Elevated levels of serum muscle enzymes in the initial phase of tick-borne encephalitis.

PURPOSE: Since some patients with tick-borne encephalitis (TBE) have pronounced myalgias, and since myositis is reported in Flavivirus diseases such as dengue, we performed systematic search for abnormalities of muscle enzymes in a group of patients in whom the presence of tick-borne encephalitis virus (TBEV) RNA in the first phase of the disease was demonstrated and who developed second phase of TBE. METHODS: Total leukocyte and platelet blood counts were determined routinely at the initial examination during the first and the second phase of TBE. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), myoglobin and troponin was determined from the available stored serum specimens; the first and second phase disease specimens were tested simultaneously. RESULTS: Of 24 patients with biphasic course of TBE, 83% had leukopenia, 65% thrombocytopenia, 83% elevated AST and 4% elevated ALT level. Furthermore, 33% had elevated serum CK, 26% myoglobin and 22% troponin activity; at least one of the muscle enzymes was elevated in 42% of patients. Leukopenia, thrombocytopenia, elevated liver enzymes and elevations of CK and myoglobin were present in the initial phase but resolve later, while troponin abnormalities were also found in the second phase of TBE. CONCLUSIONS: The present study exposes that in addition to previously known leukopenia, thrombocytopenia and increased liver enzymes activity, the initial phase of TBE is relatively often associated also with elevated muscle enzymes. Clinical relevance of these findings remains to be determined.

Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein.

Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

Platelet lumiaggregation testing: Reference intervals and the effect of acetylsalicylic acid in healthy adults.

BACKGROUND: Light transmission aggregometry with lumiaggregometry are methods commonly recommended as a first-line test in platelet dysfunction diagnostic work-up. They are poorly standardized and usually performed in specialized laboratories. For proper interpretation, each laboratory should establish its own diagnostic approach in order to recognize abnormal aggregation patterns. The aim of this study was to measure plasma lumiaggregometry with basic agonists to establish the analyzer-reagent reference intervals (RI) for adults and to test the method response to aspirin. METHODS: The Chrono-Log Model 700 lumiaggregometer using Chrono-Par and Chrono-lume reagents (Chrono-Log Corp., Havertown, PA, USA) was used to measure the maximal aggregation and adenosine triphosphate release using adenosine diphosphate (2 µmol/L), collagen (2 µg/mL), arachidonic acid (1 µmol/L), epinephrine (5.5 µmol/L) and ristocetin (1.25 mg/mL), and thrombin (1 U/mL). The effect of aspirin on platelet aggregation and granule release was inspected. RESULTS: RIs derived from 40 healthy adults were calculated using the non-parametric approach. Wider intervals and low lower limits were determined for weak agonist as well as absence or impaired aggregation in up to one of 7 healthy controls. The response of platelets to aspirin shows response comparable to previously reported study. CONCLUSIONS: Locally established RI in our study enable us to investigate platelet function in patients with a high probability of bleeding disorders. Values are agonist and equipment specific. The variability of the method can be reduced by considering standardized preanalytical and analytical variables. Pathological results must be interpreted in the context of other hemostasis test results and clinical findings.

SPTB related spherocytosis in a three-generation family presenting with kidney failure in adulthood due to co-occurrence of UMOD disease causing variant / Esferocitosis relacionada con SPTB en tres generaciones de una familia con insuficiencia cardíaca en la edad adulta debido a la presentación conjunta de la variante de UMOD causante de la enfermedad

BACKGROUND: Hereditary spherocytosis is clinically and genetically heterogeneous disorder and its clinical characteristics are spherocytosis, anaemia, jaundice and splenomegaly. The aetiology is associated to the genes encoding proteins involved in the interaction between the erythrocyte membrane and the lipid bilayer. Causative variants in BetaI-spectrin (SPTB) gene presenting as mild to moderately severe disease are responsible for approximately 25% cases in the USA and Europe. Among kidney disease, isolated cases of nephrotic syndrome due to membranoproliferative glomerulonephritis and macroscopic haematuria with proteinuria due to IgA nephropathy were previously reported in patients with SPTB deficiency. OBJECTIVE: Seven patients from the same family with spherocytosis were evaluated to assess the kidney failure presented in all affected adult patients. METHODS: Clinical, radiological and laboratory investigations were issued to evaluate the spherocytosis and kidney disease. In selected patients, we also performed genetics testing with next generation sequencing of genes related to hereditary spherocytosis, inherited glomerular disorders and tubulo-interstitial kidney disease. RESULTS: Among the family members with spherocytosis, two adults had end-stage kidney disease and one chronic kidney disease stage 4 with unspecific histopathological findings of interstitial fibrosis/tubular atrophy and glomerulosclerosis. At the time, there were no signs of kidney disease present in four paediatric patients. Novel nonsense variant in SPTB gene (NM_001024858; c.4796G>A; p.Trp1599Ter) was detected in all family members with spherocytosis and was predicted to be disease causing. Furthermore, all adult patients with kidney failure and two paediatric cousins of the index patients were heterozygous for the UMOD gene variant (NM_003361.3:c.552G > C, NP_003352.2:p.Trp184Cys) previously reported in patients with tubulo-interstitial kidney disease. UMOD variant was not present in the index patients. CONCLUSIONS: The co-occurrence of any two rare inherited disorders is extremely rare, while to our knowledge the co-occurrence of genetically confirmed HS and autosomal dominant tubulo-interstitial kidney disease (ADTKD) has previously not been reported. It is not possibly to evaluate whether the haemolytic crises due to HS are influencing the progression of the UMOD related renal disease, since the UMOD related ADTKD characteristics in general and in here presented family are extremely variable. Nevertheless, the observed kidney disease in the family is warranting the regular nephrological examinations in UMOD positive paediatric patients in the family in order to recognise hyperuricemia and treat it as early as possible. This is emphasising the importance of serum uric acid detection in routine laboratory screening of paediatric patients in order to identify early signs of tubular injury indicating possible ADTKD

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The Procoagulant Snake Venom Serine Protease Potentially Having a Dual, Blood Coagulation Factor V and X-Activating Activity.

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell's viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

SPTB related spherocytosis in a three-generation family presenting with kidney failure in adulthood due to co-occurrence of UMOD disease causing variant.

BACKGROUND: Hereditary spherocytosis is clinically and genetically heterogeneous disorder and its clinical characteristics are spherocytosis, anaemia, jaundice and splenomegaly. The aetiology is associated to the genes encoding proteins involved in the interaction between the erythrocyte membrane and the lipid bilayer. Causative variants in ßI-spectrin (SPTB) gene presenting as mild to moderately severe disease are responsible for approximately 25% cases in the USA and Europe. Among kidney disease, isolated cases of nephrotic syndrome due to membranoproliferative glomerulonephritis and macroscopic haematuria with proteinuria due to IgA nephropathy were previously reported in patients with SPTB deficiency. OBJECTIVE: Seven patients from the same family with spherocytosis were evaluated to assess the kidney failure presented in all affected adult patients. METHODS: Clinical, radiological and laboratory investigations were issued to evaluate the spherocytosis and kidney disease. In selected patients, we also performed genetics testing with next generation sequencing of genes related to hereditary spherocytosis, inherited glomerular disorders and tubulo-interstitial kidney disease. RESULTS: Among the family members with spherocytosis, two adults had end-stage kidney disease and one chronic kidney disease stage 4 with unspecific histopathological findings of interstitial fibrosis/tubular atrophy and glomerulosclerosis. At the time, there were no signs of kidney disease present in four paediatric patients. Novel nonsense variant in SPTB gene (NM_001024858; c.4796G>A; p.Trp1599Ter) was detected in all family members with spherocytosis and was predicted to be disease causing. Furthermore, all adult patients with kidney failure and two paediatric cousins of the index patients were heterozygous for the UMOD gene variant (NM_003361.3:c.552G>C, NP_003352.2:p.Trp184Cys) previously reported in patients with tubulo-interstitial kidney disease. UMOD variant was not present in the index patients. CONCLUSIONS: The co-occurrence of any two rare inherited disorders is extremely rare, while to our knowledge the co-occurrence of genetically confirmed HS and autosomal dominant tubulo-interstitial kidney disease (ADTKD) has previously not been reported. It is not possibly to evaluate whether the haemolytic crises due to HS are influencing the progression of the UMOD related renal disease, since the UMOD related ADTKD characteristics in general and in here presented family are extremely variable. Nevertheless, the observed kidney disease in the family is warranting the regular nephrological examinations in UMOD positive paediatric patients in the family in order to recognise hyperuricemia and treat it as early as possible. This is emphasising the importance of serum uric acid detection in routine laboratory screening of paediatric patients in order to identify early signs of tubular injury indicating possible ADTKD.

von Willebrand factor alloantibodies in type 3 von Willebrand disease.

: The development of neutralizing antibodies is a rare complication of von Willebrand disease treatment. In major surgical procedures for severe forms of the disease, the recognition of ineffective therapy and alternative treatment protocols are lifesaving. We report the case of a 6-year-old girl with type 3 von Willebrand disease in whom inhibitors were sought due to ineffective haemostasis together with lower than expected von Willebrand factor (VWF) recoveries after a surgical procedure. Replacement therapy first with recombinant factor VIIa and then with high doses of recombinant factor VIII in continuous infusion successfully stopped the bleeding. A high level of anti-VWF antibodies was determined by the immunological method. A frameshift mutation associated with premature termination codon (c.2435delC, p.Pro812ArgfsTer31) was determined in our patient. Although the reports on association of this mutation with inhibitor risk are inconsistent, it represents an evidence-based diagnostic and management practice in recognition of high-risk VWF genotype.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications.

UNLABELLED: Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. BIOLOGICAL SIGNIFICANCE: Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

Specific and global coagulation tests in patients with mild haemophilia A with a double mutation (Glu113Asp, Arg593Cys).

BACKGROUND: Heterogeneous bleeding phenotypes are observed in haemophilia A patients with the same mutation in the F8 gene. Specific mutations in the A2 domain of factor VIII are associated with mild haemophilia and a higher risk of inhibitor development. Double mutations in mild haemophilia A are rarely reported. In this study, we investigated the in vitro function of factor VIII, performing different specific and global coagulation assays, observed clinical characteristics and assessed the possible predictive diagnostic value of the differences. MATERIALS AND METHODS: The clinical features of haemophiliacs with a mild phenotype were reviewed. Blood samples were obtained and analysed for mutations and coagulation assays: activated partial thromboplastin time, one-stage and chromogenic factor VIII activity, factor VIII antigen and rotational thromboelastometry. RESULTS: We report on a cohort of 22 patients with double Glu113Asp, Arg593Cys mutations. All our patients have a quantitative defect of factor VIII and preserved similar functional activity. Factor VIII activities measured by the one-stage or chromogenic method were not discrepant, although the chromogenic assay resulted in 20% lower factor VIII activities. Waveform analysis showed a lower maximum value of the second derivative curve (Max2) of APTT with curve shape alternation, while thromboelastometry (INTEM) showed low sensitivity in comparison to results in a normal population. DISCUSSION: In genotyping, the coexistence of a second mutation should never be excluded, especially in cases of discordant clinical presentation. Waveform analysis correlates better with factor VIII activity than thromboelastometry and the Max2 parameter could provide additional information in managing haemophilia patients. The utility of specific factor activity and global haemostatic assays in general practice still needs to be investigated.

Comparison of tandem mass spectrometry and amino acid analyzer for phenylalanine and tyrosine monitoring--implications for clinical management of patients with hyperphenylalaninemia.

OBJECTIVES: Regular and accurate monitoring of blood phenylalanine (Phe) and tyrosine (Tyr) levels is prerequisite for a successful management of patients with hyperphenylalaninemia (HPA). We aimed to compare the tandem mass spectrometry (MS/MS) and the amino acid analyzer (AAA) as methods to measure blood Phe and Tyr levels and Phe/Tyr ratio. METHODS: Venous blood samples were collected for the AAA analysis, using Pinnacle PCX (Pickering Laboratories), with HPLC Series 1200 (Agilent). Capillary blood was spotted directly on filter paper (Whatman 903) for the MS/MS analysis, using 3200 QTrap AB SCIEX and Perkin Elmer Series 200 HPLC system. The Bland-Altman test was used to compare agreement between the methods and Pearson correlation coefficient to assess the association between the methods. RESULTS: 207 pairs of measurements were performed. The Phe levels (range 0-2500µM) obtained by the MS/MS were on average 26.1% (SD 13.9%) lower compared to those obtained by the AAA. The Tyr levels by the MS/MS were on average 15.5% (SD 20.6%) lower. The Phe/Tyr ratio by the MS/MS was on average 10.6% (SD 15.9%) lower. The Pearson correlation coefficients for Phe (range 0-2500µM), Tyr and the Phe/Tyr ratio were 0.984 (p<0.001), 0.841 (p<0.001) and 0.987 (p<0.001) respectively. CONCLUSIONS: When monitoring blood Phe and Tyr levels in patients with HPA, clinicians need to be informed about the method used. Due to the considerable inter-assay variability, a single method is preferable for long-term follow-up of patients. When using MS/MS, on average 26% lower blood Phe levels were obtained as compared to the AAA. The guidelines and recommendations on HPA management should take into consideration the differences in laboratory methods.

Hemorrhagin VaH4, a covalent heterodimeric P-III metalloproteinase from Vipera ammodytes ammodytes with a potential antitumour activity.

In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn²âº and Ca²âº ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the α-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential.

VaH3, one of the principal hemorrhagins in Vipera ammodytes ammodytes venom, is a homodimeric P-IIIc metalloproteinase.

Hemorrhage is the most potent manifestation of envenomation by Vipera ammodytes ammodytes (V. a. ammodytes) venom in man. A detailed description of the venom components contributing to this effect is thus medically very important. We have characterized a novel component, termed here VaH3, as a potently hemorrhagic snake venom metalloproteinase (SVMP). Its proteolytic activity and overall stability depend on the presence of Zn(2+) and Ca(2+) ions. The molecular mass of this slightly acidic molecule, determined by MALDI/TOF analysis, is 104 kDa. Chemical reduction and S-carbamoylmethylation result in a single monomer of 53.7 kDa. N-deglycosylation decreased this mass by 4.6 kDa. The complete amino acid sequence of VaH3 was determined by protein and cDNA sequencing, showing that each of the identical glycoprotein subunits comprise a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain, VaH3 belongs to the P-IIIc class of SVMPs. It shows strong sequence similarity to vascular endothelial cell apoptosis-inducing reprolysins. Anti-ammodytagin antibodies strongly cross-reacted with VaH3 and completely neutralized its hemorrhagic activity in rat, despite the fact that the two hemorrhagic P-III SVMPs from V. a. ammodytes venom do not share a very high degree of amino acid sequence identity. In spite of its narrow proteolytic specificity, VaH3 rapidly cleaved some basal membrane and extracellular matrix proteins, such as collagen IV, fibronectin and nidogen. Moreover, it also hydrolyzed plasma proteins involved in blood coagulation. It is an effective α-fibrinogenase that cleaves prothrombin and factor X without activating them. The degradation of these proteins likely contributes to the hemorrhagic activity of VaH3. A three-dimensional model of VaH3 was built to help explain structure-function relationships in ADAM/ADAMTS, a family of proteins having significant therapeutic potential and substantial sequence similarity to VaH3.

Pediatric thrombosis.

Thrombotic risk factors and thrombosis in children has been receiving increased attention. True idiopathic thrombosis is extremely rare in children. Most patients have a significant underlying medical condition and the presence of a central catheter is the most important risk factor. Children are more likely than adults to have one or more significant genetic abnormality or coagulation deficiency. This review discusses problems concerning the heterogeneity of thrombotic states in children and highlights the importance of understanding the concept of developmental hemostasis. Issues regarding step-wise test selection and the interpretation of results are addressed, as well as basic monitoring of anticoagulant drug effects.

Two coagulation factor X activators from Vipera a. ammodytes venom with potential to treat patients with dysfunctional factors IXa or VIIa.

Two activators of coagulation factor X, 58kDa VAFXA-I and 70kDa VAFXA-II, were purified from the venom of long-nosed viper (Vipera ammodytes ammodytes) by chromatography on gel filtration, affinity, ion-exchange and hydroxyapatite media. Both enzymes are glycoproteins composed of a heavy chain and two C-type lectin-like light chains all joined by disulphide bonds. LC-MS and LC-MS/MS analysis of their tryptic fragments demonstrated that the heavy chain consists of three domains, metalloproteinase, disintegrin-like and cysteine-rich domains. The partial amino acid sequences of VAFXAs are very similar to those of the known factor X activators, RVV-X from Vipera russelli and VLFXA from Vipera lebetina venoms, as well as to other members of the reprolysin family of metalloproteinases. The VAFXAs activate factor X in a Ca(2+)-dependent manner with the same specificity as physiological activators. The activators weakly hydrolyzed insulin B-chain, fibrinogen and some components of the extracellular matrix in vitro, but did not activate prothrombin or plasminogen. VAFXAs inhibit collagen-induced platelet aggregation in vitro. They activate coagulation factor X to Xa without toxic effects. Their application in treating patients with dysfunctional factors IXa or VIIa to restore the normal blood coagulation process is thus promising.

Ammodytase, a metalloprotease from Vipera ammodytes ammodytes venom, possesses strong fibrinolytic activity.

Ammodytase, a high molecular mass metalloproteinase with fibrinogenolytic and fibrinolytic activities, was purified from long-nosed viper (Vipera ammodytes ammodytes) venom by gel filtration, affinity and ion-exchange chromatographies. The enzyme is a single-chain glycoprotein with apparent molecular mass of 70 kDa and isoelectric point of 6.6. Ammodytase shows very weak hemorrhagic activity, and only at doses higher than 20 microg. Consistent with this, it partially degrades some components of the extracellular matrix in vitro. It cleaves the Aalpha-chain of fibrinogen preferentially at peptide bonds Glu(441)-Leu(442) and Glu(539)-Phe(540). Its preference for bulky and hydrophobic amino acids at the P1' position in substrates is demonstrated by its hydrolysis of only the Gln(4)-His(5) and Tyr(16)-Leu(17) bonds in the B-chain of insulin. Ammodytase is able to dissolve fibrin clots. It neither activates nor degrades plasminogen and prothrombin, and has no effect on collagen- or ADP-induced platelet aggregation in vitro. LC/MS and MS/MS analyses of its tryptic fragments demonstrated that ammodytase is a P-III class snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. Its similarity to hemorrhagins from V. a. ammodytes venom, accompanied by very low toxicity, makes ammodytase a promising candidate as an antigen to prepare antisera against these most dangerous components of the viper's venom. Moreover, its ability to degrade fibrin clots suggests its clinical use as an antithrombotic agent.

Thrombin inhibitors built on an azaphenylalanine scaffold.

A series of azaphenylalanine derivatives were investigated as novel thrombin inhibitors based on the prodrug principle. By systematic structural modifications we have identified optimal groups for this series that led us to potent inhibitors of thrombin incorporating the benzamidine fragment at the P1 position, and their potentially orally active benzamidoxime prodrugs. The binding modes in the thrombin active site of two representative compounds were identified by X-ray crystallographic analysis.

Novel thrombin inhibitors incorporating non-basic partially saturated heterobicyclic P1-arginine mimetics.

The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetrahydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetrahydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P(1)-arginine side-chain mimetics is described. The binding mode of the most potent candidate in the series co-crystallized with human alpha-thrombin, which exhibited an in vitro K(i) of 140nM and more that 478-fold selectivity against trypsin, is discussed.