Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization

Authors


E. Lugovskoy, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovicha Street, 01601, Kyiv, Ukraine
Fax: +38 044 2796365
Tel.: +38 044 2343354
E-mail: lougovskoy@yahoo.com

Abstract

Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10−6 m (IC50). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC50 was 1.26 × 10−6 m at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC50 = 1.31 × 10−4 m. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site ‘A’ (Aα17–19), which ordinarily initiates protofibril formation in a ‘knob-hole’ manner. This suggestion was confirmed by an HPLC assay, which showed a host–guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site ‘A’. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10−5 m and 1.10 × 10−5 m, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.

Abbreviations
1

para-hydroxyphenyl-methylene-bis-phosphonic acid

2

methylenel-bis-phosphonic acid

APTT

activated partial thromboplastin time

C-98

dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid

C-192

calix[4]arene tetrakis-methylene-bis-phosphonic acid

PT

prothrombin time

Introduction

Fibrinogen is a glycoprotein (MW ∼344 kDa) composed of two monomeric units connected by disulfide bonds. Each monomer consists of three non-identical polypeptide chains Aα, Bβ and γ, also connected by disulfide bridges [1]. The NH2-terminal ends of all six polypeptide chains are situated in the central region of fibrinogen known as the E-domain. Two peripheral regions of the molecule historically are called the D-domains. When blood coagulation system is activated, thrombin is formed from protrombin and attacks fibrinogen, splitting off two fibrinopeptides A (Aα1–16), and thereby exposing fibrin polymerization sites ‘A’ (Aα17–19) [2]. Removal of fibinopeptides A leads to a form of fibrinogen deemed ‘desAA’, which polymerizes spontaneously to form two-molecule thick protofibrils. Removal of fibrinopeptides B (‘desAABB’) encourages lateral associations that lead to fibrils [3,4]. The fibrils continue to associate, branching and forming a 3D network: the framework of the blood thrombus [5]. It is widely accepted that the initial step of fibrin polymerization (protofibril formation) is carried out by the intermolecular pairing of ‘A’ and ‘a’ polymerization sites of fibrin monomers. Site ‘a’ is a cavity (‘hole;) that includes amino acid residues γGln329, γAsp330, γHis340 and γAsp364, and is situated in the γC-domain of the fibrinogen/fibrin molecule [6].

Recently, calixarenes, comprising nanosize cup-shaped compounds, have aroused keen interest as a result of their various effects on biochemical processes [7,8]. They are widely used as the template for artificial receptors designed for the recognition and binding of bioactive compounds: biometals, amino acids, dipeptides, proteins, etc. [9,10]. Because they are capable of forming host–guest supramolecular complexes with biologically important molecules, calixarenes have the potential to influence a variety of biochemical processes and can serve as molecular platforms for drug design [11–13].

Antithrombotic properties of calixarenes were initially demonstrated in 1995 [14]. Subsequently, Da Silva et al. [15] showed that two para-sulfonato-calix[8]arenes essentially increase an activated partial thromboplastin time (APTT) and thrombin time. Recently, Coleman et al. [16] demonstrated anticoagulant activity for derivatives of two para-octanoylcalix[8]arenes. An attempt was made to elucidate the mechanism of the antithrombotic activity of these calixarene derivatives [15,16]. It was found that 49-mono-(2-carboxymethoxy)-5,11,17,23,29,35,41,47-octa-sulfonato-calix[8]arene (C8SMA) indirectly inhibited thrombin via interaction with heparin cofactor II (as dermatan sulphate) but not via interaction with antithrombin in an heparinoid-like manner. However, the effect of the calixarenes on the final step of blood coagulation (i.e. fibrin polymerization) was not investigated.

The present study aimed to investigate the anticoagulant properties of phosphorus contained calyx[n]arenes in last two steps of blood coagulation: thrombin + fibrinogen reaction and fibrin monomer polymerization. In particular, we have focused on compounds in which the molecular scaffold is decorated with methylene-bis-phosphonic acid groups. One of these, calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), has four such substituent groups. Another compound, dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98), has two such substitutents, as well as internal propyl groups (Fig. 1).

Figure 1.

 Structural formulas of C-192 and C-98.

Results and Discussion

C-192 was studied with respect to its effects on fibrin polymerization in two kinds of assay. In the first assay, the formation of fibrin was followed directly after the addition of thrombin. In the second assay, previously prepared fibrin was dispersed and then allowed to repolymerize under appropriate conditions. In both cases, fibrin formation was gauged by turbidity measurements. Turbidity analysis showed that the compound decreased the maximum rate of fibrin polymerization in the thrombin–fibrinogen reaction by 50% at a molar ratio of compound to starting fibrinogen of 1.7 : 1 (Fig. 2). The final turbidity of clots was decreased by 50% at a molar ratio of 4.3 : 1 (compound : starting fibrinogen) (Fig. 2C). The lag-time was also increased strongly in the presence of C-192, indicating either an decrease of the rate of protofibril formation or, conceivably, an increase of protofibril critical length (Fig. 2B). Similar results were obtained when calixarene C-192 inhibited the re-association of dispersed desAABB fibrin (Fig. 3A–C); in this case, IC50 = 1.26 × 10−6 m.

Figure 2.

 Turbidity analysis of the influence of C-192 on fibrin polymerization in the fibrinogen + thrombin reaction. The dependence on calixarene C-192 concentration is shown for (A) the maximum rate of fibrin polymerization Vmax, (B) the lag-time t and (C) the final turbidity of fibrin clots Δh.

Figure 3.

 Turbidity analysis of the influence of C-192 on fibrin desAABB polymerization. The dependence on calixarene C-192 concentration is shown for (A) the maximum rate of fibrin polymerization Vmax, (B) the lag-time t and (C) the final turbidity of fibrin clots Δh.

Such a strong and specific inhibition by calixarene C-192 of both the thrombin–fibrinogen reaction and the re-association of fibrin desAABB indicates that calixarene retards clotting not as a result of the inhibition of thrombin, but entirely because of the blocking of fibrin polymerization sites.

We also performed electron microscopy studies to determine the stage of fibrin polymerization that was inhibited by C-192. Transmission electron microscopy of the thrombin + fibrinogen media showed that fibrin remained in monomer state in the presence of calixarene C-192 at its final concentration of 10−5 m, whereas, at the same time, mature fibrils were formed in the absence of C-192 (Fig. 4).

Figure 4.

 Electron micrographs of fibrinogen + thrombin reaction media in the absence of C-192 (A, B), as well as in its presence (C, D). Scale bar = 100 nm.

The results of turbidity analysis and electron microscopy indicate that the inhibition by C-192 occurs at the first stage of fibrin polymerization, presumably by blocking one of the sites of protofibril formation.

We also investigated the inhibitory effects of two structural fragments of C-192: para-hydroxyphenylmethylene-bis-phosphonic acid (1) and methylene-bis-phosphonic acid (2) (Fig. 5). The results showed that these constituents inhibit fibrin polymerization with considerably less activity: the IC50 value was > 1.0 × 10−4 m for 1 and 0.72 × 10−4 m for 2.

Figure 5.

 Two structural fragments of C-192: para-hydroxyphenylmethylene-bis-phosphonic acid (1) and methylene-bis-phosphonic acid (2).

It is of interest that the inhibitory activity of C-98, which has the two methylene-bis-phosphonic acid substituents, is much less (Table 1) (IC50 = 1.31 × 10−4 m), indicating that the calixarene scaffold and the number of phosphoryl groups in the molecule play a crucial role in the inhibitory effect.

Table 1.   Concentration values of compounds that cause 50% inhibition of the maximum rate of the polymerization of fibrin produced in the fibrinogen + thrombin reaction.
CompoundIC50
C-1921.26 × 10−6 m
C-981.31 × 10−4 m
1,para-hydroxyphenyl-methylene-bis- phosphonic acid> 1.0 × 10−4 m
2,methylenel-bis-phosphonic acid0.72 × 10−4 m

Furthermore, calixarene C-192 doubles both the prothrombin time (PT) and APTT in normal human blood plasma at concentrations of 7.13 × 10−5 m and 1.10 × 10−5 m, respectively (Fig. 6). The molar ratios of C-192 to plasma fibrinogen were approximately 21 : 1 and 3.3 : 1 for the PT and APTT assays, respectively. The delays in clotting times in the blood plasma coagulation experiments are what would be expected by the inhibition of the fibrin polymerization from fibrinogen after the activation of the blood coagulation system.

Figure 6.

 The dependence of the PT and APTT ratios on the calixarene C-192 concentration.

Electron microscopy confirmed that C-192 inhibits the first stage of fibrin polymerization (i.e. the formation of protofibrils). Because this stage is fulfilled through the intermolecular binding of the complementary sites ‘A’–’a’, it appeared to be certain that this inhibition is a result of the blocking of site ‘A’ (Aα17-19, GlyProArg) by the calixarene in a ‘knob-hole’ manner. To confirm that this was the case, we employed HPLC to study complex formation between C-192 and the synthetic peptide Gly-Pro-Arg-Pro, a synthetic analogue of the A knob; the free amino acids Gly, Pro and Arg were used as controls. Association constants of calixarene C-192 complexes with amino acids Gly, Pro, Arg and tetrapeptide Gly-Pro-Arg-Pro in methanol–water mobile phase (50 : 50, v/v) were determined as previously described [17,18]. The addition of calixarene C-192 to the mobile phase decreased the capacity factor, k′, of the guest molecules (Table 2). This confirms the formation of inclusion host–guest complexes. There is linear dependence of 1/k′ versus the concentration of C-192 in the mobile phase (Fig. 7); the correlation coefficient is 0.98–0.99, indicating a 1 : 1 ratio of calixarene to Gly-Pro-Arg-Pro in the complex.

Table 2.   Values 1/k′ of the guests and association constants KA (m−1) for their complexes with calixarene C-192. RSD, relative standard deviation.
GuestCalixarene concentrationKA, M−1 (RSD, %)
0.01.482.523.545.00
1/k ′
Gly0.3020.3130.3240.3310.349280 (10)
Pro0.2940.3180.3670.3960.403814 (26)
Arg0.3110.3950.5320.5920.7942576 (21)
Gly-Pro-Arg-Pro1.2871.7542.4533.0153.6933395 (19)
Figure 7.

 Dependence of 1/k ′ for Gly, Pro, Arg and Gly-Pro-Arg-Pro on the C-192 concentration in the mobile phase.

In accordance with the molecular modelling data (Fig. 8A,B), there are two modes of C-192–Gly-Pro-Arg-Pro complexation. In the first mode (A), cooperative electrostatic interactions of two proximal negatively-charged phosphonyl groups with the Gly α-amino terminal group and the Arg guanidinium residue play a principal role in complex formation. Most of the tetrapeptide molecule is exposed outside the calixarene cavity. In the second mode (B), the hydrophobic part of Gly-Pro-Arg-Pro molecule is deeply embedded into the calixarene cavity. The complex is stabilized by P-O…H3N+ electrostatic interactions of the phosphonyl group with the Gly amino acid residue, as well as by CH-π interactions of CH2-group in the Pro residue with the calixarene aromatic ring. Hydrophobic forces can additionally stabilize the complex in a water solution.

Figure 8.

 Two modes of energy minimized structures of calixarene C-192 complexed with GlyProArgPro. (A) Cooperative electrostatic interactions of two proximal negatively charged phosphonyl groups of C-192 molecule with Gly α-amino terminal group and Arg guanidinium residue. (B) The hydrophobic part of GlyProArgPro molecule is embedded into the calixarene cavity.

Thus, we have shown for the first time that C-192 is a powerful and specific inhibitor of the final step of blood coagulation, fibrin polymerization, and can be used as the basis for the design of new class of antithrombotic agents. We found that the mechanism of C-192 inhibition involves its effect on the first step of fibrin polymerization, protofibril formation, which is carried out via intermolecular interactions of complementary polymerization sites ‘A’ and ‘a’ of fibrin molecules.

We have also shown that C-192 forms complex with synthetic peptide Gly-Pro-Arg-Pro, which imitates polymerization site ‘A’ (Aα17 Gly-Pro-Arg), suggesting that the inhibitory effect of C-192 may be a result of the blocking of site ‘A’ by this calixarene.

The results obtained in the present study suggest that the other types of calixarenes noted above [15,16] have the same mechanism of inhibitory action on blood clotting.

Materials and methods

1H and 31P NMR spectra were recorded on a VXP 300 spectrometer (Varian Inc., Palo Alto, CA, USA) operating at 300 MHz and 121.5 MHz, respectively. Chemical shifts are reported using internal tetramethylsilane and external 85% H3PO4 as references. Melting points were determined on a Boetius apparatus and are uncorrected. Bromotrimethylsilane was freshly distilled. All reactions were carried out under dry argon. Tetraformylcalix[4]arene 3 was synthesized as described previously [19].

Methylene-bis-phosphonic acid (2) was purchased from Aldrich (St Louis, MO, USA). Para-hydroxyphenylmethylene-bis-phosphonic acid (1) and tetrapropoxycalixarene bis-methylene-bis-phosphonic acid C-98 were prepared by a method described previously [20]. Calixarene tetrakis-methylene-bis-phosphonic acid C-192 was synthesized by the same method with a sequence of transformations as shown in the scheme outlined in Fig. 9. The reaction of tetraformylcalix[4]arene 3 with a large excess of (iPrO)2PONa in diisopropylphosphite/dioxane solution affords quantitatively calixarene tetrakis-methylene-bis-phosphonate 4. The standard dealkylation of calixarene phosphonate 4 with Me3SiBr and subsequent methanolysis gives quantitatively C-192.

Figure 9.

 Scheme presenting the sequence of transformations during the synthesis of C-192.

5,11,17,23-Tetrakis[bis(diisopropylphosphoryl)methyl]calix[4]arene

Sodium metal (0.69 g, 29 mmol) was carefully added in small portions to diisopropyl phosphite (15 mL) at room temperature. The solution formed was diluted with dioxane (15 mL). Tetraformylcalixarene 3 (1 g, 1.86 mmol) was added to the resulting solution, as appropriate. The reaction mixture was stirred at room temperature for 48 h and then quenched with water (100 mL) and extracted with chloroform (3 × 50 mL). The chloroform layer was concentrated under vacuum and the residue was washed with hexane and dried in vacuum. White powder, 2.9 g, yield 99%, melting point 65–67 °C (from hexane). Found: C, 53.69; H, 7.73; P, 13.65. C81H138O28P8 requires C, 53.82; H, 7.69; P, 13.71%. 1H (300 MHz; CDCl3; Me4Si) δ 7.18 (s, 8H, PhOH); 4.85 (m, 8H, OCH); 4.38 (m, 8H, OCH); 3.95 (wide s, 8H, ArCH2); 3.45 (t, 4H, JPH = 25 Hz, PCH); 1.35, 1.25, 1.18, 0.95 (four d, 30H+30H+18H+18H); 31P NMR δ 19.5; m/z (FAB MS) 1794 ([M + H]+, 100%).

5,11,17,23-Tetrakis [bis(dihydroxyphosphoryl)methyl]calix[4]arene

An eight-fold molar excess of bromotrimethylsilane per phosphonate group (5.46 g, 35 mmol) was added to a solution of tetrakis-bisphosphonate 4 (1 g, 0.55 mmol) in dry chloroform (5 mL). The reaction mixture was stirred at room temperature for 30 h and then was concentrated under reduced pressure. The residue was dissolved in absolute methanol (15 mL), the resulting mixture stirred at 50 °C for 2 h, and then concentrated and dried in vacuum (0.05 mmHg) for 10 h. Light powder, 0.59 g, yield 98%, melting point > 100 °C (decomposition). Found: C, 50.61; H, 5.12; P, 14.52. C32H40O28P8 requires C, 34.30; H, 3.60; P, 22.12%. 1H (300 MHz; DMSO-d6; Me4Si) δ 7.45 (s, 8H, PhOH); 4.25 (d, 4H, JHH = 13 Hz, ArCH2); 3.65 (d, 4H, JHH = 13 Hz, ArCH2); 3.55 (t, 4H, JPH = 25 Hz, PCH); 31P NMR δ 16.5.

Preparation of fibrinogen, fibrin desAABB

Human fibrinogen was prepared by sodium sulphate precipitation from human plasma [21] DesAABB fibrin monomer was prepared as described previously [22].

Turbidity analysis of fibrin polymerization

The effects of compounds on fibrin polymerization were studied spectrophotometrically at 350 nm as described previously [23]. The curve of increasing turbidity during fibrin clotting shows the parameters: τ, lag time, which corresponds to the time of protofibril formation; Vmax, maximum rate of fibrin polymerization, which was defined by graphic calculation of the angle of the tangent to the turbidity increase curve at the point of maximum steepness; and Δh, final turbidity of fibrin clots. The polymerization of desAABB fibrin was studied at its final concentration 0.1 mg·mL−1 in the polymerization medium containing 0.05 m ammonium acetate (pH 7.4) with 0.1 m NaCl and 1 × 10−4 m CaCl2. The polymerization of fibrin formed in the fibrinogen + thrombin reaction was investigated at a final concentration of fibrinogen of 0.1 mg·mL−1 and thrombin of 0.4 NIH units·mL−1 in the same polymerization medium.

Electron microscopy

The samples of polymerizing fibrin produced in the thrombin–fibrinogen reaction in the absence or the presence of calixarene C-192 (10−5 m) were taken out of the reaction medium at various times, placed on a carbon-coated grid for 2 min and then stained with 1% (w/v) uranyl acetate for 1 min. Transmission electron microscopy was performed with an H-600 electron microscope (Hitachi, Tokyo, Japan) operated at 75 kV. Electron micrographs were obtained at ×50 000 magnification.

The determination of association constants by the RP-HPLC method

The HPLC consisted of a high-pressure pump (type HPP 4001) (Laboratorni Pristroje, Prague, Czech Republic) connected to a Rheodyne sample 7120 injector (Rheodyne, Berkeley, CA, USA) and an ultraviolet-visible detector (type LCD 2563) (Laboratorni Pristroje). The column (150 × 3.3 mm inner diameter) was packed with Separon SGX CN (Lachema, Prague, Czech Republic). The mobile phase was a mixture of methanol–water in the ratio 50 : 50 (v/v), with the calixarene C-192 additive at a concentration in the range 1.48 × 10−4 to 5 × 10−4 m. The flow rate was 0.6 mL·min−1. The concentration of the guests/analytes in solution for analysis was 10−5 m. The solvent was identical to the mobile phase composition. The amount of the sample injected was 0.5 μL. Each of the samples was analyzed five times. All chromatograms were obtained at 20 °C.

Association constants of the calixarene complexes with amino acids Gly, Pro, Arg and tetrapeptyde Gly-Pro-Arg-Pro (280–3395 m−1) were calculated from the dependence of the 1/k′ value versus the calixarene concentration [CA] in the mobile phase by Eqn (1) (Table 1):

image(1)

where k0′ and k′ are the capacity factors determined in the absence and presence of the calixarene in the mobile phase and [CA] is the calixarene concentration in the mobile phase.

PT and APTT assays

The effects of calixarene C-192 on the coagulation of human blood plasma were studied using a coagulometer (CT 2410 ‘Solar’, Minsk, Belarus). Reagents from Renam (Moscow, Russia) were used to calculate PT and APTT. PT and APTT ratios were calculated using the formula: tc/to, where to is the time of clot formation in blood plasma without calixarene C-192 and tc is the time of clot formation in blood plasma with calixarene C-192.

Acknowledgements

We are grateful to Professor Russell Doolittle (Center for Molecular Genetics, University of California, San Diego, CA, USA) for useful discussion of the results obtained.

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