Plasma Coagulation Factors Research Articles

Frequent, short bouts of light-intensity exercises while standing decreases systolic blood pressure: Breaking Up Sitting Time after Stroke (BUST-Stroke) trial.

Stroke survivors sit for long periods each day. Uninterrupted sitting is associated with increased risk of cardiovascular disease. Breaking up uninterrupted sitting with frequent, short bouts of light-intensity physical activity has an immediate positive effect on blood pressure and plasma clotting factors in healthy, overweight, and type 2 diabetic populations. We examined the effect of frequent, short bouts of light-intensity physical activity on blood pressure and plasma fibrinogen in stroke survivors. Prespecified secondary analyses from a three-armed randomized, within-participant, crossover trial. Participants were 19 stroke survivors (nine female, aged 68 years old, 90% able to walk independently). The experimental conditions were sitting for 8 h uninterrupted, sitting with 3 min bouts of light-intensity exercise while standing every 30 min, or sitting with 3 min of walking every 30 min. Blood pressure was measured every 30 min over 8 h and plasma fibrinogen at the beginning, middle, and end of each day. Intention-to-treat analyses were performed using linear mixed models including fixed effects for condition, period, and order, and a random intercept for participant to account for repeated measures and missing data. Sitting with 3 min bouts of light-intensity exercise while standing every 30 min decreased systolic blood pressure by 3.5 mmHg (95% CI 1.7-5.4) compared with sitting for 8 h uninterrupted. For participants not taking antihypertensive medications, sitting with 3 min of walking every 30 min decreased systolic blood pressure by 5.0 mmHg (95% CI -7.9 to 2.0) and sitting with 3 min bouts light-intensity exercise while standing every 30 min decreased systolic blood pressure by 4.2 mmHg (95% CI -7.2 to -1.3) compared with sitting for 8 h uninterrupted. There was no effect of condition on diastolic blood pressure (p = 0.45) or plasma fibrinogen levels (p = 0.91). Frequent, short bouts of light-intensity physical activity decreases systolic blood pressure in stroke survivors. However, before translation into clinical practice, the optimal duration and timing of physical activity bouts needs to be determined. Australian and New Zealand Clinical Trials Registry http://www.anzctr.org.au ANZTR12615001189516.

Alterations in platelet secretion differentially affect thrombosis and hemostasis

AbstractWe genetically manipulated the major platelet vesicle-associated membrane proteins (VAMP2, VAMP3, and VAMP8) to create mice with varying degrees of disrupted platelet secretion. As previously shown, loss of VAMP8 reduced granule secretion, and this defect was exacerbated by further deletion of VAMP2 and VAMP3. VAMP2Δ3Δ8−/− platelets also had reduced VAMP7. Loss of VAMP2 and VAMP3 (VAMP2Δ3Δ) had a minimal impact on secretion when VAMP7 and VAMP8 were present. Integrin αIIbβ3 activation and aggregation were not affected, although spreading was reduced in VAMP2Δ3Δ8−/− platelets. Using these mice as tools, we asked how much secretion is needed for proper thrombosis and hemostasis in vivo. VAMP2Δ3Δ mice showed no deficiency, whereas VAMP8−/− mice had attenuated formation of occlusive thrombi upon FeCl3-induced arterial injury but no excessive bleeding upon tail transection. VAMP2Δ3Δ8−/− mice bled profusely and failed to form occlusive thrombi. Plasma-coagulation factors were normal in all of the strains, but phosphatidylserine exposure was reduced in VAMP2Δ3Δ and VAMP2Δ3Δ8−/− platelets. From our data, an ∼40% to 50% reduction in platelet secretion in vitro (dense and α granule) correlated with reduced occlusive thrombosis but no compromise in hemostasis. At a >50% reduction, thrombosis and hemostasis were defective in vivo. Our studies are the first systematic manipulation of platelet exocytic machinery to demonstrate a quantitative linkage between in vitro platelet secretion and hemostasis and thrombosis in vivo. The animals described will be invaluable tools for future investigations into how platelet secretion affects other vascular processes.

Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism.

To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP-140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson's correlation. The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.

Diabetes mellitus: endothelial dysfunction and changes in hemostasis

Diabetes mellitus involves a group of chronic metabolic disorders with elevated blood glucose concentrations. Since this disease needs lifelong treatment and care, the medical and social aspects present major public health concerns and pose a global challenge for health care providers. The number of aged patients with degenerative diseases undergoing surgical procedures is continuously increasing, resulting in an overwhelming dominance of diabetes in the perioperative care. There is a particular need for an increased awareness of diabetic patients in cardiovascular units, where the incidence of this disease reaches as high as 30-40%. The main hallmarks of the pathologic metabolic milieu of diabetes are hyperglycaemia, insulin resistance and pathologic lipid metabolism. The biochemical, cellular and organ-level pathophysiological changes lead to endothelial dysfunction including a low-grade prothrombotic balance, inflammatory state and, as a consequence, impaired micro- and macrocirculation. Diabetes is also followed by platelet dysfunction resulting from intracellular hyperglycaemia, because thrombocytes have insulin-independent glucose transporters in their cell membrane. The levels of the coagulation factors of the plasma are increased, and these factors are also modified by oxidation and glycation. Diabetes mellitus is a prothrombotic condition resulting from direct and indirect tendencies of the endothelial platelet and the plasma coagulation factors. The basic "bench to clinical basics" knowledge of the endothelial dysfunction and prothrombotic balance in diabetes may contribute to the better understanding of the clinical focuses in the perioperative care of patients with diabetes mellitus. Orv Hetil. 2018; 159(33): 1335-1345.

Application of the ISTH bleeding score in hemophilia

BackgroundHemophilia is an inherited bleeding disorder. With proper treatment and self-care, persons with hemophilia can maintain an active, productive lifestyle. Hemophilia can be mild, moderate, or severe, depending on the degree of plasma clotting factor deficiency. The aim of the study was to assess the utility of ISTH-BAT in diagnosis, determining severity of the bleeding condition in newly diagnosed and known hemophilia patients, compare the bleeding score (BS) in adult and pediatric groups and investigate its association with plasma factor levels. MethodsISTH-BAT was used to assess BS in a total of 115 patients, 78 with hemophilia A, and 37 with hemophilia B and in 100 controls. ResultsBS was significantly higher in HA and HB patients as compared to controls, with no significant difference between HA and HB. The BS was very similar in newly diagnosed compared to known hemophilia patients, lower in pediatric compared to adult and higher in severe compared to mild HA patients. ConclusionThe ISTH BAT can help identify hemophilia patients. Therefore it is a useful tool to distinguish between affected and unaffected individuals with bleeding. Moreover, an important finding of our study is that there is no major difference between the scores in known and newly diagnosed patients.

Sacciform cells in the epidermis of fugu (Takifugu rubripes) produce and secrete kalliklectin, a novel lectin found in teleosts

Kalliklectin is a novel lectin identified in the skin mucus and blood plasma of teleosts, including fugu (Takifugu rubripes). It has been found to exhibit sequence similarity to mammalian plasma kallikrein and coagulation factor XI. The objective of the present study was to clarify the cellular localization of kalliklectin using an antiserum specific to fugu kalliklectin. Immunohistochemical analysis showed that positive reactions were observed in the skin and liver, but not in other tested tissues. Several types of epidermal cells were stained by the antiserum; sacciform cells were one of the types of cells most densely stained by the antiserum in adult fugu skin, whereas mucous cells showed negative staining results. RT-PCR demonstrated that the kalliklectin gene was transcribed in the mucous cell-poor region of adult fugu skin, where sacciform cells were present. These results indicated that epidermal cells, including sacciform cells, produce kalliklectin and secrete it into the mucus.

Abnormal clotting of the intrinsic/contact pathway in Alzheimer disease patients is related to cognitive ability

AbstractAlzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular β-amyloid (Aβ) deposition. Although peripheral inflammation and cerebrovascular pathology are reported in AD, there is a lack of plasma biomarkers in this field. Because the contact system is triggered in patient plasma, we hypothesized that the hemostasis profile could be a novel biomarker in AD. Here, we assessed the clotting profile in plasma from AD patients and age-matched controls. Utilizing clinically relevant assays, thromboelastography and activated partial thromboplastin time, we found impaired clot initiation and formation rate in AD patient plasma. These coagulation end points correlated with cerebrospinal fluid neurofilament-light levels and cognition and were more profound in younger AD patients. Ex vivo intrinsic clotting of plasma from AD mice expressing human amyloid precursor protein (APP) was also delayed in an age-dependent manner, suggesting that this phenotype is related to APP, the parent protein of Aβ. Further analysis of coagulation factors in human plasma indicated that endogenous inhibitor(s) of factors XII and XI in AD plasma contribute to this delayed clotting. Together, these data suggest that delayed clotting in young AD patients is a novel biomarker and that therapies aimed to correct this phenotype might be beneficial in this patient population. Follow-up studies in additional AD patient cohorts are warranted to further evaluate these findings.

Use of Rivaroxaban for Prophylaxis of Superficial Venous Thrombosis in Klippel-Trenaunay-Weber Syndrome

Klippel-Trenaunay-Weber syndrome (KTWS) is a congenital malformation syndrome involving blood and lymph vessels and disturbed bone and soft tissue growth. Complications of KTWS include deep-vein thrombosis, pulmonary embolism, gastrointestinal bleeding, and vascular (usually lymphatic) blebs within capillary malformations. We present a case of a young male patient with KTWS who presented with superficial venous thrombosis and microangiopathic hemolytic anemia in a presentation similar to disseminated intravascular coagulation. He was ultimately maintained on prophylactic rivaroxaban to prevent recurrent thrombotic events. We performed a literature search to identify similar cases and to summarize common presenting features and treatment modalities that were offered.
 Résumé
 Le syndrome de Klippel-Trenaunay-Weber (KTWS) est un syndrome de malformation congénitale impliquant le sang et les vaisseaux lymphatiques et les os perturbés et la croissance des tissus mous. Les complications de la KTWS comprennent la thrombose veineuse profonde, l'embolie pulmonaire, les saignements gastro-intestinaux et les bulles vasculaires (habituellement lymphatiques) dans les malformations capillaires. Nous présentons un cas d'un jeune patient mâle avec KTWS qui a présenté une thrombose veineuse superficielle et une anémie hémolytique microangiopathique dans une présentation semblable à la coagulation intravasculaire disséminée. Il a finalement été maintenu sur des rivaroxaban prophylactiques pour prévenir les épisodes récurrents de purpura. Nous avons effectué une recherche documentaire pour identifier des cas similaires et pour résumer les caractéristiques communes de présentation et les modalités de traitement qui ont été offerts.
 
 

Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality.

Photodynamic treatment using methylene blue (MB) and visible light is in routine use for pathogen inactivation of human plasma in different countries. Ambient and product temperature conditions for human plasma during production may vary between production sites. The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. Plasma units equilibrated to 5±2°C, room temperature (22±2°C) or 30±2°C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. Reduced solubility of the MB pill was observed at 5±2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30±2°C. Inactivation of suid herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5±2°C than at higher temperatures. MB/light treatment affected clotting times and the activity of almost all investigated plasma proteins. Factor VIII (-17·7±8·3%, 22±2°C) and fibrinogen (-14·4±16·4%, 22±2°C) showed the highest decreases in activity. Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended.

THE PROTHROMBIN TEST AND PIVKA-II IN BREASTFEDINFANTS AGED THE FIRST 6 MONTHS OF LIFE

The aim of the research was to study vitaminK-dependent indices of the prothrombin complex of hemostasis and the content of PIVKA-II substance, as a marker of vitamin K deficiency in children receivingbreastfeeding from birth to 6 months of life.Materials and methods. The study involved 247 children. The main cohort comprised 243 infants, approximately equally boys and girls who were born on the 38-40th week of gestation in a natural way withoutbirth trauma with an assessment ву the V. Apgar score of 7-10 points and who received exclusively breastfeeding from the moment of birth and up to 6 months of life. All of them at the time of birth were found healthy. In 67 breastfed infants the capillary blood were examined forprothrombin time (PTT),prothrombin by Quick ( PT ), international normalized ratio(INR).The concentrations of PIVKA-II in the serum of venous blood were studied in 180 newborn and infants.Results. The parameters of the prothrombin complex were highly correlated with each other and with the concentration of PIVKA-II. In newborns, PTT and PT had only a tendency to exhibit hypocoagulationproperties, whether they received or did not receive a prophylactic intramuscular injection of vitaminК1in the amount of 1 mg. In children aged 1- 2 mo and 3 to 6 mo, PT was significantly higher than one prone to adults while PT was at the lower acceptable limits regardlessof the fact of vitaminК1 administration. Also the INR in neonates and especially in infants 1- 2 mo of life was characterized by values of the obvious hypocoagulation. Abnormal high plasma concentrations of PIVKA-II – (67,9 ± 10,8) Au / ml were detected in newborns who didnot receive preventive dose of vitamin K immediately after birth. In contrast children who have received a prophylactic dose of phyloqinone were almost identical to normal PIVKA-II values 45.7 ± 5.4 Au / ml. Infants aged of 1-6 mo after the course of antibacterial therapyhave demonstrated laboratory signs of significant hypocoagulation quite sufficient for the development of late neonatal hemorrhagic disease.Conclusions. Vitamin-K-dependent parameters of the prothrombin test as a INR, PTT, PT and PIVKAII in the overwhelming number of children of the first month of life who did not receive a prophylactic dose of vitamin K after birth, reveal hypocoagulation due to congenital vitamin K deficiency. In age of 1-2 months the PIVKA-II in healthy exclusively breastfed children gets normal. Simultaneously INR and PT are characterized by considerable variability and reach the value ofhypocoagulation despite the vitamin K administration after the birth.The development of intestinal microbial biocinosis contributes to the establishment of normal prothrombin test and PIVKA-II concentrations. The use of antibiotics in children of the first 6 months of life who receive breastfeeding can lead to the renewal of vitamin-K deficiency and the development of antibioticassociated disorders of plasma coagulation factors.

Using pharmacokinetics to individualize hemophilia therapy.

Prevention and treatment of bleeding in hemophilia requires that plasma clotting factor activity of the replaced factor exceeds a defined target level. Most clinical decisions in hemophilia are based on implicit or explicit application of pharmacokinetic measures. The large interindividual variability in pharmacokinetics of factor concentrates suggests that relying on the average pharmacokinetic characteristics of factor concentrates would not allow optimizing the treatment of individual patients; for example, adjusting the frequency of infusions and targeting a specific clotting factor activity level on a case-by-case basis. However, individual pharmacokinetic profiles are seldom assessed as part of routine clinical care. Population pharmacokinetics provide options for precise and convenient characterization of pharmacokinetics characteristics of factor concentrates, simplified individual pharmacokinetic profiling, and individualized dosing. Population pharmacokinetics allow for the incorporation of determinants of interpatient variability and reduces the need for extensive postinfusion plasma sampling. Barriers to the implementation of population pharmacokinetics are the need for concentrate-specific pharmacokinetic models, Bayesian calculation power, and specific expertise for production, validation, and appraisal of forecasted estimates. Population pharmacokinetics provide an important theoretical and practical contribution to tailoring the treatment of hemophilia. The need remains for prospective exploration of the clinical impact of tailoring hemophilia treatment based on individual pharmacokinetics, and for the systematic validation of existing software solutions and concentrate-specific models.

In Vitro Assessment of Nanoparticle Effects on Blood Coagulation.

Blood clotting is a complex process which involves both cellular and biochemical components. The key cellular players in the blood clotting process are thrombocytes or platelets. Other cells, including leukocytes and endothelial cells, contribute to clotting by expressing the so-called pro-coagulant activity (PCA) complex on their surface. The biochemical component of blood clotting is represented by the plasma coagulation cascade, which includes plasma proteins also known as coagulation factors. The coordinated interaction between platelets, leukocytes, endothelial cells, and plasma coagulation factors is necessary for maintaining hemostasis and for preventing excessive bleeding. Undesirable activation of all or some of these components may lead to pathological blood coagulation and life-threatening conditions such as consumptive coagulopathy or disseminated intravascular coagulation (DIC). In contrast, unintended inhibition of the coagulation pathways may lead to hemorrhage. Thrombogenicity is the property of a test material to induce blood coagulation by affecting one or more elements of the clotting process. Anticoagulant activity refers to the property of a test material to inhibit coagulation. The tendency to cause platelet aggregation, perturb plasma coagulation, and induce leukocyte PCA can serve as an in vitro measure of a nanomaterial's likelihood to be pro- or anticoagulant in vivo. This chapter describes three procedures for in vitro analyses of platelet aggregation, plasma coagulation time, and activation of leukocyte PCA. Platelet aggregation and plasma coagulation procedures have been described earlier. The revision here includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, and updated details about assay controls. The chapter is expanded to describe a method for the leukocyte PCA analysis and case studies demonstrating the performance of these in vitro assays.

Plasmatic coagulation and fibrinolysis in healthy and Otostrongylus-affected Northern elephant seals (Mirounga angustirostris).

Prepatent Otostrongylus arteritis results in hemorrhagic diathesis in free-ranging Northern elephant seals (Mirounga angustirostris) attributed to aberrant larval migration of the lungworm, Otostrongylus circumlitus. Clinical signs are often nonspecific, including lethargy, anorexia, and blepharospasm, but can progress to spontaneous frank hemorrhage and death within 72hours of onset. Previously published case reports describe coagulopathy with prolonged PT and APTT, normal to elevated platelet counts, normal antithrombin concentrations, and low concentrations of fibrinogen degradation products. Disseminated intravascular coagulation was proposed as the cause of hemorrhage, but is inconsistent with some of the reported clinicopathologic changes. The purpose of this study was to compare plasmatic coagulation and fibrinolysis in healthy and Otostrongylus-affected elephant seals, in order to identify potential therapy. We hypothesized that hyperfibrinolysis contributed to hemorrhage in these cases. Citrated plasma samples were collected from 3- to 4-month-old Northern elephant seals in a wildlife rehabilitation hospital. The sampled population included 25 healthy, prerelease seals and 32 clinically ill seals diagnosed with presumptive Otostrongylus arteritis. Twenty-one of the included seals had Otostrongylus infestation confirmed at necropsy. Standard coagulation tests and plasma thromboelastography were performed for a complete assessment of coagulation and fibrinolysis. Northern elephant seals with definitive Otostrongylus infestation were hypocoagulable and hypofibrinolytic compared to healthy controls. Results were most consistent with disseminated intravascular coagulation. Treatment with antifibrinolytic drugs to control hemorrhage may be unrewarding; alternative therapies such as plasma transfusions or coagulation factor concentrates should be investigated.

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIb\u03b2 expression

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1−/− mice, but not Epac2−/− mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1−/− mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1−/− mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bβ and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1−/− mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bβ level was decreased also in Epac1−/− bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1−/− mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1−/− is connected to secondary hemostasis.

Significant inhibition of the triggering mechanism of the extrinsic system of blood coagulation in rats by thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4

INTRODUCTION : The role of the thymus gland and the effects of thymic peptides on the coagulation status are relatively insufficiently studied. Recent data indicate controversial and not persuasive results due to in­vestigation of single hemostasis parameters and application of various pathological models. AIM : The present study aimed to elucidate the influence of thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4 applied s.c. on intact rats, on key triggering factors of blood coagulation via the extrin­sic pathway: plasma concentration of tissue factor (TF); concentration and activity of free TFPI; and the ac­tivity of plasma clotting factor VII, as well as on prothrombin time (PT) - an integral clinical parameter of the intrinsic pathway. MATERIALS AND METHODS : The study was performed on 52 male Wistar strain rats, weighing 200-220 g and bred under standard conditions at dark/light cycle 12/12 h. The rats were divided into four equal groups (n=13) and injected s.c. once daily in the time interval 08.00-09.00 h in the course of three consecutive days as follows: 1st group (controls) - with saline (the solvent of the peptides); 2nd group - with thymulin (Sigma Aldrich, 0.4 mg/kg b.m.); 3rd group - with thymosin alpha 1 (Sigma Aldrich, 0.3 mg/kg b.m.); and the 4th group - with thymosin beta 4 (Sigma Aldrich, 0.3 mg/kg b.m.). RESULTS : Results indicate that thymulin and thymosin alpha 1 expressively decrease TF and increase TFPI concentrations. All three thymic peptides reduce FVIIa activity, increase TFPI activity, and significantly elongate PT. CONCLUSIONS : Thymus peptides thymulin, thymosin alpha 1 and thymosin beta 4 significantly suppress blood coagulation through the extrinsic pathway and generate a distinct tendency for hypocoagulability.

Development of aptameric affinity ligands specific to human plasma coagulation factor VIII using SEC-SELEX

Protein specific aptamers are highly applicable affinity ligands in different fields of research and clinical applications. They have been developed against various targets, in particular, bio-macromolecules such as proteins. Among human proteins, the coagulation factors are the most attractive targets for aptamer selection and their specific aptamers had valuable characteristics in therapeutic and analytical applications. In this study, a plasma derived coagulation factor VIII was considered as the protein target for DNA aptamer selection using size exclusion chromatography-SELEX. Potential aptameric oligonucleotides with high affinity and specificity were achieved during eight rounds of selection. Binding affinity constant of selected aptamer and aptameric enriched pool were in nanomolar range that was comparable to monoclonal antibodies. Further improvement studies can result in aptamers that are more promising as an industrial affinity ligand for the purification of anti-hemophilia factor from plasma source.

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1–4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. SignificanceFull sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and compared. A novel, simplified phylogenetic tree based on the sequences of GiSPs and their closely related orthologs from other pitvipers reveals six major subtypes and offers a better understanding of vSP duplication and evolution in pitvipers of both the Old and New Worlds. It is well known that specific vSPs are potential therapeutic or diagnostic agents that target the plasma proteins or coagulation factors. Our results not only render deeper insights into the variation and evolution of vSPs, but may help to choose right venoms for the development of better therapeutic leads.

Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease

Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances.

Opposite effects of Agrimonia pilosa Ledeb aqueous extracts on blood coagulation function

Agrimonia pilosa Ledeb (APL) has showed anticoagulant and antithrombotic activities in some studies, whereas its actual effects on blood coagulation are still unclear. This study was designed to observe the in vitro effects of APL aqueous extracts on blood coagulation, as well as to investigate the underlying mechanisms. Studies were divided into four groups: 0, 4, 20, and 80 g/L of APL aqueous extracts mixed with plasma or whole blood samples. Clotting time of whole blood, plasma coagulation tests, activities of plasma coagulation factors, plasma calcium ion, platelet aggregation test, and platelet fibrinogen receptor as well as the blood viscosity were measured. It was observed that the APL aqueous extracts in 4 g/L significantly prolonged the whole blood clotting time and activated partial thromboplastin time, shortened prothrombin time, decreased activities of coagulation factor VIII, IX and XI, and levels of platelet aggregation and fibrinogen receptor expression. However, coagulation factor VII activity, and blood viscosity were increased after the extracts treatment. And the effects of APL extracts were in a concentration-dependent manner (0-80 g/L). The results suggest that APL aqueous extracts have a total anticoagulant activity, whereas they exhibit opposite effects of greater anticoagulant activity than pro-coagulant activity.

Influences of ABO blood group, age and gender on plasma coagulation factor VIII, fibrinogen, von Willebrand factor and ADAMTS13 levels in a Chinese population.

BackgroundABO blood group is a hereditary factor of plasma levels of coagulation factor VIII (FVIII) and von Willebrand factor (VWF). Age and gender have been shown to influence FVIII, VWF, fibrinogen (Fbg), and ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 motif, 13). We investigated the effects of ABO type, age, and gender on plasma levels of FVIII, Fbg, VWF, and ADAMTS13 in a Chinese population.MethodsA total of 290 healthy volunteers were eligible for this study. ABO blood group was determined by indirect technique. FVIII:C and Fbg were measured by clotting assays. VWF antigen (VWF:Ag), collagen-binding activity (VWF:CBA), and ADAMTS13 antigen were assessed by ELISA, whereas VWF ristocetin cofactor activity (VWF:Rcof) was performed by agglutination of platelets with ristocetin.ResultsMean FVIII:C and VWF levels (VWF:Ag, VWF:CBA, and VWF:Rcof) were significantly higher in non-O than in O type subjects (p < 0.05 for all comparison). ADAMTS13 antigen decreased with increasing age, whereas the other parameters increased. Other than ADAMTS13 (p < 0.01), no gender-related variations were observed in the other parameters. Moreover, FVIII:C, Fbg, VWF:Ag, VWF:CBA, and VWF:Rcof showed significant and positive relationships with age (r = 0.421, 0.445, 0.410, 0.401, and 0.589, resp.; all p < 0.001), whereas a negative relationship was observed for ADAMTS13 antigen (r = 0.306; p = 0.006). Furthermore, FVIII:C were strongly correlated with VWF:Ag, VWF:CBA, and VWF:Rcof (r = 0.746, r = 0.746, and r = 0.576, resp.; p < 0.0001). VWF parameters were also strongly correlated with each other (r = 0.0.847 for VWF:Ag and VWF:CBA; r = 0.722 for VWF:Ag and VWF:Rcof; p < 0.0001).ConclusionsABO blood group, age, and gender showed different effects on plasma levels of FVIII:C, Fbg, VWF:Ag, VWF:CBA, VWF:Rcof, and ADAMTS13 antigen. These new data on a Chinese population are quite helpful to compare with other ethnic groups.