The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.     

Anophelin: kinetics and mechanism of thrombin inhibition

Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an alpha-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of alpha-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of alpha-thrombin, displaying a competitive type of inhibition. The binding of anophelin to alpha-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 +/- 1.46 pM, a calculated association rate constant (k(1)) of 2.11 +/- 0.06 x 10(8) M(-1) s(-1), and a dissociation rate constant (k(-1)) of 4.05 +/- 0.97 x 10(-4) s(-1). In the presence of 0.15 and 0.4 M NaCl, a 17.6- and 207-fold increase in the K(i) of anophelin-alpha-thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-alpha-thrombin complex formation. Incubation of alpha-thrombin with C-terminal hirudin fragment 54-65 that binds to alpha-thrombin anion binding exosite 1 (TABE1) attenuates alpha-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin-mediated proteolysis of alpha-thrombin. Using gamma-thrombin, an alpha-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of gamma-thrombin hydrolysis of small chromogenic substrate (K(i) = 0. 694 +/- 0.063 nM). In addition, anophelin-gamma-thrombin complex formation is prevented by treatment of the enzyme with D-Phe-Pro-Arg-chloromethyl ketone (PPACK), a reagent that irreversibly blocks the catalytic site of thrombin. It is concluded that anophelin is a potent dual inhibitor of alpha-thrombin because it binds both to TABE1 and to the catalytic site, optimal binding being dependent on the availability of both domains. Finally, anophelin inhibits clot-bound alpha-thrombin with an IC(50) of 45 nM and increases the lag phase that precedes explosive in vitro alpha-thrombin generation after activation of intrinsic pathway of blood coagulation. Because of its unique primary sequence, anophelin may be used as a novel reagent to study the structure and function of alpha-thrombin.     

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.     

A kinetic model for the alpha-thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III

In this study we report a kinetic model for the alpha-thrombin-catalyzed production of fibrin I and fibrin II at pH 7.4, 37 degrees C, gamma/2 0.17. The fibrin is produced by the action of human alpha-thrombin on plasma levels of human fibrinogen in the presence of the major inhibitor of alpha-thrombin in plasma, antithrombin III (AT). This model quantitatively accounts for the time dependence of alpha-thrombin-catalyzed release of fibrinopeptides A and B concurrent with the inactivation of alpha-thrombin by AT and delineates the concerted interactions of alpha-thrombin, fibrin(ogen), and AT during the production of a fibrin clot. The model also provides a method for estimating the concentration of alpha-thrombin required to produce a clot of known composition and predicts a direct relationship between the plasma concentration of fibrinogen and the amount of fibrin produced by a bolus of alpha-thrombin. The predicted relationship between the concentration of fibrinogen and the amount of fibrin produced in plasma provides a plausible explanation for the observed linkage between plasma concentrations of fibrinogen and the risk for ischemic heart disease.     

Thrombin active site inhibitors: chemical synthesis, in vitro and in vivo pharmacological profile of a novel and selective agent BMS-189090 and analogues.

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure- activity relationships (SAR), selectivity and activity in vivo. BMS-189090 (5) is identified as a potent, selective, and reversible inhibitor of human alpha-thrombin that is efficacious in vivo in a mice lethality model, and in inhibiting both arterial and venous thrombosis in a rat model.     

Reformable intramolecular cross-linking of the N-terminal domain of heparin cofactor II: effects on enzyme inhibition.

The crystal structure of a heparin cofactor II (HCII)-thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of alpha-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease of alpha-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards alpha-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and alpha-thrombin.     

Alpha-thrombin-catalyzed hydrolysis of fibrin I. Alternative binding modes and the accessibility of the active site in fibrin I-bound alpha-thrombin

Steady-state kinetic parameters were determined for the action of human alpha-thrombin on human fibrin I polymer, an intermediate in the alpha-thrombin-catalyzed conversion of fibrinogen to the fibrin matrix of blood clots during the terminal phase of the blood clotting cascade. Values of 49 s-1 and 7.5 microM were determined (at 37 degrees C, pH 7.4, gamma/2 0.17) for kcat and Km, respectively. Studies of the effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of the fluorogenic substrate N-p-Tos-Gly-L-Pro-L-Arg-7-amido-4-methylcoumarin (tos-GPR-amc) and the effect of fibrin I on the reaction of alpha-thrombin with antithrombin III (AT) were presented which indicate that the active site of alpha-thrombin is accessible while it is bound to its substrate fibrin I. Fibrin I inhibited alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc in a manner inconsistent with the pure competitive inhibition expected for an alternative substrate, whereas fibrinogen, an alpha-thrombin substrate, behaved as a pure competitive inhibitor of the alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc. The effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc was shown to be consistent with alpha-thrombin binding to fibrin I in alternative orientations. In one orientation both the active site and a site distinct from the active site (an exosite) of alpha-thrombin are occupied by fibrin I. In the other orientation only the exosite of alpha-thrombin is occupied and the active site is freely accessible to other substrates. The values of both kcat (21 s-1) and Km (less than 0.23 microM) determined for fibrin I-bound alpha-thrombin acting on tos-GPR-amc were decreased relative to the values of kcat (180 s-1) and Km (7.3 microM) observed for the action of uncomplexed alpha-thrombin on tos-GPR-amc. This observation suggests that the active site of alpha-thrombin is altered in fibrin I-bound alpha-thrombin. Studies of the effect of fibrin I on the reaction of AT with alpha-thrombin (at 37 degrees C, pH 7.4, gamma/2 0.17) indicated that when alpha-thrombin is bound to fibrin I in an orientation where the active site of alpha-thrombin is accessible, AT reacts with alpha-thrombin with a rate constant (greater than 4.2 x 10(4) M-1 s-1) that is greater than the rate constant (1.5 x 10(4) M-1 s-1) for reaction of AT with the free enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.     

Novel morpholinone-based D-Phe-Pro-Arg mimics as potential thrombin inhibitors: design, synthesis, and X-ray crystal structure of an enzyme inhibitor complex

A morpholinone structural motif derived from D(+)- and L(-)-malic acid has been used as a mimic of D-Phe-Pro in the thrombin inhibiting tripeptide D-Phe-Pro-Arg. In place of Arg the more rigid P1 truncated p-amidinobenzylamine (Pab) or 2-amino-5-aminomethyl-3-methyl-pyridine have been utilized. The synthetic strategy developed readily delivers these novel thrombin inhibitors used to probe the alpha-thrombin inhibitor binding site. The best candidate in this series of thrombin inhibitors exhibits an in vitro IC(50) of 720 nM. The X-ray crystal structure of this candidate co-crystallized with alpha-thrombin is discussed.     

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.     

Activation of cyclic nucleotide formation in murine neuroblastoma N1E-115 cells by modified human thrombins

The activity of alpha-thrombin and chemically modified derivatives of this enzyme in stimulating cGMP formation in murine neuroblastoma clone N1E-115 cells is reported. The rank order potency of the compounds falls into three classes: 1) alpha-thrombin is the most potent and effective; 2) the catalytically active enzymes gamma-thrombin, trypsin, and nitro-alpha-thrombin are approximately 50-fold less potent than alpha-thrombin; and 3) active site blocked derivatives of alpha-thrombin are 100 to 1000-fold less potent than alpha-thrombin. Native alpha-thrombin consistently produces the most effective response, usually 1.5 to 3-fold greater than any of the other compounds tested. Preincubation of cells with quinacrine, an inhibitor of phospholipase A2, or with the lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid prior to thrombin challenge resulted in a concentration-dependent attenuation of the response. Indomethacin was without effect in modifying the response. These results suggest that thrombin stimulation of neuroblastoma cells involves the release of arachidonic acid and its metabolism by lipoxygenase. These results clearly demonstrate the activity of alpha-thrombin in stimulating a receptor-mediated response in cultured nerve cells. The results are discussed in relation to the interaction of endogenous alpha-thrombin with nerve cells following invasive trauma and to the possible presence of endogenous proteinases with a neurotransmitter-like function.     

Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys47), was found to be a competitive inhibitor of human alpha-thrombin with respect to peptidyl p-nitroanilide substrates. These results contrast with those of Degryse and coworkers that suggest that recombinant hirudin variant-2(Lys47) inhibited thrombin by a noncompetitive mechanism [Degryse et al. (1989) Protein Engng, 2, 459-465]. gamma-Thrombin, which can arise from alpha-thrombin by autolysis, was shown to have an affinity for recombinant hirudin variant-2(Lys47) that was four orders of magnitude lower than that of alpha-thrombin. It was demonstrated that the apparent noncompetitive mechanism observed previously was probably caused by a contamination of the thrombin preparation by gamma-thrombin. Comparison of the inhibition of alpha-thrombin by recombinant hirudins variant-2(Lys47) and variant-1, which differ from one another in eight out of 65 amino acids, indicated that the two variants have essentially the same kinetic parameters.     

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor.

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the "tethered-ligand" thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.     

Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.     

The hirudin-binding site of human alpha-thrombin. Identification of lysyl residues which participate in the combining site of hirudin-thrombin complex

Chemical modification of 6 lysyl residues within the B-chain of alpha-thrombin was decreased on binding of equal molar hirudin, a thrombin-specific inhibitor. They are Lys-B21 (87% protected), Lys-B52 (80%), Lys-B65 (greater than 95%), Lys-B106 (71%), Lys-B107 (71%), and Lys-B154 (80%). The data suggest that these lysyl residues are situated within or in close proximity to the hirudin-binding site of human alpha-thrombin.     

Amphiregulin is a potent mitogen for the vascular smooth muscle cell line,A7r5

The regulation of amphiregulin, an epidermal growth factor (EGF) family member, and its effect on vascular smooth muscle cells (VSMC) were examined. Amphiregulin mRNA was upregulated by amphiregulin itself as well as alpha-thrombin. Amphiregulin caused an approximate 3-fold increase in DNA synthesis. Its effect on growth was compared with those of other mitogens, and was found to be approximately 3.5-, 2.4-, and 1.0-fold greater than those of endothelin-I (ET-I), alpha-thrombin, and platelet-derived growth factor-AB (PDGF-AB), respectively. As evidenced by Western blot analysis, amphiregulin stimulated the phosphorylation of p42/p44-mitogen-activated protein kinase (MAPK), p38-MAPK, c-Jun NH2-terminal protein kinase (JNK), and Akt/protein kinase B (PKB), respectively. By statistical analysis, the amphiregulin-induced growth effect was significantly decreased by the MAP kinase/ extracellular regulated kinase kinase-1 (MEK-1) inhibitor PD98059, p38-MAPK inhibitor SB203580, and phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, respectively, but was not decreased by JNK inhibitor SP600125. These results suggest that amphiregulin is the most potent mitogen of the mitogens tested, and its growth effect is mediated at least in part through the p42/p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB pathways in VSMC.     

Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies

Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human alpha-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC50 = 40 microM), and the thrombin-induced fibrinogen clotting (IC50 = 20 microM). The latter is completely reversed by albumin (30 mg mL(-1)) suggesting that, at therapeutic concentrations, suramin is unable to affect alpha-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing Vmax without changing the Km for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in alpha-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in alpha-thrombin tertiary structure, without affecting the secondary structure content. Interaction with alpha-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to alpha-thrombin inhibition at extra-vascular sites.