rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii.

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 µM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

Towards Development of an Anti-Vampire Bat Vaccine for Rabies Management: Inoculation of Vampire Bat Saliva Induces Immune-Mediated Resistance.

The common vampire bat (Desmodus rotundus) is a hematophagous species responsible for paralytic rabies and bite damage that affects livestock, humans and wildlife from Mexico to Argentina. Current measures to control vampires, based upon coumarin-derived poisons, are not used extensively due in part to the high cost of application, risks for bats that share roosts with vampires and residual environmental contamination. Observations that vampire bat bites may induce resistance in livestock against vampire bat salivary anticoagulants encourage research into novel vaccine-based alternatives particularly focused upon increasing livestock resistance to vampire salivary components. We evaluated the action of vampire bat saliva-Freund's incomplete adjuvant administered to sheep with anticoagulant responses induced by repeated vampire bites in a control group and examined characteristics of vampire bat salivary secretion. We observed that injections induced a response against vampire bat salivary anticoagulants stronger than by repeated vampire bat bites. Based upon these preliminary findings, we hypothesize the utility of developing a control technique based on induction of an immunologically mediated resistance against vampire bat anticoagulants and rabies virus via dual delivery of appropriate host and pathogen antigens. Fundamental characteristics of host biology favor alternative strategies than simple culling by poisons for practical, economical, and ecologically relevant management of vampire populations within a One Health context.

Warfarin drug interaction with vitamin K and other foodstuffs / Interacción de la droga warfarina con la vitamina K y otros productos alimenticios

Vitamin K is found in higher concentrations in dark green plant and in vegetable oils. The adequate intake of vitamin K is 90 and 120ug/day for adult elderly men and women, respectively. The main function of vitamin K is to act as an enzymatic cofactor for hepatic prothrombin synthesis, blood coagulation factors, and anticoagulant proteins. Prominent among the many available anticoagulants is warfarin, an antagonist of vitamin K, which exerts its anticoagulant effects by inhibiting the synthesis of vitamin K1 and vitamin KH2. From the beginning of the therapy it is necessary that the patients carry out the monitoring through the prothrombin time and the international normalized ratio. However, it is known that very low intake and/or fluctuations in vitamin K intake are as harmful as high consumption. In addition, other foods can interact with warfarin, despite their content of vitamin K. The aim of this study was to gather information on the drug interaction of warfarin with vitamin K and with dietary supplements and other foods.

La vitamina K se encuentra en concentraciones más altas en plantas de color verde oscuro y en aceites vegetales. La ingesta adecuada de vitamina K es de 90 y 120 ug/día para hombres y mujeres adultos mayores, respectivamente. La función principal de la vitamina K es actuar como un cofactor enzimático para la síntesis de protrombina hepática, factores de coagulación de la sangre y proteínas anticoagulantes. Entre los muchos anticoagulantes disponibles destaca la warfarina, un antagonista de la vitamina K, que ejerce sus efectos anticoagulantes al inhibir la síntesis de la vitamina K1 y la vitamina KH2. Desde el inicio de la terapia, es necesario que los pacientes realicen el monitoreo a través del tiempo de protrombina y la proporción normalizada internacional. Sin embargo, se sabe que una ingesta muy baja y/o fluctuaciones en la ingesta de vitamina K son tan dañinas como un consumo alto. Además, otros alimentos pueden interactuar con la warfarina, a pesar de su contenido de vitamina K. El objetivo de este estudio fue recopilar información sobre la interacción de los medicamentos de la warfarina con la vitamina K y con los suplementos dietéticos y otros alimentos.

Tunicate Heparan Sulfate Enriched in 2-Sulfated ß-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin.

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Blood anticlotting activity of a Rhipicephalus microplus cathepsin L-like enzyme.

In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ±â€¯11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.

Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied's false boa; Xenodontinae; Pseudoboini).

In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 µg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 µg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

Warfarin Safety: A Cross-Sectional Study of the Factors Associated with the Consumption of Medicinal Plants in a Brazilian Anticoagulation Clinic.

OBJECTIVE: The aim of this study was to analyze factors associated with the consumption of medicinal plants by patients being treated with warfarin in a Brazilian anticoagulation clinic and to study the safety of medicinal plant use in patients on warfarin therapy. METHODS: The study was performed as an observational cross-sectional analysis. Study participants were outpatients on long-term warfarin therapy for at least 2 months for atrial fibrillation or prosthetic cardiac valves. Interviews were carried out concerning information about the habits of medicinal herb consumption, and logistic regression analysis was performed to identify factors associated with the consumption of herbs. The scientific names of the medicinal plants were identified to search for information on the effects on the hemostasis of the interactions between the medicinal herbs reported and warfarin. RESULTS: The mean age of the 273 patients included was 60.8 years; 58.7% were women. Medicinal plants were used by 67% of the participants. No association between demographic and clinical data and the use of medicinal plants was identified. Patients reported a total of 64 different plants, primarily consumed in the form of tea. The plants were mainly used to treat respiratory tract and central nervous system disorders. About 40% of the plants cited have been reported to potentially interfere with the anticoagulation therapy, principally by potentiating the effects of warfarin, which could, increase the risk of bleeding. CONCLUSION: The use of medicinal plants was highly common and widespread in patients receiving warfarin as an anticoagulation therapy. Univariate analysis of variables associated with the consumption of herbs showed no statistically significant difference in the consumption of medicinal plants for any of the sociodemographic and clinical data. The medicinal plants that were reportedly consumed by the patients could affect hemostasis. This study reinforces the need for further studies evaluating the habits of patients consuming medicinal plants and their clinical implications, and will help to design strategies to manage the risks associated with warfarin-herbal interactions.

Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D 1H and 2D 15N-1H NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.

"In-House" Production of Single Stranded Oligodeoxyribonucleotides.

The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.

Intra- and Extracellular Biosynthesis and Characterization of Iron Nanoparticles from Prokaryotic Microorganisms with Anticoagulant Activity.

BACKGROUND: The use of microorganisms for the synthesis of nanoparticles (NPs) is relatively new in basic research and technology areas. PURPOSE: This work was conducted to optimized the biosynthesis of iron NPs intra- and extracellular by Escherichia coli or Pseudomonas aeruginosa and to evaluate their anticoagulant activity. STUDY DESIGN/METHODS: The structures and properties of the iron NPs were investigated by Ultraviolet-visible (UV-vis) spectroscopy, Zeta potential, Dynamic light scattering (DLS), Field emission scanning electron microscope (FESEM)/ Energy dispersive X-ray (EDX) and transmission electron microscopy (TEM). Anticoagulant activity was determined by conducting trials of Thrombin Time (TT), Activated Partial Prothrombin Time (APTT) and Prothrombin Time (PT). RESULTS: UV-vis spectrum of the aqueous medium containing iron NPs showed a peak at 275 nm. The forming of iron NPs was confirmed by FESEM/ EDX, and TEM. The morphology was spherical shapes mostly with low polydispersity and the average particle diameter was 23 ± 1 nm. Iron NPs showed anticoagulant activity by the activation of extrinsic pathway. CONCLUSION: The eco-friendly process of biosynthesis of iron NPs employing prokaryotic microorganisms presents a good anticoagulant activity. This could be explored as promising candidates for a variety of biomedical and pharmaceutical applications.

Study of warfarin utilization in hospitalized patients: analysis of possible drug interactions.

Background Drug-drug interactions in patients taking warfarin may contribute to a higher risk of adverse events. Objective To identify and evaluate the prevalence and characteristics of potential DDIs with warfarin. Methods A cross-sectional study was performed in a Brazilian tertiary hospital. The electronic prescriptions of the patients receiving warfarin between January 2004 and December 2010 were analyzed. Socio-demographic, clinical, and therapeutic variables were collected. Warfarin drug-drug interactions were classified as either risk A, B, C, D, or X according to the Lexi-Interact™ Online database. Results A total of 3048 patients were identified who were prescribed warfarin. Of the 154,161 total drug prescriptions issued, 42,120 (27.3 %) were for warfarin. Evaluation of the prescriptions showed that 63.1 and 0.1 % of patients received concomitant drugs classified as having class D or X risk. It was found that 20,539 (48.7 %) prescriptions had at least one drug with a D or X risk. Patients were prescribed an average of 1.4 (±0.4) concomitant medications with a class D or X warfarin-DDI risk, the most frequent being acetylsalicylic acid and amiodarone. Conclusion The results demonstrate a high prevalence of concomitant drug prescriptions with the potential for clinically relevant DDIs with warfarin, the most frequent being acetylsalicylic acid and amiodarone.

Study of Anticoagulant, Procoagulant, and Fibrinolytic Pathways in Mexican Patients With Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: In Mexico, the frequency of thromboembolic events associated to paroxysmal nocturnal hemoglobinuria is 3%; a clone size > 50% in granulocytes has been associated with a higher risk of thromboembolic events. METHODS: Between 2001 and 2012, 40 patients with paroxysmal nocturnal hemoglobinuria were studied. In 12 cases anticoagulant, procoagulant, and fibrinolytic pathways were analyzed. RESULTS: Only two of 40 patients (5%) developed a thromboembolic event over a 25.5-year follow-up period. From 12 patients, 91.7% had a paroxysmal nocturnal hemoglobinuria clone > 50% in granulocytes and 83.3% a clone > 50 % in monocytes. Five of 12 cases had elevated FV levels and four showed increased FVIII, von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity and FX. Protein S and protein C were decreased in nine and three patients, respectively. Only antithrombin correlated positively with paroxysmal nocturnal hemoglobinuria clone size in monocytes (p = 0.0442), whereas von Willebrand factor ristocetin cofactor correlated negatively with lactic dehydrogenase levels (p = 0.0186). No statistically significant associations were recorded with all other factors. CONCLUSION: The low frequency of thromboembolic events in Mexican patients could partly be explained by the associations between anticoagulant system (antithrombin) with paroxysmal nocturnal hemoglobinuria monocyte clone size, and procoagulant system (von Willebrand factor ristocetin cofactor) with lactic dehydrogenase levels.

Biochemical and biological analysis of Philodryas baroni (Baron's green racer; Dipsadidae) venom: relevance to the findings of human risk assessment.

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bß- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 µg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Genetic contribution of CYP2C9, CYP2C19, and APOE variants in acenocoumarol response.

Oral anticoagulants of the coumarin type have an inconveniently narrow therapeutic window, making their use difficult. In Mexico, genetic variables that participate in the heterogeneity of the therapeutic response remain poorly investigated. With the focus on warfarin, extensive pharmacogenomic studies have been performed, including those on the CYP450 family and APOE. The objective of this study was to determine the contribution of CYP2C9, CYP2C19, and APOE polymorphisms to the variations in response to the doses of acenocoumarol, which is the main anticoagulant prescribed to the Mexican population. The polymerase chain reaction-restriction fragment length polymorphism method was applied to identify 2 and 3 of CYP2C9, 2 of CYP2C19, and APOE variants. The genetic distribution of every polymorphism tested showed high variability when compared with other populations worldwide. Our results showed statistical differences only in the CYP2C19 gene between the 1 1 and 1 2 groups, with effective acenocoumarol doses of 2.56 ± 1.34 mg/day vs 1.35 ± 0.84 mg/day (P = 0.005), respectively. Multiple regression analysis, including patient age and both the CYP2C9 and CYP2C19 genes, showed that these variables explained more than 20% of the dose variations. This is the first report in Mexico searching for the relationship between CYP450 and APOE polymorphisms and the dose requirements of acenocoumarol. Our results suggest that, in the Mexican population, CYP2C19 is more involved in acenocoumarol metabolism than CYP2C9 and APOE. Besides considering the age factor, pharmacogenetic testing for CYP2C19 2 before initiating acenocoumarol treatment could lead to a safer anticoagulation therapy in Mexican patients.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva.

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Alterations of fibrin network structure mediated by dermatan sulfate.

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 µg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.

Jararhagin, a hemorrhagic snake venom metalloproteinase from Bothrops jararaca.

Jararhagin is a metalloproteinase isolated from Bothrops jararaca snake venom, which has been extensively studied. These studies showed its involvement on most of the systemic and local damaging effects of snakebite envenomings. In this review we comment on the major targets of jararhagin as the vascular endothelium, platelets and coagulation factors and also its action on other cell systems as inflammatory cells and their mediators, cancer and cell signaling. The mechanisms of jararhagin action are discussed together with structural features essential for the expression of its biological activities. The studies reviewed here denote jararhagin as a prototype for studies of snake venom metalloproteinases, bringing new insights into cellular-matrix interactions and adding for the improvement of snakebite treatment.

Heparin from bovine intestinal mucosa: glycans with multiple sulfation patterns and anticoagulant effects.

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression.

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.