Higher Coagulation Activity Research Articles

Partitioning purification, biochemical characterization, and milk coagulation efficiency of protease from a newly Streptomyces sp. isolate.

An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60°C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60°C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.

New Coagulant Proteases for Cheesemaking from Leaves and Latex of the Spontaneous Plant Pergularia tomentosa: Biochemical Characterization of Coagulants and Sensorial Evaluation of Cheese

The purpose of this study was to evaluate the caseinolytic and milk-clotting activities of aqueous crude extracts from leaves and latex of the Pergularia tomentosa, to determine their suitability as a rennet substitute. These extracts were subjected to a series of biochemical tests before being used in the production of cheese. The results showed that the enzymatic latex extract had a higher coagulant activity than the leaf extract. However, under different clotting conditions (pH, temperature, and CaCl2 concentration), both coagulants behaved similarly in the coagulation of Berridge substrate. The SDS-PAGE and zymographic analysis revealed identical protein bands with a single active zone in both extracts, corresponding to a molecular weight of 26.98 kDa and 26.03 kDa in the extract of leaf and latex, respectively. Both extracts were stable to different effectors but strongly inhibited by iodoacetamide and Hg, suggesting it to be a cysteine protease. Both extracts were able to hydrolyze casein and generate peptides of 14 kDa, with excessive hydrolysis of the other casein fractions. The physicochemical parameters of cheese made from latex and leaf extract evolved similarly to control cheese. According to the sensory evaluation, cheese made with latex had a mildly bitter flavor but showed a high acceptance rate (>80%).

Establishment of normal reference range for thromboelastography based on 17,708 cases in Beijing, China.

BackgroundThromboelastography (TEG) can dynamically evaluate the interaction between platelets and coagulation cascade and the effects of other cellular components on the activities of plasma factors, to comprehensively analyze the whole process of blood coagulation and dissolution. Due to differences in both the incidence rates and the coagulation state of related diseases, many studies have highlighted the necessity of establishing the normal reference ranges for TEG for local regions. The aim of the present study was to determine the local normal reference ranges according to the TEG results of 17,708 volunteers in Beijing, to explore the coagulation characteristics related to the age and sex of the study population.MethodsReference ranges of reaction time (R), coagulation time (K), coagulation angle (α-angle), and maximum thrombus consistency (MA) for TEG in healthy adults in Beijing were determined in the physical examination of 17,708 Beijing volunteers (5,319 women and 12,389 men). The volunteers were divided into the elderly group (≥60 years old) and young and middle-aged group (20–59 years old), and the reference ranges of each group were calculated according to sex.ResultsBased on the TEG results of the 17,708 volunteers who underwent physical examination, the 95% reference ranges of R, K, α-angle, and MA for TEG in Beijing were 5.1–10 min, 1.3–3.8 min, 44.9–70.2°, and 50.4–71 min, respectively. The results of R, K, α-angle, and MA for TEG between the young and middle-aged group and the elderly group, as well as between women and men were significantly different (P<0.001). Finally, reference ranges for TEG in the young and middle-aged group and the elderly group were obtained.ConclusionsCompared with the reference standards provided by previous reagent manufacturers, the coagulation factor and fibrinogen function of TEG tend to be hypocoagulable in Beijing population. We found that the young and middle-aged group had lower coagulation activity than the elderly group, and women had higher coagulation activity than men in the same group.

MILK CLOTTING ACTIVITY OF RECOMBINANT CAMEL CHYMOSIN

One of the earliest biotechnological applications of enzymes is cheese production. The high specific milk-clotting activity of chymosin gives it an advantage over other proteases in the cheese industry. The action of chymosin is based on hydrolysis of the bond between the amino acid residues Phe105 and Met106 in the κ-casein molecule, which causes destabilisation of casein micelles, coagulation of milk and its separation into a clot and a whey. A recombinant chymosin of the camel Camelus Bactrianus was obtained using the yeast producer strain Pichia pastoris GS115/pGAPZαA/ProchymCB. The recombinant chymosin was purified by ion exchange chromatography. The milk-clotting activity of recombinant camelian chymosin against mare's milk was studied as a function of pH and temperature. It was found that the optimum value at which the maximum activity of recombinant camel's chymosin with respect to mare's milk is observed is pH 4.0 and the optimum temperature is 70°C. The activity of recombinant camel chymosin on mare, cow and goat milk was 123, 9605 and 4650 units/mg, respectively. Recombinant camel chymosin was shown to have high coagulation activity, sufficient to produce curd and cheese. The yield of cheese clot from 6 l of goat milk was 936 grams. Recombinant camel chymosin has prospects for use in cow's and goat's milk processing and cheese production technologies.

A high efficient FVIII variant corrects bleeding in hemophilia A mouse model

Hemophilia A is a bleeding disorder caused by quantitative or qualitative deficiencies in coagulation factor VIII (FVIII). Low FVIII expression due to its unstable mRNA and binding with immunoglobulin-binding protein (BiP) compromises gene therapy endeavors in hemophilia A. Site-directed mutagenesis have demonstrated an improvement in the expression of FVIII proteins. In this study, a targeted point mutation of Pro at position 290 to Thr (P290T) enhances the in vitro specific activity of B-domain-deleted factor VIII (BDD-FVIII). Hydrodynamic gene delivery of P290T cDNA into FVIII-deficient (FVIII−/-) mice corrected bleeding symptoms. P290T variant resulted in high plasma FVIII coagulant activity 24 h post-gene delivery. Furthermore, bleeding time and average blood loss was significantly reduced in FVIII−/- mice injected with P290T variant, whereas BDD-FVIII and PBS-injected mice experienced prolonged bleeding and excessive blood loss. Histological analysis of the liver biopsies revealed no apparent signs of liver damage. No signs of potential toxicity were seen in mice following mice bodyweights assessment. Altogether, our results demonstrate that the introduction of P290T mutation increases both in vitro and in vivo FVIII coagulant activity, supporting ongoing efforts to develop more effective replacement therapy for hemophilia A.

Protein C or Protein S deficiency associates with paradoxically impaired platelet‐dependent thrombus and fibrin formation under flow

BackgroundLow plasma levels of protein C or protein S are associated with venous thromboembolism rather than myocardial infarction. The high coagulant activity in patients with thrombophilia with a (familial) defect in protein C or S is explained by defective protein C activation, involving thrombomodulin and protein S. This causes increased plasmatic thrombin generation. ObjectiveAssess the role of platelets in the thrombus‐ and fibrin‐forming potential in patients with familial protein C or protein S deficiency under high‐shear flow conditions. Patients/MethodsWhole blood from 23 patients and 15 control subjects was perfused over six glycoprotein VI–dependent microspot surfaces. By real‐time multicolor microscopic imaging, kinetics of platelet thrombus and fibrin formation were characterized in 49 parameters. Results and ConclusionWhole‐blood flow perfusion over collagen, collagen‐like peptide, and fibrin surfaces with low or high GPVI dependency indicated an unexpected impairment of platelet activation, thrombus phenotype, and fibrin formation but unchanged platelet adhesion, observed in patients with protein C deficiency and to a lesser extent protein S deficiency, when compared to controls. The defect extended from diminished phosphatidylserine exposure and thrombus contraction to delayed and suppressed fibrin formation. The mechanism was thrombomodulin independent, and may involve negative platelet priming by plasma components. [Display omitted]

Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

Nejayote biopolyelectrolytes multifunctionality (glucurono ferulauted arabinoxylans) in the separation of hazardous metal ions from industrial wastewater

The scarcity and contamination of water by suspended nanoparticles and hazardous metal ions demand more performant technologies to reduce space, time and cost of wastewater treatment. For example, it is desirable to find eco-friendly chemicals that warranty simultaneous separation of colloidal particles and hazardous metallic contaminants for a more sustainable recovery of water quality.As the second part of a series of physicochemical studies about multifunctional biopolyelectrolytes (BPEs), in this paper, we present three of the main features of the glucurono ferulauted arabinoxylans (GFAXs) from nejayote: simple extraction, physicochemical characterization and multifunctional performance when used in a wastewater treatment unit. The polyelectrolytic and macrochelating properties of the GFAXs were studied through zeta potential measurements to predict and understand the multifunctional performance of GFAXs in a conventional coagulation-flocculation unitary operation. Two real cases were approached: the wastewater from silicon wafer cutting process (SWCP) and the wastewater from electroplating baths (EPB). Nejayote GFAXs showed high coagulant, flocculant and macrochelating activity in the simultaneous separation of Si4+, Al3+, Cr3+, Ni2+, Mg2+, Ca2+, Ni2+, Cd2+, Pb2+, Cu2+, Zn2+ and Fe2+. The water treatment tests demonstrate the high performance of the GFAXs in the coagulation-flocculation of the wastewater according to the following parameters: EPB wastewater at pH = 5.0 (optimal dose = 55–65 mg GFAXs/L, clarification = 380 T%/h and sedimentation kinetics = 110.4 mm/h); SWCP wastewater at pH = 5.5 (optimal dose = 30.0–32.5 mg GFAXs/L, clarification = 295 T%/h and sedimentation kinetics = 89.4 mm/h). With these results, nejayote GFAXs showed to be a sustainable solution for the wastewater treatment from semiconductor and metal finishing industry with high content of hazardous metal ions and SiO2 nanoparticles.

Higher coagulation activity in hip fracture patients: A case-control study using rotational thromboelastometry.

Trauma-induced coagulopathy has been extensively investigated in the multitrauma setting, but only sparsely following moderate orthopedic trauma. The purpose of this study was to evaluate changes in the hemostatic profile of patients with hip fractures, using rotational thromboelastometry (ROTEM). 198 patients with hip fractures who underwent surgery were included in the study. A matched group of 52 healthy individuals was also enrolled. Demographics, conventional laboratory assays, and ROTEM parameters were recorded and compared between patients and healthy adults. The preoperative and postoperative ROTEM values of fractured patients were also compared. The conventional coagulation assays were similar for the 2 groups. However, several ROTEM parameters including EXTEM MCF (P<.001), EXTEM alpha angle (P<.001), INTEM MCF (P<.001), INTEM A10 (P<.001), and INTEM alpha angle (P<.001) significantly differed between the 2 groups indicating a higher coagulation potential following hip fractures. Also, fractured patients had significantly lower INTEM and EXTEM CT values (P=.008 and P=.012, respectively) and significantly lower INTEM and EXTEM CFT values (P<.001). Adjusted analysis for confounders further confirmed the direct relationship between hip fracture and higher coagulation activity. Last, INTEM CT and CFT significantly decreased (P=.008 and P<.001, respectively), while INTEM MCF, A10, and alpha angle significantly increased (P<.001) postoperatively, indicating that surgery further increases the coagulation potential. A higher coagulation activity following hip fractures and surgical treatment can be detected by ROTEM shortly after the injury, even when this is undetectable by conventional coagulation assays.

Paroxysmal atrial fibrillation is associated with early coagulation activity regardless of risk factors for embolism.

Paroxysmal atrial fibrillation (PAF) is associated with an increased incidence of embolic events, even in patients with no embologenic risk factors. This fact raises the question for the hypercoagulability in PAF as a state closely related to the arrhythmia itself, independent of other well established embologenic risk factors. The scarce data on that topic predisposed our aim that was to study coagulation activity in the early hours (up to the twenty-fourth hour) of the disease. Fifty-one non-anticoagulated patients (26 men, 25 women; mean age 59.84±1.60 years) and 52 controls (26 men, 26 women; mean age 59.50±1.46 years) were consequently selected for the study. Plasma coagulation activity of factor II (FII), factor V (FV) and factor X (FX) was examined. In the PAF group, there was a higher activity of FII (167.81±9.12% vs. 100.43±5.77%, P<0.001), FV (198.47±10.88% vs. 121.53±4.79%, P<0.001) and FX (193.20±11.85 vs. 116.20±5.86, P<0.001). The deviations were independent of age, sex, body mass index, presence of hypertension and CHA<inf>2</inf>DS<inf>2</inf>-VASc risk score (P>0.05). PAF duration was a significant predictor of FII activity (r=0.83, P<0.001) but no correlation was established between FV and FX activity and the arrhythmia duration (r=0.10, P>0.05; r=0.11, P>0.05, respectively). We established high coagulation activity during the first twenty-four hours of PAF. The observed deviations in the studied parameters give us reason to consider PAF a procoagulant state independent of the well-established prothrombotic risk factors, even in its early clinical manifestation.

Factor VIIa‐induced interaction with integrin controls the release of tissue factor on extracellular vesicles from endothelial cells

Essentials Prothrombotic extracellular vesicles (EV) carry agonist pathway-specific proteomes Agonists for protease activated receptor (PAR) 2 signaling have distinct effects on EV composition PAR2 signaling rapidly generates prothrombotic EV and slowly EV with inactive tissue factor (TF) FVIIa integrin ligation restricts TF incorporation into EV from endothelial cells SUMMARY: Background Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EVs occurs in concert with the release of cell adhesion receptors, including integrinβ1 heterodimers, which control trafficking of the TF-activated factorVII (FVIIa) complex. Activation of the TF signaling partner, protease-activated receptor (PAR)2, also triggers TF release on integrinβ1+ EVs from endothelial cells, but the physiological signals for PAR2-dependent EV generation at the vascular interface remain unknown. Objective To define relevant protease ligands of TF contributing to PAR2-dependent release on EVs from endothelial cells. Methods In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EVs by using a combination of activity and antigen assays, immunocapture, and confocal imaging. Results and Conclusions PAR2 stimulation generated time-dependent release of distinct TF+ EVs with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF-FVIIa-activated FX complex triggered TF+ EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced formation of a complex of TF with integrinα5 β1 . Internalization of TF by FVIIa or anti-TF and activating antibodies against integrinβ1 prevented PAR2 agonist-induced release of TF on EVs. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrinβ1 regulates TF availability for release on procoagulant EVs.

Evaluation of Moringa oleifera seed extract by extraction time: effect on coagulation efficiency and extract characteristic.

The seed of Moringa oleifera (MO) is a well-known coagulant used in water and wastewater treatment, especially in developing countries. The main mechanism of MO seed extract in coagulation is the positive protein component for charge neutralization. The method for efficient extraction of MO seed is very important for high coagulation activity. In this study, the effects of extraction mixing speed and extraction time of MO on coagulation activity were evaluated using a distilled water extraction method. Although the rotation per minute for extraction did not affect the coagulation efficiency, the extraction time strongly affected the coagulation efficiency of the extract. To evaluate the characteristic change of MO extract by extraction time, the charge of MO extract and protein characteristic in MO extract were analysed. As the extraction time was short, more positive charge and higher protein content were observed. For detailed protein analysis, the fluorescence spectroscopic study (EEM analysis) was performed. The tryptophan-like peak increased at longer extraction times. For efficient extraction of MO seed, a short extraction time is strongly recommended.

Performance of Natural Coagulant Extracted from Plantago ovata Seed for the Treatment of Turbid Water

Present study investigates the coagulation ability of Plantago ovata (P. ovata) seed extracts for turbidity removal. The active coagulant agents were successfully extracted from P. ovata seeds using different solvents such as distilled water (PO-DW), tap water (PO-TW), NaCl (PO-NaCl), and ammonium acetate (PO-AA). Experiments were conducted in batch mode for initial turbidity such as 500 NTU (high), 150 NTU (medium), and 50 NTU (low). Results demonstrated that P. ovata extracts are less efficient in low turbidities, while PO-NaCl was found to provide high coagulation activity in all initial turbidity concentrations compared to other extracts. PO-NaCl was able to remove 98.2, 94.9, and 80.2% of turbidity from water having in initial turbidities of 500, 150, and 50 NTU, respectively. Coagulation activity of the extract was the best when the extraction was performed for 50 min at room temperature. Jar test procedure with the coagulation time of 1 min and flocculation time of 30 min was optimized, irrespective of the initial turbidity. The optimum settling time for 500, 150, and 50 NTU water samples were 20, 30, and 90 min, respectively. PO-NaCl was used in different pH turbid solutions and it was found to be working very efficiently in alkaline conditions. The coagulation efficiency of the coagulant stored in refrigerator was higher than that stored at room temperature. Thus, the natural coagulants extracted from P. ovata seeds revealed to be effective for turbidity removal.

Dynamic change of plasma D-dimer level in patients with acute craniocerebral injury and cerebral hemorrhage and its significance

Objective To explore the dynamic change and clinical significance of plasma D-dimer level in patients with cerebral hemorrhage and acute craniocerebral injury. Methods 50 patients with cerebral hemorrhage and 40 patients with acute craniocerebral injury were selected, The enzyme-linked immunosorbent assay (ELISA) was used to measure plasma D-dimer level in two groups of patients after onset, and the results were compared with 40 healthy controls. Results The levels of plasma D-dimer in the patients with cerebral hemorrhage were 1.59mg/L, 2.10mg/L, 1.03mg/L, 0.82mg/L at 3h, 6h, 12h, 2d after onset, which in the patients with acute craniocerebral injury were 1.61mg/L, 2.02mg/L, 1.01mg/L and 0.67mg/L, respectively.And the plasma D-dimer levels were 0.50mg/L, 0.49mg/L, 0.47mg/L, 0.48mg/L in the control group at 3h, 6h, 12h and 2d after onset.The levels of plasma D-dimer in the patients with acute craniocerebral injury were significantly higher than those in the control group, and the differences were statistically significant (t=9.35, 12.17, 4.03, 3.05, all P 0.05). Conclusion Cerebral hemorrhage patients and acute craniocerebral injury patients have high coagulation and fibrinolytic activity in brief increase trend, dynamic observation of plasma D-dimer level in patients with cerebral hemorrhage and acute craniocerebral injury is helpful to determine courses, condition and evaluate prognosis. Key words: Craniocerebral trauma; Cerebral hemorrhage; D-dimer

Real-time investigation of dynamic morphology of live platelets and generation of platelet microparticles using hopping probe ion conductance microscopy

Platelets are rapidly activated by activators and produce a large number of platelet microparticles (PMPs) with high coagulation activity, resulting in coagulation dysfunction. However, the generation mechanism of PMPs is still not clear. Hopping probe ion conductance microscopy (HPICM) has special technical advantages in non-contact, real-time, high-resolution imaging of living cells under physiological conditions. Using HPICM, this study monitored the processes of platelet activation and generation of PMPs in real time in the presence of calcium ionophore A23187 and cytochalasin D (CD), respectively. The results proved that the intracellular calcium concentration and the cytoskeletal proteins played important roles in the platelet activation and the generation of PMPs. Compared with the low density spread shape platelets (LDSS), the high density bubble shape platelets (HDBS) were more sensitive to the calcium ionophore A23187 and cytochalasin D. This research has a guiding significance for the further study on the relationship between platelet activation and coagulation function using HPICM.

The effect of exercise on coagulation and fibrinolysis factors in patients with peripheral arterial disease.

Peripheral arterial disease is a widely prevalent atherosclerotic occlusive disorder. Symptoms commence with exercise-induced pain in the lower extremities, known as claudication. Despite the fact that exercise has been shown to improve fibrinolytic profile some patients, the effect of exercise on coagulation and fibrinolysis cascades in claudicants has not been comprehensively defined. Literature search in English language yielded 13 studies of exercise on claudicants, including 420 patients. Claudicants tend to have a higher coagulation activity at rest compared to healthy individuals, a trend that persists even after exercise. Post-exercise coagulation activity of claudicants is increased when compared to their respective baseline levels, but it is so in a non-consistent manner. From the available data, it has been suggested that claudicants have a functional and effective fibrinolytic mechanism in place, operating continuously at a relatively higher activity level compared to healthy individuals. Fibrinolysis seems to be activated by exercise; a positive outcome with a prolonged effect as shown by a few of the studies. A final conclusion whether coagulation or fibrinolysis activity is affected mostly by exercise type and intensity in claudicants could not be answered. All conclusions regarding the effect of exercise on the coagulation and fibrinolysis mechanisms should be taken under cautious consideration, due to the limited number of studies, the small number of patients and the different exercise strategies employed in each study. Further randomized studies with similar exercise protocols could provide safer conclusions in the future.

The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity

Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies.In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. DisclosuresNo relevant conflicts of interest to declare.

Evaluación de la producción experimental de enzimas coagulantes de leche utilizando cepas de Rhizomucor spp

&lt;p&gt;&lt;strong&gt;Título en ingles: Evaluation of experimental production of milk-clotting enzymes using &lt;em&gt;Rhizomucor&lt;/em&gt; spp&lt;em&gt; &lt;/em&gt;strains&lt;/strong&gt;&lt;strong&gt;&lt;/strong&gt;&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Título corto: Producción de enzimas coagulantes de leche&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Resumen&lt;/strong&gt;: La producción experimental de enzimas coagulante de leche se llevó a cabo&lt;em&gt; &lt;/em&gt;en un medio de cultivo de laboratorio durante 190 h de incubación, utilizando tres cepas certificadas de &lt;em&gt;Rhizomucor pusillus, &lt;/em&gt; &lt;em&gt;R.&lt;/em&gt; &lt;em&gt;miehei&lt;/em&gt; y dos especies nativas de &lt;em&gt;Rhizomucor spp. &lt;/em&gt;BIOMI-12 y 13. La evaluación se realizó midiendo la concentración de glucosa y proteína durante la incubación, estimación de la productividad, actividad específica, índice fuerza de cuajo/actividad proteolítica en los extractos enzimáticos crudos, determinación de los pesos moleculares y actividad proteolítica en los extractos enzimáticos parcialmente purificado. Todas las cepas mostraron un consumo de glucosa similar, el mismo comportamiento se observó en el contenido de proteína, excepto la cepa BIOM-13. Los incrementos en el contenido de proteínas después del descenso, coincidieron con la máxima actividad coagulante registrada por cada cepa, siendo el extracto crudo de la cepa BIOMI-13 la de mayor actividad coagulante (148,15 FC), productividad (3,09 FC/h), índice fuerza de cuajo/actividad proteolítica (142,60 FC/U) y actividad específica (1.062,00 FC/mg). Los extractos enzimáticos parcialmente purificados de las cepas &lt;em&gt;R miehei&lt;/em&gt; 37, &lt;em&gt;Rhizomucor spp&lt;/em&gt; BIOM-12 y 13, presentaron proteínas con pesos moleculares en aproximadamente 22,6 y 46,52 KDa, mientras el extracto &lt;em&gt;R pusillus&lt;/em&gt; 39 presentó una banda adicional de 39,6 KDa. En el zimograma se observó para todas las cepas actividad proteolítica en las bandas comprendidas entre 40-50 KDa y 20-22 KDa, no así para el &lt;em&gt;R pusillus&lt;/em&gt; 36, donde fue escasa. Finalmente se determinó que la cepa&lt;strong&gt; &lt;/strong&gt;BIOMI-13, tiene la mayor capacidad para producir enzimas coagulantes de la leche.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Palabras clave: &lt;/strong&gt;renina microbiana, fuerza de&lt;strong&gt; &lt;/strong&gt;cuajo, actividad proteolítica&lt;em&gt;, &lt;/em&gt;productividad.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Abstract: &lt;/strong&gt;Experimental production of milk clotting enzymes was conducted on a laboratory culture medium for 190 h incubation, using three certified strains of &lt;em&gt;Rhizomucor pusillus&lt;/em&gt;, &lt;em&gt;miehei &lt;/em&gt;and two&lt;em&gt; &lt;/em&gt;native &lt;em&gt;Rhizomucor &lt;/em&gt;spp. BIOMI-12 and 13. The evaluation was performed by measuring the concentration of glucose and protein during incubation, estimate productivity, specific activity, rennet strength/proteolytic activity index in the crude enzyme extracts, determining the molecular weights and proteolytic activity in the partially purified enzyme extracts. All strains showed consumption rates of glucose, the same behavior observed protein content, except strain BIOM-13. The increase in protein content after descent coincided with the recorded maximum coagulant activity each strains, being the crude extract of strain BIOMI-13 higher coagulant activity (148,15 FC), productivity (3.09 HR / h), rennet strength/proteolytic activity index (142,60 FC/U) and specific activity (1,062 FC/mg). The partially purified enzyme extracts from strains &lt;em&gt;R miehei&lt;/em&gt; 37, &lt;em&gt;Rhizomucor &lt;/em&gt;spp BIOM-12 and 13, presented proteins with molecular weights in approximately 22,6 kDa and 46.52, while the extract &lt;em&gt;R pusillus&lt;/em&gt; 39 present an additional band of 39,6 KDa. In the zymogram was observed for all strains, proteolytic activity in the bands between 40-50 KDa and 20-22 KDa, but not for the &lt;em&gt;R pusillus&lt;/em&gt; 36, where activity was very dim. Finally it was determined that the strain BIOMI-13, has the greatest capacity to produce milk clotting enzymes.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Key words&lt;/strong&gt;: microbial rennet, rennet strength, proteolitic activity,&lt;strong&gt; &lt;/strong&gt;productivity.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Recibido: &lt;/strong&gt;mayo 14 de 2014&lt;strong&gt; Aprobado: &lt;/strong&gt;abril 21 de 2015&lt;/p&gt;

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway.

A New Microparticle Coagulant Activity Assay to Predict Venous Thromboembolism in Patients with Pancreatic Cancer

Introduction: Patients with pancreatic cancer are at very high risk of venous thromboembolism (VTE). While the etiology of the prothrombotic state of these patients is not fully understood, emerging evidence suggests a causal role for circulating tumor-derived, tissue factor bearing microparticles (TFMP). We aim to assess whether a new, practical TFMP coagulant activity assay – the fibrin generation test (FGT) – is able to identify pancreatic cancer patients at the highest risk of VTE as they would benefit most from pharmaceutical thromboprophylaxis.Methods: In this ongoing prospective cohort study, patients with pancreatic cancer who have recently started neoadjuvant or palliative chemotherapy are enrolled after informed consent. Exclusion criteria are VTE within 3 months prior to inclusion and current anticoagulant use. Within 30 minutes from standardized blood withdrawal, the FGT is performed on fresh, essentially cell-free, citrated plasma. The time to fibrin formation is measured by optical densitometry after incubation of the samples with either saline or anti-factor VIIa, both in duplicate. The prolongation in the time to fibrin formation in the presence of anti-factor VIIa compared to saline reflects the contribution of the TFMP to coagulation. A predefined cut-off of 13% was used to indicate high TFMP coagulant activity. The primary outcome is objectively confirmed symptomatic or asymptomatic deep vein thrombosis (DVT) or pulmonary embolism (PE) during 6 months follow-up. We use multiple imputation techniques to avoid bias from excluding patients in whom the FGT result is unavailable due to non-adherence to the standardized blood withdrawal. We perform competing risk regression analysis to deal with death as competing event for the occurrence of VTE.Results: At present, 88 patients with pancreatic cancer have been enrolled. Baseline characteristics and follow-up data are presented in the Table. The TFMP coagulant activity as assessed with the FGT was high in 43 patients (48.9%). Fourteen percent (6/43) of patients with a high FGT result developed VTE, as compared to 2.6% (1/39) in patients with a low FGT result (OR 6.0; 95%CI 0.74-48.99). In the multivariate competing risk model including sex, age, tumor extent, and histology the adjusted OR was 6.04 (95%CI 0.75-48.8).Conclusion: Our preliminary results suggest that the FGT, a practical TFMP coagulant activity assay, has predictive value for VTE in patients with pancreatic cancer. Although the number of events at present is too low to draw definite conclusions, the FGT is a promising biomarker that could easily be implemented in clinical practice. If these results will be confirmed, the FGT may be used as a risk stratification tool to identify pancreatic cancer patients at high-risk for VTE and install thrombosis prophylaxis in these patients.TableBaseline characteristics and follow-up dataCharacteristicAll patients (N=88)Median age, years (IQR)62.6 (55.1-68.0)Mean BMI, kg/m2 (SD)26.3 (20)Male49 (55.7%)Performance statusWHO 050 (56.8%)WHO 132 (36.4%)WHO 26 (6.8%)Antiplatelet therapy12 (13.6%)Cancer detailsExtentLocal disease36 (40.9%)Resectable2 (2.3%)Borderline resectable8 (9.1%)Irresectable26 (29.5%)Distant metastasis52 (59.1%)Recurrent cancer8 (9.1%)Adenocarcinoma80 (90.9%)Median time from cancer diagnosis to inclusion, days (IQR)70 (47-103.5)ChemotherapyGemcitabin68 (77%)Neoadjuvant chemotherapy10 (11.4%)Median time from start chemotherapy to inclusion, days (IQR)14 (7-45)Median FGT, % (IQR)14.9 (0-42.9)FGT result unavailable6 (6.8%)Median follow-up, days (IQR)123 (4-180)Follow-up statusWithdrawn alive57 (64.8%)Death24 (27.3%)DVT or PE7 (8.0%) DisclosuresNo relevant conflicts of interest to declare.