'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Activated coagulation during open and endovascular abdominal aortic aneurysm repair

OBJECTIVE: The study was conducted to determine activation of coagulation in patients undergoing open and endovascular infrarenal abdominal aortic aneurysm repair (EVAR). METHODS: In a prospective, comparative study, 30 consecutive patients undergoing open repair (n = 15) or EVAR (n = 15) were investigated. Blood samples to determine fibrinopeptide A, fibrin monomer, thrombin-antithrombin complex, and D-dimer were taken up to 5 days postoperatively. Routine hematologic and hematochemical parameters as well as clinical data were collected. RESULTS: Both groups showed comparable demographic variables. Operating time was longer in open repair (249 +/- 77 minutes vs 186 +/- 69 minutes, P < .05). Perioperatively elevated markers of coagulation were measured in both groups. Fibrinopeptide A levels did not differ significantly between the groups (P = .55). The levels of fibrin monomer and thrombin-antithrombin complex were significantly higher in patients undergoing EVAR (P < .0001), reflecting increased thrombin activity and thrombin formation compared with open surgery. The D-dimer level did not differ significantly between the groups. These results were also valid after correction for hemodilution. CONCLUSION: These data suggest increased procoagulant activity in EVAR compared with open surgery. A procoagulant state may favor possible morbidity derived from micro- and macrovascular thrombosis, such as in myocardial infarction, multiple organ dysfunction, venous thrombosis and thromboembolism, or disseminated intravascular coagulation.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Off-pump coronary artery bypass operation does not increase procoagulant and fibrinolytic activity: preliminary results

BACKGROUND: This study analyzes the effects on coagulation and fibrinolysis comparing off-pump coronary artery bypass (OPCAB) and on-pump CABG operations. METHODS: In a prospective, nonrandomized, comparative evaluation, patients scheduled for elective myocardial revascularization were studied. Due to possible confounding factors patients with postoperative retransfusion of mediastinal shed blood were excluded. Nine patients underwent OPCAB operation and 16 underwent on-pump CABG. Activated clotting time (ACT) was adjusted to 250 seconds in OPCAB (81 +/- 18 [mean +/- SD] IU/kg heparin) and to more than 480 seconds in on-pump CABG (400 IU/kg heparin, additional 10,000 IU in pump prime). Perioperatively blood samples were collected and hematologic and hemostatic variables including fibrinopeptide A (FPA), fibrin monomer (FM), thrombin-antithrombin complex (TAT), and D-dimer were analyzed. RESULTS: Both groups showed comparable demographic variables. Number of grafts per patient was slightly higher in the on-pump group (3.6 +/- 0.6 versus 3.0 +/- 1.1, p = 0.23). The FPA levels did not differ significantly between the groups. The FM, TAT, and D-dimer values were significantly higher in on-pump CABG (p < 0.0001, p < 0.01, and p < 0.0001, respectively), reflecting increased coagulant and fibrinolytic activity. This was also the case when values were corrected for hemodilution. CONCLUSIONS: Despite lower systemic anticoagulation activation of coagulation and fibrinolysis is reduced in OPCAB compared with on-pump CABG. Reduced thrombin generation and reduced fibrinolytic activity in OPCAB indicates better preservation of hemostasis. We suggest the term "preserved hemostasis" instead of "hypercoagulant activity" with respect to OPCAB.

Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes.

In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.