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Peptidomimetic inhibitors for activated protein C: implications for hemophilia management

2006; Elsevier BV; Volume: 4; Issue: 11 Linguagem: Inglês

10.1111/j.1538-7836.2006.02226.x

1538-7933

Saulius Butenas, Thomas Orfeo, Michael Kalafatis, Kenneth G. Mann,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Journal of Thrombosis and HaemostasisVolume 4, Issue 11 p. 2411-2416 Free Access Peptidomimetic inhibitors for activated protein C: implications for hemophilia management S. BUTENAS, S. BUTENAS Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this authorT. ORFEO, T. ORFEO Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this authorM. KALAFATIS, M. KALAFATIS Department of Chemistry, Cleveland State University, Cleveland, OH, USASearch for more papers by this authorK. G. MANN, K. G. MANN Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this author S. BUTENAS, S. BUTENAS Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this authorT. ORFEO, T. ORFEO Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this authorM. KALAFATIS, M. KALAFATIS Department of Chemistry, Cleveland State University, Cleveland, OH, USASearch for more papers by this authorK. G. MANN, K. G. MANN Department of Biochemistry, University of Vermont, Colchester, VTSearch for more papers by this author First published: 16 October 2006 https://doi.org/10.1111/j.1538-7836.2006.02226.xCitations: 19 S. Butenas, Department of Biochemistry, University of Vermont, 208 South Park Dr, Suite 2, Room T227, Colchester, VT 05446, USA.Tel.: +1 802 656 0350; e-mail: sbutenas@uvm.edu AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Summary. Background: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. Objective: To test the effect of APC inhibition on thrombin generation in hemophilia. Methods: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. Results: Peptide-based APC inhibitors, which mimic the P4–P4′ residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1–2 μm; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. Conclusions: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment. Introduction The most common blood coagulation defects leading to impaired hemostasis are those caused by factor (F) VIII (hemophilia A) or FIX (hemophilia B) deficiency. Both deficiencies lead to a bleeding diathesis, the severity of which is largely dependent upon the concentration of FVIII and FIX [1]. In contrast to hemophilia, defects in the protein C pathway, which include genetic protein C and protein S deficiencies, and a relatively common mutation in the factor V gene, Arg506 → Gln (FV Leiden), often lead to prothrombotic consequences [2]. Several clinical studies and a study using transgenic hemophilia/FV Leiden mice demonstrated that the combination of these two opposing coagulation disorders, i.e. the simultaneous presence of FV Leiden and hemophilia A or B, leads to a decrease in the severity of the bleeding diathesis in patients deficient in FVIII or FIX [3-6]. The mechanism underlying this compensatory effect of FV Leiden is presumably related to the interplay between the processes leading to thrombin generation and clot formation and the inhibitory regulation of those processes [7]. The blood coagulation cascade is initiated when tissue factor (TF) is expressed/exposed to the circulating blood and binds plasma FVIIa. The resulting FVIIa–TF complex triggers a cascade of enzymatic reactions, which lead to thrombin generation and consequent fibrin–platelet clot formation. This coagulation cascade is downregulated by the stoichiometric inhibitors antithrombin-III (AT-III) and TF pathway inhibitor (TFPI), and by the dynamic protein C system [8]. Dynamic negative feedback inhibition of coagulation is initiated when thrombin bound to its cofactor thrombomodulin activates protein C to activated protein C (APC) [9]. APC downregulates blood coagulation by inactivating FVa and FVIIIa, and the procofactors FV and FVIII [10-14]. FV/Va is inactivated in a phospholipid-dependent manner following cleavages at Arg306, Arg506 and Arg679 of the heavy chain [11]. The complete loss of activity is connected with the cleavage at Arg306, whereas the more rapid cleavage at Arg506 of FVa decreases cofactor activity. Deficiencies in the components of the FVIIIa–FIXa complex (hemophilia A and B) delay and suppress robust thrombin generation [15-17]. Thrombin generation can be (partially) restored by extending the lifetime of the prothrombinase complex. Prolongation of FVa function due to resistance to APC cleavage has been observed for the naturally occurring FV Leiden mutant [18], leading to increased thrombin generation in both complete and FVIII- or FIX-deficient systems [3, 6, 7, 16]. The increased stability of FVa Leiden provides a mechanistic explanation for the observed compensatory mechanism of this FV mutant in hemophilia A and hemophilia B. An alternative to a naturally occurring FV mutation leading to APC resistance could be an efficient and specific APC inhibitor. APC is inhibited by the majority of serine protease inhibitors (serpins) circulating in vivo, including protein C inhibitor (PCI) [19, 20], protease nexin I [20], α2-macroglobulin [21], α2-antiplasmin [22], α1-antitrypsin [23, 24], and plasminogen activator inhibitor 1 [25]. The inhibition of APC by serpins (including PCI) is slow and inefficient, due to their low affinity, especially when compared with their affinity for the abundant procoagulant serine proteases [19, 20, 26-29]. This allows APC time to inactivate FVa and FVIIIa before inhibition occurs. A large number of various synthetic and naturally occurring non-plasmatic derivatives have been suggested as APC inhibitors [30-34]. However, all of these molecules have a serious shortcoming in common with the natural inhibitors – they are not specific for APC. In the current study, we present the synthesis of specific APC inhibitors, an evaluation of their protection of FVa, and their ability to support thrombin generation in hemophilia. Materials and methods Chemicals Amino acids and their derivatives were purchased from Bachem (King of Prussia, PA, USA). Other reagents and solvents for inhibitor syntheses and biochemical assays were purchased from Sigma (St Louis, MO, USA). Dioleoyl-sn-glycero-3-phospho-l-serine (PS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Phospholipid vesicles (PCPS) composed of 25% PS and 75% PC were prepared as described [35]. Spectrozyme TH and Spectrozyme fXa were purchased from American Diagnostica, Inc. (Greenwich, CT, USA). Proteins Human FVII, FX and FIX, protein C and prothrombin were isolated from fresh frozen plasma using the methods of Bajaj et al. [36], and FXI using monoclonal anti-FXI antibody α-FXI-2. The proteins were purged of trace contaminants and traces of active enzymes as previously described [37]. Human FV and AT-III were isolated from freshly frozen citrated plasma [38, 39]. Human FXa, α-thrombin, APC, FXIa and Lys–plasmin were gifts from Dr Richard Jenny from Hematologic Technologies, Inc. (Essex Junction, VT, USA). Recombinant FVIII and recombinant TF1–242 (expressed in Escherichia coli) were gifts from Drs Shu Len Liu and Roger Lundblad (Hyland division, Baxter Healthcare Corp, Duarte, CA, USA). Recombinant human FVIIa was a gift from Dr Ulla Hedner (Novo Nordisk, Denmark). Recombinant full-length TFPI was a gift from Dr K. Johnson (Chiron Corp, Emeryville, CA, USA). Recombinant soluble thrombomodulin (Solulin) was a gift from Dr J. Morser (Berlex, Richmond, CA). Preparation of the TF/PCPS reagent (molar ratio 1 : 5000) was as previously described [15]. Methods Syntheses of inhibitors Inhibitors were synthesized using the conventional methods of peptide synthesis as previously described [40]. Inhibition constant (Ki) determination The assays were performed in 20 m HEPES, 150 mm NaCl (pH 7.4) (HBS) with 2 mm CaCl2 at 25 °C using a THERMOmax microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA). The final volume for all assays was 200 μL. Inhibitors were dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 10 mm, and this solution was used for FXa and thrombin inhibition assays. For the APC inhibition assays, a 10 mm solution was diluted in HBS to 50 μm. Final concentrations of inhibitors varied from 0 to 25 μm in the APC inhibition assays and from 0 to 450 μm in the FXa and thrombin inhibition assays. The substrates Spectrozyme TH (for APC or thrombin) or Spectrozyme fXa (for FXa) were added (100, 200 and 400 μm final concentrations), and the corresponding enzyme was then added at a final concentration of 5 nm. The rate of substrate hydrolysis was monitored for 5 min at 405 nm. The inhibition constants were calculated graphically using Dixon plots. The influence of inhibitor VI on the enzymatic activity of FVIIa–TF, FXIa and Lys–plasmin was determined in the chromogenic assay using 10 nm enzyme and 40 μm inhibitor. APC inhibition in the prothrombinase assay Human APC (40 nm) was incubated in HBS/5 mm CaCl2 with 100 or 200 μm inhibitor VI or corresponding amounts of DMSO (control) for 30 min at 37 °C. This solution was added to 400 nm FVa and 15 μm PCPS (final concentrations) in HBS/5 mm CaCl2, and the mixture was incubated at 37 °C. The final concentration of APC was 4.4 nm and that of inhibitor VI was 11 or 22 μm. At selected time intervals (0–120 min), 10-μL aliquots were taken and added to a cuvette containing 1.4 μm prothrombin, 3 μm dansylarginine-N-[3-ethyl-1,5-pentanediyl]amide, 20 μm PCPS and 10 nm FXa. The final volume of the mixture was 2 mL. An increase in fluorescence intensity over time was monitored using a Perkin-Elmer MPF-44A (Perkin-Elmer, Wellesley, MA, USA) fluorescence spectrophotometer with λex 280 nm and λem 550 nm. Fluorescence intensity was proportional to the concentration of thrombin formed. Under these conditions, the initial rate of thrombin formation is linearly related to the active FVa concentration, and it was calculated as described previously [41]. Synthetic coagulation proteome The procedure used was a modification of those of Lawson et al. [42] and van 't Veer et al. [37]. Relipidated TF at 5 pm final concentration was added to the mixture of FV, FVIII, FVII, FVIIa, FIX, FX, FXI, TFPI, prothrombin, protein C and AT-III (all at mean physiologic concentrations) [43] and thrombomodulin at 10 nm in HBS/2 mm CaCl2 containing 2 μm PCPS. When desired, FVIII was omitted and 10 μm inhibitor VI were added. Thrombin generation over time was measured in a chromogenic assay using Spectrozyme TH and a THERMOmax microplate reader. Results Inhibitor structure and affinity for APC, FXa and thrombin The general structure of the compounds synthesized is presented in Fig. 1. The amino acid sequences of all compounds (Fig. 1A) are identical and mimic that of the APC FVa cleavage site at Arg306 (Fig. 1B). P4–P2, P3′ and P4′ residues of the synthesized compounds (Lys-Lys-Thr and Lys-Lys, respectively) correspond to those of the Arg306 cleavage site. The P1 Arg is substituted with homoarginine (hArg; 16 compounds), d-Arg (one compound) or β-homoarginine (β-hArg; 1 compound). This prevents cleavage of the compounds by serine proteases. The P1′ and P2′ residues of the 306 cleavage site are replaced by 2,6-aminonaphthalenesulfonamide [44]. Variations in the nature, location and number of protecting groups for amino acids of the synthesized compounds led to 14 compounds with identical amino acid sequences but quite distinct inhibitory properties. Additionally, two compounds studied had truncated sequences in either the P or the P′ structure. Figure 1Open in figure viewerPowerPoint General structure of synthesized activated protein C (APC) inhibitors (A) and P4–P4′ sequence of the factor Va cleavage site at Arg306 by APC. (B) The majority of the synthesized compounds contain homoarginine (hArg) in the P1 position. The vertical dashed line indicates the scissile bond. The inhibition constants (Ki) for 18 compounds are presented in Table 1. All compounds are competitive inhibitors of APC and FXa (see Fig. 2 for APC inhibition by compound VI). The Ki for APC varies from 1.1 μm for compound XVII to 280 μm for compound V. The Ki values show strong dependence on an inhibitor's P and P′ structure, particularly on the location of blocking groups. A truncated form of the synthesized compounds, which contains only hArg in the P structure (compound I), is a poor inhibitor of APC, with a relatively high Ki (90 μm). Similarly, compound V, which contains the complete P1–P4 structure without blocking groups and 2,6-aminonaphthalenebenzylsulfonamide in the P′ structure, shows an even lower affinity for APC, with a Ki of 280 μm. Compounds II, III and IV, which contain the P1–P4, P3′ and P4′ amino acids representing the FVa cleavage site at Arg306, with all the lysine functional groups blocked, have Ki values for APC in the range 28–39 μm. The chemical nature of the blocking groups appears to have little effect on the affinity for APC. Elimination of these groups from the amino and carboxy moieties of both the main and side chains does not increase the affinity for APC (compound XVI; Ki = 33 μm). However, selective elimination of blocking groups leads to increased efficiency. Thus, compounds XIV and XV, which have only the C-terminus blocked, have higher affinities for APC (Ki 15 μm and 7.9 μm, respectively) than their completely blocked or completely unblocked analogs. Additionally, a comparison of these compounds (XIV and XV) shows that an aromatic benzyl blocking group is preferable to the aliphatic methyl group. Further analysis of the influence of blocking groups on inhibitor affinity for APC indicates that compounds with at least one Lys with an unblocked side chain in the P structure and completely blocked functional groups in the P′ structure have the highest affinity for APC (compounds VI–X). The Ki values of these inhibitors for APC vary in a narrow range from 1.3 μm for compound IX to 2.1 μm for compound VI, and are not influenced by the location of a P3 or P4 Lys with an unblocked side chain (compare compounds VIII and X). Moreover, elimination of the blocking groups from both lysines (compound IX) or complete removal of blocking groups from the P structure of synthesized inhibitors (compound VI) has little, if any, effect on an inhibitor's affinity for APC. However, when the side chains of both P lysines are blocked, the Ki of the inhibitor for APC increases to 4.5 μm, despite an unblocked N-terminus (compound XI). Blocking of the P2 Thr side chain with a benzoyl protecting group has no effect on the affinity for APC (compare compounds IX and XII). Substitution of l-hArg in the P1 position of compound IX by d-Arg (compound XVII) or β-l-hArg (compound XVIII) has little effect on the affinity of these compounds for APC (the Ki values vary in the range 1.1–2.3 μm). Table 1. Inhibition constants (Ki) of synthesized compounds* Compound R1 R2 R3 R4 R5 R6 R7 K i (μm) APC FXa Thrombin I – – – – Z Z Bzl† 90 – – II Boc† Boc Boc H Z Z Bzl 39 – – III Boc Boc Boc H Boc Z Bzl 28 – – IV Boc Boc Boc H Boc Boc Me† 29 – – V H H H H – – – 280 – – VI H H H H Z† Z Bzl 2.1 400 NI‡ VII H Z H H Z Z Bzl 1.6 440 NI VIII Z Z H H Z Z Bzl 1.8 450 NI IX Z H H H Z Z Bzl 1.3 740 NI X Z H Z H Z Z Bzl 1.5 230 NI XI H Z Z H Z Z Bzl 4.5 330 NI XII Z H H Bz† Z Z Bzl 1.2 660 NI XIII H H H H H Z Bzl 8.8 2200 NI XIV H H H H H H Me 15 170 NI XV H H H H H H Bzl 7.9 280 3100 XVI H H H H H H H 33 – – XVII§ Z H H H Z Z Bzl 1.1 280 NI XVIII¶ Z H H H Z Z Bzl 2.3 – – *Positions of R1–R7 are indicated in Fig. 2A. †Bzl, benzyl; Bz, benzoyl; Boc, t-butyloxycarbonyl; Me, methyl; Z, benzyloxycarbonyl. ‡NI: Ki > 5 mm. §Contains d-Arg in the P1 position. ¶Contains β-hArg in the P1 position. Figure 2Open in figure viewerPowerPoint Graphical evaluation (Dixon plot) of inhibition constant Ki of compound VI for activated protein C (APC). Concentrations of chromogenic substrate Spectrozyme TH: •, 100 μm; , 200 μm; , 400 μm. The compounds with a relatively high affinity for APC (compounds VI–XV) were tested for their inhibition of FXa and thrombin. All were poor inhibitors of thrombin, with Ki values exceeding 5 mm for 10 of the 11 compounds. A somewhat higher affinity (in the sub-millimolar range) was observed for FXa. The Ki values for FXa vary from 170 μm for compound XIV to 2.2 mm for compound XIII. The former compound is the least specific for APC with respect to factor Xa; the Ki ratio (FXa/APC) was ca 11. However, for the majority of the inhibitors, this ratio was > 100. After identifying a subset of compounds as direct, competitive and specific APC inhibitors with inhibition constants in the low micromolar range, we evaluated one of these compounds (compound VI) in the prothrombinase assay and in more complex systems, i.e. in TF-initiated thrombin generation using numerical simulation and blood coagulation proteome models. Compound VI was selected for these experiments, due to its higher solubility in aqueous buffers than that of other synthesized compounds with similar affinity for APC. This compound is a poor inhibitor of FXa (Ki ratio FXa/APC ca 200) and does not inhibit thrombin, FXIa, the FVIIa–TF complex and Lys–plasmin. Inhibition of APC in the prothrombinase assay Under the test conditions chosen, when FVa is incubated with APC prior to the assembly of prothrombinase, it loses > 50% of its cofactor activity in 1 min and 87% of its activity in 5 min in the absence of APC inhibitors (Fig. 3). Preincubation of APC with inhibitor VI decreases the ability of the enzyme to cleave and inactivate FVa in a concentration-dependent manner. At an inhibitor concentration of 11 μm (ca 5 × Ki) (Fig. 3), 15 min is required for FVa activity to be decreased by 50%, and 60 min for it to be decreased by 87%. At 10 × Ki (22 μm; Fig. 3), FVa retains over 60% of its cofactor activity after 30 min of treatment with APC, and ca 40% after 60 min. Figure 3Open in figure viewerPowerPoint The effect of activated protein C (APC) inhibition on factor Va activity evaluated in the prothrombinase assay. , APC is present, inhibitor is absent (control); , APC is present, inhibitor VI is present at 11 μm; , APC is present, inhibitor VI is present at 22 μm. Synthetic coagulation proteome Figure 4 shows thrombin generation initiated with 5 pm relipidated TF. In the presence of 2 μm PCPS and coagulation proteins and inhibitors at mean physiologic concentrations, the duration of the initiation phase of thrombin generation is ca 300 s. During the propagation phase, the maximum rate of thrombin generation reaches 2.1 nm s−1, and the maximum concentration of active thrombin observed is 300 nm. When protein C at a mean physiologic concentration (65 nm) and 10 nm thrombomodulin are added (Fig. 4), the duration of the initiation phase is almost unaltered. Thrombin generation during the propagation phase, however, is suppressed with respect to both the maximum rate and maximum concentration of thrombin (to 1.1 nm s−1 and 165 nm, respectively). In the absence of FVIII and in the presence of protein C pathway proteins (hemophilia A), the initiation phase is only slightly prolonged (to 360 s), whereas both the maximum rate of thrombin generation and the maximum level of thrombin are further suppressed (0.2 nm s−1 and 15 nm, respectively). Under these conditions and in the presence of 10.5 μm (5 × Ki) of this compound, the initiation phase of thrombin generation is decreased to 240 s, the maximum rate of thrombin generation is increased to 0.5 nm s−1 and the maximum concentration of active thrombin detected is 73 nm. A similar effect caused by compound VI is observed for FIX deficiency as well (data not shown). The potency of this compound in restoring thrombin generation was unexpected. Numerical simulations (not shown) indicated that in order for compound VI to cause this level of alteration in thrombin generation, i.e. to increase it to that observed in the absence of the protein C pathway, concentrations in excess of 20 × Ki (> 42 μm) would be required. Figure 4Open in figure viewerPowerPoint Coagulation proteome. The influence of compound VI on the protein C pathway in hemophilia A. □, Normal control without protein C and thrombomodulin; , normal control with protein C and 10 nm thrombomodulin; ○, hemophilia A with protein C and 10 nm thrombomodulin; , hemophilia A with protein C, 10 nm thrombomodulin and 10.5 μm compound VI. Discussion The data obtained in the current study indicate that the synthesized compounds are efficient and specific inhibitors of APC. Compound VI is able to compensate for the effect of FVIII or FIX deficiencies in TF-triggered thrombin generation. Thus, APC inhibitors may be useful as potential adjuvants for hemophilia treatment. Such inhibitors increase the hemostatic potential in the synthetic hemophilia proteome and would be predicted to decrease the concentration of FVIII that must be maintained to achieve normal hemostasis. As a consequence, specific APC inhibitors hold the promise of reducing the consumption of FVIII and FIX products used for replacement therapy. It has been established that the propagation phase of TF-triggered thrombin generation is primarily related to the production of FXa by the intrinsic FXase (FVIIIa–FIXa complex) [42, 45]. Thus, the propagation phase is significantly suppressed in FVIII and FIX deficiency, i.e. in hemophilia A and B [15-17, 42]. A further decrease is caused by the depletion of FVIIIa and FVa by APC. Elevated concentrations of functional FVa and FVIIIa can be maintained either by increasing the stability of these cofactors to APC or by decreasing the concentration of active APC. The former situation is observed for some naturally occurring FVa mutants [2], particularly for FV Leiden. However, coinheritance of FV Leiden and hemophilia A [3, 4] or B [5] is relatively rare. No FVIII mutants with FV Leiden-type resistance to APC have been reported. The second approach, a decrease in APC activity, can be realized by the use of specific and efficient APC inhibitors. The data obtained in the current study indicate that specific APC inhibitors can increase thrombin generation in severe hemophilia to a level similar to that observed for a severe hemophilia A proteome in combination with FV Leiden [7, 16]. The design of potential APC inhibitors is based upon identified sequences of APC natural substrate FVa cleavage sites by this serine protease [11]. To increase the affinity of synthesized peptidomimetic compounds for APC and to prevent their hydrolysis by serine proteases, several modifications in their structure have been made (Fig. 1). Inhibitors synthesized on the basis of the FVa amino acid sequence flanking Arg306, i.e. one of the cleavage sites for APC, have the highest affinity and specificity for this serine protease. Our numerical simulations suggested that APC inhibitors could partially compensate for severe hemophilia but would require concentrations in excess of 20 × Ki. However, coagulation proteome experiments showed that the influence of inhibitor VI on thrombin generation in the hemophilia proteome is more pronounced than predicted. These data suggest that this compound has influences beyond inhibiting the protein C pathway, or is able to be more effective in the dynamic environment of procoagulant and anticoagulant processes. The elucidation of the mechanism of this additional procoagulant effect will be the subject of future studies. Acknowledgements We would like to thank Craig Partin and Matthew Gissel for their technical support. We also thank Dr Richard Jenny for providing FXa, α-thrombin, APC, FXIa and Lys–plasmin, Drs Shu Len Liu and Roger Lundblad for providing FVIII and TF, Dr Ulla Hedner for providing FVIIa, Dr John Morser for providing Solulin, and Dr Kirk Johnson for providing TFPI. This work was supported by NIH Grant RO1 HL 34575 (to K.G. Mann). 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