[Development and Periodicity of Human Exposures in Suicidal Intention Reported to the Centre Erfurt from 2004-2013]. / Entwicklung und Periodizität humaner Expositionen in suizidaler Absicht im Einzugsbereich des Giftnotrufs Erfurt von 2004­2013.

Aim of the Study: Exposures in suicide attempts are demanding for hospitals and poisons information centres (PICs). Therefore, the time characteristics of their frequency were studied. Methods: A retrospective analysis was undertaken of all human exposures reported to Centre Erfurt from the beginning of 2004 to the end of 2013 according to their frequency in the respective year, season, month, weekday, time of the weekday, circumstances of exposure, age and gender. Results: 59.7% of all exposures (n=137 104) were accidental, 23.4% occurred in suicide attempts and 3.3% in substance abuse. 0.3% of the suicide attempts resulted in death. Their number continuously increased from n=2 422 in 2004 to n=3 458 in 2013, but their relative frequency remained almost constant at 23.4%. Their highest numbers were reached in the spring and summer with maxima in July and August and minima in February and September. During the week, the most suicide attempts were observed between Sunday and Tuesday and the least on Friday. Highest rate of suicide attempts was seen at 10 pm and lowest at 6 am. The median of age was 39 years (first quartile 24 years, third quartile 50 years). The female proportion was almost twice as high as the male. Conclusions: Hospitals the Centre Erfurt is serving should be particularly prepared for exposures in suicide attempts in the spring and summer (especially in July and August), at the beginning of the week and shortly before midnight.

[Human Single Drug Exposures to Non-opioid Analgesics Reported to the Poisons Information Centre Erfurt from 2003 to 2012]. / Humane nichtopioide Analgetika-Monoexpositionen im Einzugsbereich des Giftnotrufes Erfurt von 2003-2012.

AIM OF THE STUDY: Because of their frequency, non-opioid analgesics (NOA) single drug exposures registered by Poisons Information Centre (PIC) Erfurt have been studied over a decade. METHODS: A retrospective analysis of frequencies, circumstances of exposure, symptom severity, and age groups in NOA single drug exposures received by the PIC Erfurt from the beginning of 2003 to the end of 2012 was undertaken. RESULTS: Of all 4749 NOA single drug exposures, the 10 most frequent were caused by paracetamol (n=1 686), ibuprofen (n=1 439), acetylsalicylic acid (n=456), dipyrone (n=274), diclofenac (n=267), flupirtine (n=138), naproxen (n=41), etoricoxib (n=36), indomethacin (n=24), and dexketoprofen (n=19). Paracetamol single drug exposures increased from 158 in 2003 to 216 in 2007 and fell afterwards to 133 in 2012. Ibuprofen single drug exposures continously rose from 57 in 2003 to 258 in 2012. Adults were more often involved in NOA (53.8%) and all single drug exposures (54.1%) than children (45.9% and 45.6%, respectively). Suicidal attempts were more frequent in NOA (43.1%) than in all single drug exposures (34.2%), whereas accidental exposures or exposures in abuse were less often (33.4 and 0.2%, 46.0 and 0.9% respectively). NOA single drug exposures resulted mostly in none to minor symptoms (77.0%) and rarely in moderate (2.1%) or severe symptoms (1.0%). One adult was found dead after probable ingestion of 32 g of acetylsalicylic acid in suicidal intention. CONCLUSIONS: Because many NOA are over-the-counter drugs, it is difficult to obtain data on their use. PIC data could provide information on the NOA use in the population.

[Poisonings due to Substance Abuse Reported to the Poisons Information Centre Erfurt from 2002 to 2011]. / Substanzmissbrauch - Entwicklung des Vergiftungsgeschehens im Einzugsbereich des Giftnotrufes Erfurt 2002-2011.

AIM OF THE STUDY: Because of their importance for clinical toxicology, developments of sub-stance abuse reported to the Poisons Information Centre (PIC) Erfurt were investigated and compared to other reasons of human exposures. METHODS: A retrospective analysis of all human exposures (exposures of humans to substances in abuse, accidental and unknown circumstances, and suicide attempts) (n=125,130) from the beginning of 2002 to the end of 2011 was undertaken according to substance classes, reasons of exposures, symptom severity, age groups, and gender. RESULTS: Cases of substance abuse (3,760, 3.0% of all exposures) continuously increased from 252 (92 with one and 160 with multiple substances) in 2002 to 507 in 2011 (239 with one and 268 with multiple substances). In relation to all exposures, only the abuse of multiple substances rose significantly (p<0.001). In comparison to all substances of exposure, ethanol, amphetamine-type stimulants, benzodiazepines/analogues, and liquid ecstasy abuse significantly (p<0.005) increased while cannabis and Brugmansia/Datura species abuse significantly (p<0.05) decreased. Substance abuse significantly (p<0.001) more often caused moderate (23.7%) and severe symptoms (6.1%) than in suicide attempts (9.6%; 4.4%). First legal highs exposures were registered in 2010 and led significantly (p<0.001) more often to moderate symptoms (50%) than cannabis exposures (19.4%). CONCLUSIONS: The clinical significance of substance abuse is shown by the fact that it resulted more often in moderate and severe symptoms than suicide attempts. Data on substance abuse from PICs could supplement official annual drug reports in aspects of clinical toxicology.

[Human exposures to veterinary medicines reported to the Poisons Information Centre Erfurt from 2003 to 2012]. / Humane Tierarzneimittelexpositionen im Einzugsbereich des Giftnotrufes Erfurt 2003-2012.

AIM OF THE STUDY: The purpose of this study was to get information on all human exposures to veterinary medicines (HEVM) reported to the Poisons Information Centre (PIC) over a 10-year period. METHODS: A retrospective analysis of all HEVM was undertaken and a comparison was made to all human exposures (HE) registered by the PIC from the beginning of 2003 to the end of 2012 according to frequencies, circumstances of exposure, symptom severity, age groups, and substances involved in HEVM. RESULTS: In total, 389 cases of HEVM with 409 veterinary medicines were registered (0.30% of all HE, 360 monoexposures). The relative frequency of children and adults in HEVM (children: 52.4%, adults: 46.0%) and all HE (children: 48.7%, adults 48.7%) was the same with significant (p<0.05) differences in some age subgroups. The portion of accidental exposures was significantly (p<0.05) higher in HEVM (83.3%) than in all exposures (59.3%), whereas the portion of suicidal exposures was significantly (p<0.05) lower (HEVM: 6.4%, all exposures: 23.6%). Most frequent veterinary medicines (ATCvet) in HEVM were antiparasitic substances, insecticides and repellents (n=185), substances for the nervous system (n=48), substances for the cardiovascular system (n=35), and immunologicals (n=35). HEVM mostly resulted in no or mild symptoms (83.8%) and rarely in moderate (10/389, 2.6%) or even severe symptoms (5/389, 1.3%). In 4 of 5 cases of HEVM with severe symptoms, veterinary surgeons used products for animal euthanasia (n=3) or methadone (n=1). Once, self-medication with anthelmintics for several days by a goatherd resulted in transient blindness. CONCLUSIONS: In comparison to other HE, HEVM are rare. Most accidental HEVM in laymen result only in none to mild symptoms. If veterinary surgeons, however, swallow or inject products for animal euthanasia or opioids in suicidal intention, severe symptoms can be expected.

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.

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.

Inhibition of activated protein C by benzamidine derivatives.

In studies on the inhibition of activated protein C (APC) by benzamidine derivatives potent inhibitors of APC were found among anilides of 4-amidinophenyl-alpha-aminobutyric acid (Ki = 0.58 mumol/l). Several bis-benzamidine derivatives containing a cycloalkanone linking bridge inhibit APC with Ki values near the micromolar range. Potent and selective inhibitors of thrombin derived from 3- and 4-amidinophenylalanine do not inhibit APC. This is of great importance for further development of these inhibitors as potential anticoagulant drugs.

Blood coagulation and fibrinolysis after extreme short-term exercise.

INTRODUCTION: Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s. METHODS: A total of 15 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of 15, 45, and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, 15 min, and 1 h after completion of exercise. For the investigation of blood coagulation, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complex (TAT), intrinsic and extrinsic total (TTPin+ex), and endogenous thrombin potential (ETPin+ex) were measured. For testing fibrinolysis, determinations of plasmin-alpha(2)-antiplasmin complex (PAP), tissue-type plasminogen activator (tPA)-antigen, plasminogen activator inhibitor (PAI)-1-antigen and D-dimer were used. RESULTS: Immediately after the exercise tests, only F1+2 (15- and 90-s test) and TTPin (45 and 90 s) showed a moderate increase (p<0.05), while TAT and ETP was unchanged. In contrast, a clear increase in PAP and tPA-antigen already after 15 s maximal exercise in relation to the exercise duration time could be investigated. These effects were not totally reversed to baseline 15 min after exercise; D-dimer and PAI-1-antigen still remained unchanged after these types of exercise. CONCLUSIONS: Maximal short-term exercise does not lead to a relevant activation of blood coagulation in healthy young subjects, it is only slightly altered within the normal range. In contrast, fibrinolysis is clearly activated, and the increase is directly dependent on exercise duration. Additionally, it could be shown for the first time that fibrinolysis is already activated after 15 s maximal exercise duration.

Novel plasma kallikrein inhibitors of the benzamidine type.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with an amidino moiety are inhibitors of trypsin-like enzymes. However, in most cases ordinary benzamidine derivatives are not selective inhibitors. Relatively selective inhibitors of some enzymes were found among amino acids containing a benzamidine moiety at the side chain. For example, derivatives of phenyl-alpha-aminobutyric acid with one or two amidino moieties such as the 4'-amidinoanilide of N alpha-[4-amidinophenylsulfonyl]-phenyl-alpha- aminobutyric acid are potent and selective inhibitors of plasma kallikrein (Ki = 0.15 microM). Using N alpha-arylsulfonylated 3-amidinophenylalanine as the key building block, novel inhibitors of plasma kallikrein were developed. Several esters and amides of the nipecotic acid derivative were synthesized which inhibit plasma kallikrein competitively with Ki values between 0.1 and 1.0 microM. The compounds prolonged aPTT in micromolar concentration, indicating an inhibition of the intrinsic coagulation pathway.

[Inhibitors of factor XIIIa]. / Hemmstoffe von Faktor XIIIa.

Factor XIIIa (FXIIIa) catalyzes the covalent crosslinking of fibrin polymers and incorporation of proteins into the fibrin network and thus confers on the thrombus additional structural stability and relative resistance to plasmin-mediated degradation. Moreover, FXIIIa is involved in other physiological and pathophysiological processes such as wound healing and arteriosclerosis. Selective FXIIIa inhibitors may be a valuable tool for evaluation of the various functions of FXIIIa and their pharmacological control. This paper presents an overview of the inhibitors of FXIIIa. Analogues of natural FXIIIa substrates - including glutamine containing peptides and low molecular weight substituted alkylamines - are incorporated into the fibrin network and thus prevent crosslinking of fibrin. Naturally occurring, direct inhibitors of FXIIIa have been isolated from a leech species and microorganisms. With effective concentrations in the nanomolar range the peptide tridegin is the most potent FXIIIa inhibitor up to now. The majority of the synthetic, low molecular Weight inhibitors bind covalently to Cys314 at the active site of FXIIIa. Besides the relatively nonspecific thiol reagents, azol derivatives, azolium salts and related substances are described as specific inhibitors of FXIIIa. They inhibit the activity of FXIIIa at nanomolar concentrations. Animal experiments have demonstrated improved thrombolysis by a plasminogen activator in combination with a FXIIIa inhibitor.

Calcium channel antagonist exposures reported to the Poisons Information Center Erfurt.

BACKGROUND: We investigated the toxicity profile of the three main groups of calcium channel antagonists (CCA) and compared mixed CCA exposures (CCA plus another drug) with mono CCA exposures. METHODS: All CCA exposures reported to the PIC Erfurt from 2000 to 2009 were analyzed retrospectively. RESULTS: In total, 727 (230 mono and 497 mixed) CCA exposures were registered. Although CCA exposures increased almost twofold from 56 in 2000 to 108 in 2009 their relative frequency to all exposures remained constant. The five CCAs most frequently involved in exposures were the five most frequently prescribed ones in Germany over the same period. In mono and mixed CCA exposures, none or minor symptoms were most often seen with dihydropyridines (mono: 84.7%; mixed: 68.0%) followed by diltiazem (mono: 71.4%; mixed: 62.5%) and verapamil (mono: 57.1%; mixed: 50.0%). Highest rates of moderate (mono: 8.6%: mixed: 20.2%) and severe symptoms (mono: 18.6%; mixed: 23.7%) were observed after verapamil ingestions. Death most frequently occurred with diltiazem (mono: 28.6%; mixed: 12.5%). Rates of moderate symptoms were higher in mixed (13.3%) than in mono CCA exposures (4.8%). No distinct differences were seen regarding the relative frequency of none or minor symptoms, severe symptoms, and death between mono and mixed CCA exposures. CONCLUSION: Exposures to verapamil more often resulted in moderate and severe symptoms than with dihydropyridines. Death mainly occurred with diltiazem. Moderate symptoms were more frequent in mixed than in mono CCA exposures. The frequency of CCAs involved in exposure was related to their prescription.

Inhibition of glandular and plasma kallikrein by benzamidine derivatives.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with other cationic groups are inhibitors of trypsin-like serine proteinases. Particularly aromatic ring structures with an amidino moiety have high affinity for these enzymes. In most cases ordinary benzamidine derivatives are no selective inhibitors, however, among derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-amino-alkylcarboxylic acids selective competitive inhibitors of several enzymes were found. Amides of phenyl-alpha-aminobutyric acid containing an amidino moiety are inhibitors of plasma kallikrein. The p-amidinoanilide of 2-tosylamino-4-phenylbutyric acid inhibits selectively plasma kallikrein with a Ki of 0.70 mumol/l. In contrast, potent and selective inhibitors of glandular kallikrein were hardly found among benzamidines.

Inhibition of human mast cell tryptase by benzamidine derivatives.

Considerable circumstantial evidence has been provided by in vitro studies that tryptase (EC 3.4.21.59), a neutral serine proteinase stored in large amounts in mast cell granules, may play an important pathogenetic role in mast cell-dependent diseases. However, a definitive role has not yet been ascribed to this trypsin-like enzyme with restricted substrate specificity as natural or synthetic inhibitors of tryptase applicable for in vivo studies are not available so far. Therefore, we have studied structure-activity relationships for inhibition of tryptase by benzamidine derivatives, compounds known to be potent inhibitors of various trypsin-like enzymes. Among the benzamidine derivatives 4-amidinophenylpyruvic acid exerts a striking inhibitory activity with a Ki of 0.71 mumol/l. Several additional inhibitors of tryptase with Ki values in the micromolar range were found among bis-benzamidines. Derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-aminoalkylcarboxylic acids are only weak inhibitors of tryptase, although members of this group are potent and selective inhibitors of several other trypsin-like enzymes. Comparison of the inhibition of tryptase and trypsin revealed that the affinities of the benzamidine derivatives to both proteinases are closely correlated (correlation coefficient r = 0.702; n = 37; p < 0.001). These results demonstrate that 4-amidinophenylpyruvic acid may be useful as a pharmacologic tool for the investigation of the (patho)physiological role of tryptase. In addition, benzamidine derivatives may be applicable to probe the active site topography of tryptase isoenzymes.

Structure-activity relationships of inhibitors derived from 3-amidinophenylalanine.

Thrombin is the key enzyme in coagulation and its inhibitors are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine piperidide (NAPAP). However, NAPAP and other substances designed so far do not fulfill the pharmacological requirements. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties, such as absorption after oral application and a sustained elimination. Novel derivatives of 3-amidinophenylalanine as key building block were synthesized. The amidino moiety and both the N alpha- and the C-terminal substituents were widely varied. Some of the newly synthesized compounds are potent inhibitors of thrombin and exert improved pharmacokinetic properties.

Thrombin potential and thrombin generation after exhaustive exercise.

Exhaustive exercise leads to an activation of blood coagulation, but the implications of this activation are still unclear. The aim of this study was to investigate if a hypercoagulant stage exists after exhaustive treadmill- or cycle exercise; intrinsic and extrinsic endogenous thrombin potential (ETP) were measured by using the method of Hemker et al. Thirteen healthy male subjects underwent an exhaustive treadmill (TR) or cycle (CY) ergometer test and a control-day in random order. Blood samples were taken, repeatedly, after a 30 min rest, immediately before and after, and 1 h after exercise for measuring intrinsic and extrinsic total thrombin potential (TTPin, TTPex) (including free and alpha 2 -macroglobulin-bound thrombin) and endogenous thrombin potential (ETPin, ETPex), aPTT, PT, F1 + 2 and TAT. In comparison to the pre-value taken immediately before the exercise, the intrinsic TTP was significantly (p < 0.05) increased directly after exercise (TR-TTPin, + 11.6 %; CY-TTPin, + 11.5 %). In contrast, ETPin remained unchanged after both exercises. Additionally for TTPex and ETPex, no changes after exercise were detectable. aPTT was significantly (p < 0.05) shorter after exercise (TR-aPTT, - 16.2 %; CY-aPTT - 17.5 %), F1 + 2-concentrations were higher (p < 0.05) (TR-F1 + 2, + 21.2 %; CY-F1 + 2, + 9.8 %), but TAT remained unchanged. Differences between TR or CY could not be determined. These results show the expected shortening of aPTT and the increase of F1 + 2 indicating an activation of the coagulation system during exercise. However, the unchanged intrinsic and extrinsic ETP lead to the conclusion that in healthy young male subjects the potential for thrombin generation is insignificant, is directly counterbalanced by alpha 2-macroglobulin and is independent of the type of exhaustive exercise done.

Structural mapping of the active site specificity determinants of human tissue-type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors.

The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg174, which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors.