantly increase the intrinsic rate of inhibition of APC provided it reacts more slowly than PCI and the k value of PCI in the presence of therapeutic levels of heparin gives some indication of the magnitude of k that would be appropriate. Using the minimum k value for thrombin and the maximum k value, it is possible to calculate a required selectivity ratio k(thrombin)/k(APC) (= k(thrombin)/k(APC)) of 5000. LS-AT was the only recombinant serpin for which this selectivity ratio was achieved (). However, the concentration of LS-AT that would be required for an antithrombotic effect was 22 µM (1/(4.6 10M) which is equivalent to 1 g/liter and is probably too high to make its use as an antithrombotic agent feasible. The calculated values for LS-Pro-AT are more encouraging. Its selectivity ratio is only 8-fold higher than the suggested target value and as noted above, it was calculated that a concentration of about 0.1 g/liter of LS-Pro-AT should be antithrombotic. Thus, the kinetic parameters of LS-Pro-AT with thrombin and APC are sufficiently promising to warrant further investigation of its antithrombotic potential in animal models of thrombosis. The plasma half-life of -antitrypsin is severely affected by its glycosylation state (40, 41) , thus material produced in E. coli could have a short plasma half-life and may be antigenic. This situation may be alleviated by producing -antitrypsin variants in the mammary glands of sheep as described previously (42) which produces fully glycosylated -antitrypsin.

  
Table: Association rate constants of natural and mutant serpins with thrombin and APC

Mutants were made and their association constants against thrombin and APC were determined in the absence of heparin as described under ``Experimental Procedures.'' All estimates of k had standard errors of less than 5%. Each of the values were determined at least twice and the weighted means of the estimates are given. Protease nexin 1 is abbreviated to PN1.



FOOTNOTES

*
This work was supported by the British Heart Foundation, Wellcome Trust, and Pharmaceutical Proteins Ltd. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Haematology, University of Cambridge, MRC Centre, Hills Rd., Cambridge CB2 2QH, United Kingdom. Tel.: +44-1223-336826; Fax: +44-1223-336827.

Elmore student.

The abbreviations used are: APC, activated protein C; site-specific mutants of recombinant -antitrypsin are identified by the sequence of their P-P-P` residues, e.g. PRS-AT and ART-AT represent recombinant -antitrypsin with the P-P-P` sequences Pro-Arg-Ser and Ala-Arg-Thr, respectively; LS-AT; recombinant -antitrypsin with the P to P` residues of the reactive site loop replaced with those from the antithrombin loop; LS-Pro-AT, LS-AT with the P glycine mutated to proline; PCR, polymerase chain reaction; PCI, protein C inhibitor.


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