Preservation of in vitro function of platelets stored in the presence of a synthetic dual inhibitor of factor Xa and thrombin.

BACKGROUND: The use of citrate anticoagulant limits the clinical significance of platelet function tests. Thrombin inhibitors cannot prevent thrombin-induced platelet activation completely. We examined the influence of benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA), a dual inhibitor of Factor Xa (FXa) and thrombin, on platelet responsiveness to agonists when measured between 2 and 24 h after venipuncture. METHODS: Blood samples from 36 individuals were anticoagulated with citrate and BAPA, respectively. Turbidimetric platelet aggregometry (TPA) and impedance platelet aggregometry (IPA), a whole blood platelet counting assay for measuring platelet aggregation (PCA), and Platelet Function Anlayzer-100 (PFA-100 closure times (CTs) were determined after whole blood storage between 2 and 24 h after venipuncture. Native whole blood was studied over 48 h to determine the inhibition of thrombin generation by BAPA, hirudin and melagatran. RESULTS: BAPA inhibited thrombin generation completely for 48 h, while hirudin and melagatran did not. The use of citrate resulted in significantly reduced TPA induced by arachidonic acid (AA) or adenosine 5'-diphosphate (ADP), and significantly reduced IPA regardless of agonist when measured 10 and 24 h after blood collection. PCA ratios in citrated blood also dropped significantly 10 and 24 h after venipuncture. The length of storage of BAPA-anticoagulated blood samples over 24 h had no significant influence on any platelet response. The reproducibility of platelet function assay results obtained from BAPA-anticoagulated samples was significantly better than corresponding data from citrated blood. CONCLUSION: TPA, IPA, PCA or PFA-100 CTs remain stable for 24 h when whole blood is anticoagulated with a dual inhibitor of FXa and thrombin. This would greatly simplify the shipment of samples for platelet function testing.

Trypsinogen activation in rat pancreatic acinar cells hyperstimulated by caerulein.

Inappropriate trypsinogen activation is discussed as an early intracellular event in the secretagogue-induced model of acute pancreatitis. However, the mechanisms by which trypsinogen is activated are not well characterized. In the present work, trypsinogen activation was studied in intact acinar cells using bis-(CBZ-arginyl)-Rhodamine 110 [(CBZ-Arg)2-Rho 110] as a cell-permeant substrate for trypsin and also independently via the formation of trypsinogen activation peptide (TAP). Preincubation with 10 nM caerulein increased the Rho 110-substrate cleavage more than threefold. This proteolytic activity was fully sensitive to a benzamidine (BA)-type serine protease inhibitor. The appearance of enzymatic activity was paralleled by the formation of TAP. The lack of effect of the high-molecular soybean trypsin inhibitor indicates an intracellular substrate cleavage. The cathepsin B inhibitor CA-074 prevented neither the caerulein-induced formation of TAP nor the (CBZ-Arg)2-Rho 110-cleaving activity. BA inhibited the Rho 110-substrate cleavage and significantly reduced the TAP formation. These results show that trypsinogen activation in caerulein-hyperstimulated acinar cells may occur independently of the activity of cathepsin B. On the contrary, the effect of BA suggests the role of a serine protease in trypsinogen activation.

Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition.

The binding of a series of low molecular weight ligands towards trypsin and thrombin has been studied by isothermal titration calorimetry and protein crystallography. In a series of congeneric ligands, surprising changes of protonation states occur and are overlaid on the binding process. They result from induced pK(a) shifts depending on the local environment experienced by the ligand and protein functional groups in the complex (induced dielectric fit). They involve additional heat effects that must be corrected before any conclusion on the binding enthalpy (DeltaH) and entropy (DeltaS) can be drawn. After correction, trends in both contributions can be interpreted in structural terms with respect to the hydrogen bond inventory or residual ligand motions. For all inhibitors studied, a strong negative heat capacity change (DeltaC(p)) is detected, thus binding becomes more exothermic and entropically less favourable with increasing temperature. Due to a mutual compensation, Gibbs free energy remains virtually unchanged. The strong negative DeltaC(p) value cannot solely be explained by the removal of hydrophobic surface portions of the protein or ligand from water exposure. Additional contributions must be considered, presumably arising from modulations of the local water structure, changes in vibrational modes or other ordering parameters. For thrombin, smaller negative DeltaC(p) values are observed for ligand binding in the presence of sodium ions compared to the other alkali ions, probably due to stabilising effects on the protein or changes in the bound water structure.

Crystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds.

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.

Geometry of binding of the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino phenylalanine to thrombin and trypsin. X-ray crystal structures of their trypsin complexes and modeling of their thrombin complexes.

The X-ray crystal structures of the complexes formed with bovine trypsin and the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino-D,L-phenylalanine (3-TAPAP, 4-TAPAP and 4-TGPAP) were determined with data to 1.8 A resolution. The L-stereoisomer of 3-TAPAP binds as a compact entity into the active site of trypsin, with the amidino and the carbonyl groups of the central amidinophenylalanyl residue hydrogen-bonded to Gly216 of trypsin. According to modeling and energy minimization, 3-TAPAP fits perfectly in this conformation to the more restrictive thrombin active site also (Bajusz et al. (1978) Int. J. Pept. Prot. Res. 12, 217-221); the piperidine moiety extends into the cage-like S2 subsite of thrombin, but leaves room for additional substituents which might help to improve binding and pharmacological properties. In contrast, 4-TAPAP and 4-TGPAP bind only weakly and in an extended conformation to trypsin; their considerably enhanced affinities for thrombin would suggest a more compact binding to thrombin.

Dramatic enhancement of the catalytic activity of coagulation factor IXa by alcohols.

The coagulation factor IXa (FIXa) exhibits a very weak proteolytic activity towards natural or synthetic substrates. Upon complex formation with its cofactor FVIIIa and Ca2+-mediated binding to phospholipid membranes, FIXa becomes a very potent activator of FX. The presence of FVIIIa has no effect on the cleavage of peptide substrates by FIXa, however. We found that several alcohols dramatically enhance the catalytic activity of human FIXa towards synthetic substrates. Substrates with the tripeptidyl moiety R-D-Xxx-Gly-Arg are especially susceptible to the enhanced FIXa catalysis. Maximal increase up to 20-fold has been measured in the presence of ethylene glycol. We suggest that alcohols modify the conformation of FIXa rendering the active-site cleft more easily accessible to tripeptide substrates with a hydrophobic residue in the P3-position.

Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine.

Thrombin is the key enzyme in the blood coagulation system, and inhibitors of its proteolytic activity are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-[(2-naphthylsulfonyl)glycyl]-4-amidinophenylalanylpiperid ide (NAPAP). However, NAPAP and other benzamidine derivatives do not show favorable pharmacological properties; above all, they have very low systemic bioavailability after oral administration. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties. Piperazide derivatives of 3-amidinophenylalanine as the key building block were synthesized. The piperazine moiety opened the possibility to introduce quite different substituents on the second nitrogen using common synthetic procedures. Some of the newly synthesized compounds are potent inhibitors of thrombin and offer an approach to study structure-function relationships for inhibition of thrombin and related enzymes and for the improvement of their pharmacokinetic properties.

Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase.

The trypsin-like serine proteinase superfamily contains a number of potential therapeutic targets, many of which are unsuitable for routine X-ray crystallographic studies. We have cocrystallized a selection of benzamidine-based inhibitors with bovine trypsin and solved their structures to a resolution of up to 1.7 A. Despite similar chemical formulas, the inhibitors exhibit a range of diverse binding modes that reflect their inhibitory spectra against the serine proteinases trypsin, thrombin, factor Xa, tissue-type plasminogen activator (tPA) and urokinase (uPA). In contrast to the compact folded conformations of thrombin inhibitors which allow optimal binding in the well-defined hydrophobic S2/S4 pocket of thrombin, those effective against factor Xa exhibit an extended conformation that allows occupation of the S3/S4 region, where hydrophobic and electrostatic interactions can stabilize the conformation. One group of inhibitors containing an N-terminal 2,4, 6-triisopropylphenylsulfonyl (TIPPS) moiety show little or no penetration into the S3/S4 subsites of trypsin. These latter sites are occluded in uPA, explaining why this class of compounds is effective against uPA. Despite presenting an extensive hydrophobic surface toward the solvent, the Ki values for TIPPS-containing compounds against trypsin is in the range 10(-7) to 10(-8) M. Comparison of the binding of a bis-benzamidine inhibitor in trypsin and tPA indicate that a shift in potency can be induced by relatively minor changes in binding mode. Implications for the inhibition of these proteinases are discussed.

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

The ability of thrombin inhibitors to reduce the thrombin activity generated in plasma on extrinsic and intrinsic activation.

In a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action of influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced. By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki < or = 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1-0.6 microM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.

Inhibition of thrombin generation in plasma by inhibitors of factor Xa.

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Trypsin- and SLIGRL-induced vascular relaxation and the inhibition by benzamidine derivatives.

Serine proteinases are involved in several physiological processes and elicit profound cellular effects in a variety of tissues. Besides the thrombin receptor a second receptor, activated by trypsin, the proteinase-activated receptor 2 (PAR-2), was cloned and characterized. Both enzymes generate a new extracellular N-terminus by limited proteolytic cleavage which functions as tethered ligand to activate the receptor. Synthetic peptides corresponding to the sequences of the newly generated N-terminus are able to mimic the effects of the enzymes. In porcine pulmonary arteries trypsin and the receptor-derived peptide SLIGRL elicited an endothelium-dependent transient relaxation of PGF2alpha-precontracted vessels. The EC50 values for trypsin and SLIGRL amounted to 1.1 +/- 0.2 nM and 5.4 +/- 0.6 microM, respectively. Trypsin and SLIGRL caused a homologous desensitization but thrombin and the thrombin receptor-activating peptide SFLLRN were still able to elicit pronounced relaxant effects. The trypsin- and SLIGRL-induced relaxant responses were markedly diminished after blockade of the nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (200 microM) and were absent in endothelium-denuded vessels. Indomethacin and hirudin did not influence the relaxant effects. The effect of trypsin but not that of SLIGRL was blocked by the proteinase inhibitor aprotinin suggesting that only proteolytically active trypsin activates the receptor. Benzamidine derivatives of the 3-amidinophenylalanine type with different affinity for trypsin and thrombin inhibited the vascular effects of trypsin (IC50 0.007-0.7 microM) correlating with its antitrypsin activity. The data suggest that the vascular effects of trypsin and SLIGRL are mediated through activation of PAR-2 which differs from the thrombin receptor.

Comparison of the anticoagulant and antithrombotic effects of synthetic thrombin and factor Xa inhibitors.

The anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors. In vivo, in a venous stasis thrombosis model and a thrombo-plastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar Ki value for the respective enzyme were not effective at equimolar dosage. The results are discussed in the light of the different prerequisites and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Pharmacokinetics and anticoagulant effect of hirudin in man.

The pharmacokinetics and the effects on the haemostatic system of hirudin were assessed in six healthy subjects after single intravenous or subcutaneous dose (1000 AT-U/kg). When hirudin was given intravenously first-order elimination kinetics followed the initial distribution phase. The decline in plasma hirudin concentration was most adequately expressed by a biexponential equation describing a two compartment model. A mean elimination half-life of 0.84 hr and a mean volume of distribution of 12.9 l were calculated. After subcutaneous injection a low hirudin level (approximately 0.5 AT-U/ml) was maintained for a prolonged period of time. In the 24 hour-urine up to 50 per cent of the administered amount of hirudin was excreted in active form. Thrombin time, partial thromboplastin time and prothrombin time measured in plasma samples ex vivo were prolonged dependent on the hirudin plasma level. Platelet counts, fibrinogen level and the fibrinolytic system were unchanged. Bleeding time was prolonged twice at maximum. Subcutaneous or intravenous administration of pure hirudin was tolerated without side-effects.

Retardation of preovulatory desensitization to negative oestrogen feedback: mechanism of the effect of progesterone in 5-day cyclic rats.

Recent studies have shown that oestrogen can induce desensitization to its own gonadotrophin-inhibiting effect in female rats by an action on the medial preoptic area (MPOA). Probably as a consequence of this action, sensitivity to the negative oestrogen feedback declines markedly between metoestrus and dioestrus of the 4-day ovarian cycle. To study this desensitization process in 5-day cyclic rats, females exhibiting regular 5-day vaginal cyclicity were ovariectomized on consecutive days of the cycle, injected with oestradiol benzoate (OB) or oil on the day of ovariectomy and autopsied 24 h after the injection. Estimation of the serum concentration of LH revealed that desensitization to negative oestrogen feedback occurred only between day 2 of dioestrus and pro-oestrus, i.e. 2 days later than in females with a 4-day cycle. In the latter animals, an injection of progesterone in metoestrus or early dioestrus, which induced lengthening of the ovarian cycle for 1 day, delayed the onset of desensitization to a degree similar to that found in spontaneously 5-day cyclic rats. In acutely ovariectomized females, progesterone implants placed in the MPOA, but not those located in the mediobasal hypothalamus, increased the LH-inhibiting effect of low doses of OB. The results suggest that the prolonged secretion of progesterone recorded in 5-day cyclic rats retards follicle maturation and delays the forthcoming ovulation by acting, at least partly, on the MPOA and antagonizing the desensitizing effect of oestrogen. In this way, inhibition of gonadotrophin secretion by oestrogen is enhanced and the increase in tonic LH secretion necessary for the completion of follicle maturation is retarded.

Advances in the development of thrombin inhibitors.

Thromboembolic diseases are a major cause of morbidity and mortality, particularly in the Western world, which has stimulated enormous research efforts by the pharmaceutical industry to introduce new antithrombotic therapies. One strategy is the development of direct inhibitors of the serine protease thrombin, which holds a central position in the final steps of the blood coagulation cascade and in platelet activation. At present there is only limited clinical use of some parenteral preparations of thrombin inhibitors in acute situations, especially when the common antithrombotic drugs heparin, warfarin and aspirin are ineffective or associated with side effects. However, for use in prophylaxis of thrombotic diseases such inhibitors should be orally available, must be safe to avoid bleeding complications and should have an appropriate half-life, allowing once or twice daily dosing to maintain adequate antithrombotically effective blood levels. Details of several new and potent thrombin inhibitors have been published during the last years. For some of them oral bioavailability is claimed and they are effective in in vitro coagulation assays. However, most of them showed only limited efficacy in animal studies with respect to the doses administered. For that reason, effort is concentrated on the evaluation and optimisation of the overall physicochemical characteristics of the inhibitors in order to improve the pharmacokinetics and, thus, the development of promising drug candidates. Nevertheless, only careful clinical studies can give clear answers about the true therapeutical benefit of new developments in this field. This review summarises the current status of direct thrombin inhibitors which are already in clinical use and clinical development and gives an overview on recently published and promising new compounds.

Inhibition of tumour cell invasion by protease inhibitors: correlation with the protease profile.

The invasive potential of eight established human tumour cell lines of different origin has been studied in the Matrigel assay. Between 25% and 70% of the cells migrated through the Matrigel layer within 24 h, indicating that invasiveness varies with the cell type. Semiquantitative measurements of the proteases MMP-2 and MMP-9, and cathepsins B and L were performed in these cell lines and the cell culture media. High invasive potential was found in those cell lines expressing high levels of cathepsins B and L or matrix metal proteases (MMP), either alone or in combination. Overexpression of one of these enzymes is enough to explain a high invasive potential of a cell line. Selective protease inhibitors at 10 nM concentration in the culture medium were used to inhibit the migration of tumour cells in the Matrigel assay. The MMP inhibitor Batimastat reduced the invasive potential of all cell lines studied independently of the MMP expression. The effect of cysteine protease inhibitors was strongly correlated with the protease profile of the tumour cell line. Our findings support the hypothesis of a very complex activation cascade of matrix-degrading proteolytic enzymes and they underline the need to analyse the protease profile of any tumour before beginning an antiproteolytic tumour treatment.