Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization Eduard V. Lugovskoy 1 , Pavel G. Gritsenko 1 , Tatyana A. Koshel 1 , Ievgen O. Koliesnik 1 , Serhey O. Cherenok 2 , Olga I. Kalchenko 2 , Vitaliy I. Kalchenko 2 and Serhey V. Komisarenko 1 1 Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, Kyiv, Ukraine 2 Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine Introduction Fibrinogen is a glycoprotein (MW $344 kDa) com- posed of two monomeric units connected by disulfide bonds. Each monomer consists of three non-identical polypeptide chains Aa,Bb and c, also connected by disulfide bridges [1]. The NH 2 -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 (Aa1– 16), and thereby exposing fibrin polymerization sites ‘A’ (Aa17–19) [2]. Removal of fibinopeptides A leads to a form of fibrinogen deemed ‘desAA’, which poly- merizes spontaneously to form two-molecule thick protofibrils. Removal of fibrinopeptides B (‘desA- Keywords calix[4]arene methylenebisphosphonic acid; fibrin; fibrinogen; inhibition; polymerization Correspondence 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 (Received 30 September 2010, revised 23 December 2010, accepted 2 February 2011) doi:10.1111/j.1742-4658.2011.08045.x 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]ar- ene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maxi- mum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 · 10 )6 M (IC 50 ). At this con- centration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC 50 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 poly- merization with an IC 50 = 1.31 · 10 )4 M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site ‘A’ (Aa17– 19), which ordinarily initiates protofibril formation in a ‘knob-hole’ man- ner. 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. Calix- arene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentra- tions 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 anti- thrombotic 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. 1244 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS ABB’) 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 ini- tial step of fibrin polymerization (protofibril forma- tion) 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 res- idues cGln329, cAsp330, cHis340 and cAsp364, and is situated in the cC-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, dipep- tides, 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 pro- cesses and can serve as molecular platforms for drug design [11–13]. Antithrombotic properties of calixarenes were ini- tially 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 anticoagu- lant activity for derivatives of two para-octanoylca- lix[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 anticoag- ulant properties of phosphorus contained calyx[n]ar- enes 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 deco- rated with methylene-bis-phosphonic acid groups. One of these, calix[4]arene tetrakis-methylene-bis-phosphon- ic acid (C-192), has four such substituent groups. Another compound, dipropoxycalix[4]arene bis-methy- lene-bis-phosphonic acid (C-98), has two such substitu- tents, as well as internal propyl groups (Fig. 1). 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 mea- surements. Turbidity analysis showed that the com- pound decreased the maximum rate of fibrin polymerization in the thrombin–fibrinogen reaction by 50% at a molar ratio of compound to starting fibrino- gen of 1.7 : 1 (Fig. 2). The final turbidity of clots was decreased by 50% at a molar ratio of 4.3 : 1 (com- pound : 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, IC 50 = 1.26 · 10 )6 m. 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 cal- ixarene 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 calixa- rene 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). Fig. 1. Structural formulas of C-192 and C-98. E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1245 Fig. 2. Turbidity analysis of the influence of C-192 on fibrin polymerization in the fibrinogen + thrombin reaction. The dependence on calixar- ene 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 Dh. Fig. 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 Dh. A 30 s B 85 s D 200 s C 85 s Fig. 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. Calix[4]arene methylenebisphosphonic acids E. V. Lugovskoy et al. 1246 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS The results of turbidity analysis and electron micros- copy 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-hydroxyphenylm- ethylene-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 IC 50 value was > 1.0 · 10 )4 m for 1 and 0.72 · 10 )4 m for 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) (IC 50 = 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. 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, respec- tively. 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. Electron microscopy confirmed that C-192 inhibits the first stage of fibrin polymerization (i.e. the forma- tion of protofibrils). Because this stage is fulfilled through the intermolecular binding of the complemen- tary sites ‘A’–’a’, it appeared to be certain that this inhibition is a result of the blocking of site ‘A’ (Aa17-19, 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 addi- tion 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. Fig. 5. Two structural fragments of C-192: para-hydroxyphenyl- methylene-bis-phosphonic acid (1) and methylene-bis-phosphonic acid (2). Table 1. Concentration values of compounds that cause 50% inhi- bition of the maximum rate of the polymerization of fibrin produced in the fibrinogen + thrombin reaction. Compound IC 50 C-192 1.26 · 10 )6 M C-98 1.31 · 10 )4 M 1,para-hydroxyphenyl-methylene-bis- phosphonic acid > 1.0 · 10 )4 M 2,methylenel-bis-phosphonic acid 0.72 · 10 )4 M Fig. 6. The dependence of the PT and APTT ratios on the calixa- rene C-192 concentration. Table 2. Values 1 ⁄ k¢ of the guests and association constants K A (M )1 ) for their complexes with calixarene C-192. RSD, relative stan- dard deviation. Guest Calixarene concentration K A ,M )1 (RSD, %) 0.0 1.48 2.52 3.54 5.00 1 ⁄ k ¢ Gly 0.302 0.313 0.324 0.331 0.349 280 (10) Pro 0.294 0.318 0.367 0.396 0.403 814 (26) Arg 0.311 0.395 0.532 0.592 0.794 2576 (21) Gly-Pro-Arg-Pro 1.287 1.754 2.453 3.015 3.693 3395 (19) E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1247 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 a-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 dee- ply embedded into the calixarene cavity. The complex is stabilized by P-O ) H 3 N + electrostatic interactions of the phosphonyl group with the Gly amino acid resi- due, as well as by CH-p interactions of CH 2 -group in the Pro residue with the calixarene aromatic ring. Hydrophobic forces can additionally stabilize the com- plex in a water solution. 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 anti- thrombotic 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 comple- mentary polymerization sites ‘A’ and ‘a’ of fibrin mole- cules. We have also shown that C-192 forms complex with synthetic peptide Gly-Pro-Arg-Pro, which imitates polymerization site ‘A’ (Aa17 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 1 H and 31 P 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% H 3 PO 4 as references. Melting points were determined on a Boetius apparatus and are uncorrected. Bromotrimeth- ylsilane was freshly distilled. All reactions were carried out under dry argon. Tetraformylcalix[4]arene 3 was synthe- sized as described previously [19]. Methylene-bis-phosphonic acid (2) was purchased from Aldrich (St Louis, MO, USA). Para-hydroxyphenylmethyl- ene-bis-phosphonic acid (1) and tetrapropoxycalixarene bis- methylene-bis-phosphonic acid C-98 were prepared by a method described previously [20]. Calixarene tetrakis-meth- ylene-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 tetra- formylcalix[4]arene 3 with a large excess of (iPrO) 2 PONa in diisopropylphosphite ⁄ dioxane solution affords quantita- tively calixarene tetrakis-methylene-bis-phosphonate 4. The standard dealkylation of calixarene phosphonate 4 with Fig. 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 a-amino terminal group and Arg guanidinium residue. (B) The hydrophobic part of GlyProArgPro molecule is embedded into the calixarene cavity. Fig. 7. Dependence of 1 ⁄ k ¢ for Gly, Pro, Arg and Gly-Pro-Arg-Pro on the C-192 concentration in the mobile phase. Calix[4]arene methylenebisphosphonic acids E. V. Lugovskoy et al. 1248 FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS Me 3 SiBr and subsequent methanolysis gives quantitatively 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 reac- tion 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 concen- trated under vacuum and the residue was washed with hex- ane 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. C 81 H 138 O 2 8P 8 requires C, 53.82; H, 7.69; P, 13.71%. 1 H (300 MHz; CDCl 3 ;Me 4 Si) d 7.18 (s, 8H, PhOH); 4.85 (m, 8H, OCH); 4.38 (m, 8H, OCH); 3.95 (wide s, 8H, ArCH 2 ); 3.45 (t, 4H, J PH = 25 Hz, PCH); 1.35, 1.25, 1.18, 0.95 (four d, 30H+30H+18H+18H); 31 P NMR d 19.5; m ⁄ z (FAB MS) 1794 ([M + H] + , 100%). 5,11,17,23-Tetrakis [bis(dihydroxyphospho- ryl)methyl]calix[4]arene An eight-fold molar excess of bromotrimethylsilane per phosphonate group (5.46 g, 35 mmol) was added to a solu- tion 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 abso- lute 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. C 32 H 40 O 28 P 8 requires C, 34.30; H, 3.60; P, 22.12%. 1 H (300 MHz; DMSO-d 6 ;Me 4 Si) d 7.45 (s, 8H, PhOH); 4.25 (d, 4H, J HH = 13 Hz, ArCH 2 ); 3.65 (d, 4H, J HH = 13 Hz, ArCH 2 ); 3.55 (t, 4H, J PH = 25 Hz, PCH); 31 P NMR d 16.5. Preparation of fibrinogen, fibrin desAABB Human fibrinogen was prepared by sodium sulphate precip- itation 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 pre- viously [23]. The curve of increasing turbidity during fibrin clotting shows the parameters: s, lag time, which corre- sponds to the time of protofibril formation; V max , maxi- mum rate of fibrin polymerization, which was defined by graphic calculation of the angle of the tangent to the tur- bidity increase curve at the point of maximum steepness; and Dh, final turbidity of fibrin clots. The polymerization of desAABB fibrin was studied at its final concentration 0.1 mgÆmL )1 in the polymerization medium containing 0.05 m ammonium acetate (pH 7.4) with 0.1 m NaCl and 1 · 10 )4 m CaCl 2 . The polymerization of fibrin formed in the fibrinogen + thrombin reaction was investigated at a final concentration of fibrinogen of 0.1 mgÆmL )1 and thrombin of 0.4 NIH unitsÆmL )1 in the same polymeriza- tion medium. Electron microscopy The samples of polymerizing fibrin produced in the throm- bin–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. Fig. 9. Scheme presenting the sequence of transformations during the synthesis of C-192. E. V. Lugovskoy et al. Calix[4]arene methylenebisphosphonic acids FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1249 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) con- nected 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 mLÆmin )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 sam- ple injected was 0.5 lL. 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): K A ¼ k 0 0 ð1=k 0 À 1=k 0 0 Þ ½CA ð1Þ where k 0 ¢ 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: t c ⁄ t o , where t o is the time of clot formation in blood plasma without calixarene C-192 and t c is the time of clot forma- tion 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. References 1 Blomback B (1996) Fibrinogen and fibrin – proteins with complex roles in haemostasis and thrombosis. Thromb Res 83, 1–75. 2 Laudano AP & Doolittle RF (1978) Synthetic peptide derivatives that bind to fibrinogen and prevent the poly- merization of fibrin monomers. Proc Natl Acad Sci USA 75, 3085–3089. 3 Fowler WE, Hantgan RR, Hermans J & Erickson HP (1981) Structure of the fibrin protofibril. 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Calix[4]arene methylenebisphosphonic acids FEBS Journal 278 (2011) 1244–1251 ª 2011 The Authors Journal compilation ª 2011 FEBS 1251 . ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC 50 was 1.26 · 10 )6 M at a molar ratio of C-192 to fibrin. 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,