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1.
Int J Mol Sci ; 22(2)2021 Jan 06.
Article in English | MEDLINE | ID: mdl-33419227

ABSTRACT

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.


Subject(s)
Antithrombin Proteins/chemistry , Antithrombin Proteins/metabolism , Protein Conformation , Protein Processing, Post-Translational , Antithrombin III/chemistry , Antithrombin III/genetics , Antithrombin III/metabolism , Antithrombin Proteins/genetics , Circular Dichroism , Glycosylation , Humans , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Thrombosis
2.
Biotechnol Lett ; 42(1): 103-114, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31686286

ABSTRACT

OBJECTIVE: Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris. RESULT: Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 µg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay. CONCLUSION: Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.


Subject(s)
Metalloendopeptidases/metabolism , Pichia/metabolism , Recombinant Fusion Proteins/metabolism , Thrombolytic Therapy/methods , Antithrombin Proteins/chemistry , Antithrombin Proteins/genetics , Antithrombin Proteins/metabolism , Humans , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Molecular Dynamics Simulation , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/metabolism , Pichia/genetics , Protein Conformation , Recombinant Fusion Proteins/genetics
3.
Pediatr Blood Cancer ; 66(7): e27729, 2019 07.
Article in English | MEDLINE | ID: mdl-30938058

ABSTRACT

BACKGROUND: L-asparaginase is a cornerstone treatment for children with acute lymphoblastic leukemia (ALL). However, immune reaction to the drug may increase the clearance or impair the function of L-asparaginase and reduces its therapeutic efficacy. The objective of this study was to identify potential plasma proteins that could be used as proxies for L-asparaginase activity. METHODS: Fibrinogen, von Willebrand factor antigen (VWF:Ag), total protein, and albumin levels as well as antithrombin (AT) and L-asparaginase activities were measured in 97 children with ALL treated for prolonged period of time with L-asparaginase. Binary logistic regression and a receiver operating characteristic (ROC) curve analysis were performed to evaluate the predictive value of plasma proteins for L-asparaginase activity. RESULTS: Median E. coli L-asparaginase activity was 220 IU/L (range, 0-1308) throughout the treatment period. L-asparaginase activity was below 100 IU/L in 23% of measured samples. L-asparaginase activity was inversely associated with AT activity, fibrinogen, total protein, and albumin levels (r = -0.63, -0.62, -0.57, and -0.45, respectively; P < 0.0001), but not with VWF:Ag. ROC curve analyses showed an intermediate accuracy of AT activity (area under the ROC curve [AUC] = 0.77) to detect specimens with subtherapeutic level of L-asparaginase. An optimal accuracy was found when AT and fibrinogen were combined (AUC = 0.82; sensitivity = 75%; specificity = 82%; positive predictive value = 55%; negative predictive value = 92%) with cutoff values of 0.73 IU/mL and 1.85 g/L, respectively. CONCLUSIONS: AT combined with fibrinogen levels could be used as a proxy to identify patients with therapeutic level of L-asparaginase activity in the absence of real-time asparaginase measurement during prolonged exposure to L-asparaginase.


Subject(s)
Antithrombin Proteins/metabolism , Asparaginase/administration & dosage , Fibrinogen/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Time Factors
4.
J Clin Pathol ; 71(7): 614-619, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29331940

ABSTRACT

AIMS: Dysfunctional prothrombin residue Arg596 associated mutation has been found to precipitate venous thromboembolism (VTE). In the current study we investigated the prevalence of Arg596 associated mutations in Chinese patients with VTE and explored the functional impact of Arg596Gln mutation on coagulation function in affected patients. METHODS: Prothrombin clotting activity was measured in 267 unrelated patients with unprovoked VTE. Patients with moderately decreased activities underwent further analysis of the F2 gene. Prothrombin amidolytic activity and antigen levels were detected in mutation carriers. Specific family members were investigated about their VTE histories and clinical phenotypes. The thrombin generation test (TGT) was used to evaluate thrombin function and antithrombin resistance assay was applied to assess the extent of impaired antithrombin inhibition of mutation carriers. RESULTS: Two heterozygous mutation carriers of prothrombin Arg596Gln were identified, both of whom had moderately decreased clotting activities but normal amidolytic activities and antigen levels. Among the families of the two probands, nine out of 13 mutation carriers experienced episodes of VTE. TGTs showed that patients had elevated endogenous thrombin potential and prolonged start tail time. Thrombin generation could be inhibited in the presence of thrombomodulin. The thrombin Arg596Gln variant in patients' plasma presented strong resistance to antithrombin inhibition. CONCLUSION: Prothrombin Arg596Gln mutation is a risk factor for Chinese patients with VTE due to its moderately decreased clotting activity but strong resistance to antithrombin inhibition. Prothrombin clotting activity screening and its encoding gene sequencing should be considered in patients with VTE when other established risk factors are absent.


Subject(s)
Blood Coagulation/genetics , DNA Mutational Analysis , Genetic Testing/methods , Mutation , Prothrombin/genetics , Venous Thromboembolism/genetics , Adult , Antithrombin Proteins/metabolism , Asian People/genetics , Blood Coagulation Tests , Case-Control Studies , China/epidemiology , Female , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Heredity , Heterozygote , Humans , Male , Middle Aged , Pedigree , Phenotype , Risk Factors , Venous Thromboembolism/blood , Venous Thromboembolism/diagnosis , Venous Thromboembolism/ethnology
5.
Int J Lab Hematol ; 40(3): 251-257, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29356352

ABSTRACT

INTRODUCTION: Factor VII activation occurs postprandially. A proportion of activated factor VII (VIIa) circulates in complex with antithrombin (VIIaAT). Our primary objective was to assess the effects of postprandial lipemia on circulating VIIaAT, procoagulant phospholipid (PPL) activity, and thrombin generation. METHODS: Plasma samples from postmyocardial infarction patients (n = 40) and controls (n = 39) were taken before and at 3 and 6 hours during a standardized oral fat tolerance test (OFTT). Fasting PPL activity measurements were also made in a second cohort of 108 postinfarction patients and 109 controls. VIIaAT was analyzed with the Asserachrom VIIaAT ELISA, PPL activity with the STA-Procoag-PPL kit, and thrombin generation with calibrated automated thrombogram with PRP-Reagent as trigger (all Diagnostica Stago products). RESULTS: Postprandially, VIIaAT increased in all samples without significant case-control differences in the overall response during the OFTT. Thrombin generation measures peak height and velocity, and PPL activity, were marginally affected by the test meal in the controls. Levels of all patient baseline measures were significantly different from controls, indicating a more hypercoagulable state, and these differences were maintained throughout the OFTT. Fasting samples from cases showed higher PPL activity than control samples. CONCLUSION: Viewing VIIaAT quantitation as a surrogate for TF activity measurement, postprandial increase in VIIaAT may reflect a mechanism that adds to the cardiovascular risk associated with postprandial lipemia. On the other hand, the impact of postprandial lipemia on PPL activity and thrombin generation seems to be minor.


Subject(s)
Antithrombin III/metabolism , Factor VIIa/metabolism , Hyperlipidemias/metabolism , Phospholipids/metabolism , Postprandial Period , Thrombin/biosynthesis , Adult , Antithrombin Proteins/metabolism , Cardiovascular Diseases/etiology , Case-Control Studies , Female , Humans , Hyperlipidemias/complications , Male , Middle Aged , Myocardial Infarction , Protein Binding
6.
Int J Hematol ; 107(4): 490-494, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29071478

ABSTRACT

Inherited antithrombin (AT) deficiency is one of the most clinically significant forms of congenital thrombophilia and follows an autosomal dominant mode of inheritance. We analyzed SERPINC1 in a patient who developed deep-vein thrombosis and low AT activity during pregnancy, and identified a novel missense mutation c.259A>G (p.Asn87Asp; N87D). Surprisingly, analysis of the parents' DNA showed that they did not possess this mutant, and thus, it may have been due to a de novo mutation. We also expressed this mutant AT protein in COS-1 cells and compared its intracellular localization and intracellular and extracellular antigen levels with that of wild-type AT. The expression experiment did not reveal a significant difference in the antigen levels of the mutant and wild-type AT in the cell lysate, but the mutant AT antigen level was markedly lower than that of its wild-type counterpart in the COS-1 cell supernatant. Immunofluorescence did not indicate any difference between the mutant and wild-type AT in terms of cytoplasmic localization of fluorescence signals. Our findings suggest that the patient's AT deficiency may have been caused by impaired extracellular secretion of mutant AT protein p.Asn87Asp.


Subject(s)
Antigens/metabolism , Antithrombin III Deficiency/genetics , Antithrombin III/genetics , Antithrombin Proteins/genetics , Antithrombin Proteins/physiology , Mutation, Missense , Pregnancy Complications/genetics , Adult , Animals , Antithrombin Proteins/immunology , Antithrombin Proteins/metabolism , COS Cells , Chlorocebus aethiops , Female , Humans , Pregnancy , Thrombophilia/genetics , Venous Thrombosis
7.
J Biol Chem ; 292(40): 16513-16520, 2017 10 06.
Article in English | MEDLINE | ID: mdl-28743742

ABSTRACT

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.


Subject(s)
Antithrombin Proteins/chemistry , Blood Coagulation Disorders, Inherited , Factor Xa/chemistry , Molecular Docking Simulation , Thrombin/chemistry , Amino Acid Substitution , Antithrombin Proteins/genetics , Antithrombin Proteins/metabolism , Catalytic Domain , Factor Xa/genetics , Factor Xa/metabolism , Female , Humans , Male , Mutation, Missense , Protein Domains , Protein Structure, Secondary , Thrombin/genetics , Thrombin/metabolism
8.
BMC Vet Res ; 13(1): 182, 2017 Jun 19.
Article in English | MEDLINE | ID: mdl-28629364

ABSTRACT

BACKGROUND: Septic arthritis is a common and potentially devastating disease characterized by severe intra-articular (IA) inflammation and fibrin deposition. Research into equine joint pathologies has focused on inflammation, but recent research in humans suggests that both haemostatic and inflammatory pathways are activated in the joint compartment in arthritic conditions. The aim of this study was to characterize the IA haemostatic and inflammatory responses in horses with experimental lipopolysaccharide (LPS)-induced joint inflammation. Inflammation was induced by IA injection of LPS into one antebrachiocarpal joint of six horses. Horses were evaluated clinically with subjective grading of lameness, and blood and synovial fluid (SF) samples were collected at post injection hours (PIH) -120, -96, -24, 0, 2, 4, 8, 16, 24, 36, 48, 72 and 144. Total protein (TP), white blood cell counts (WBC), serum amyloid A (SAA), haptoglobin, iron, fibrinogen, thrombin-antithrombin (TAT) and d-dimer concentrations were assessed in blood and SF. RESULTS: Intra-articular injection of LPS caused local and systemic signs of inflammation including increased rectal temperature, lameness and increased joint circumference and skin temperature. Most of the biomarkers (TP, WBC, haptoglobin, fibrinogen and TAT) measured in SF increased quickly after LPS injection (at PIH 2-4), whereas SAA and d-dimer levels increased more slowly (at PIH 16 and 144, respectively). SF iron concentrations did not change statistically significantly. Blood WBC, SAA, haptoglobin and fibrinogen increased and iron decreased significantly in response to the IA LPS injection, while TAT and d-dimer concentrations did not change. Repeated pre-injection arthrocenteses caused significant changes in SF concentrations of TP, WBC and haptoglobin. CONCLUSION: Similar to inflammatory joint disease in humans, joint inflammation in horses was accompanied by an IA haemostatic response with changes in fibrinogen, TAT and d-dimer concentrations. Inflammatory and haemostatic responses were induced simultaneously and may likely interact. Further studies of interactions between the two responses are needed for a better understanding of pathogenesis of joint disease in horses. Knowledge of effects of repeated arthrocenteses on levels of SF biomarkers may be of value when markers are used for diagnostic purposes.


Subject(s)
Arthritis, Experimental/veterinary , Biomarkers/metabolism , Horse Diseases/metabolism , Synovial Fluid/metabolism , Animals , Antithrombin Proteins/metabolism , Arthritis, Experimental/blood , Arthritis, Experimental/metabolism , Arthrocentesis/veterinary , Biomarkers/blood , Female , Fibrin Fibrinogen Degradation Products/metabolism , Fibrinogen/metabolism , Hemostasis/drug effects , Horse Diseases/immunology , Horses , Inflammation/metabolism , Injections, Intra-Articular , Lameness, Animal/chemically induced , Lameness, Animal/metabolism , Lipopolysaccharides , Male , Thrombin/metabolism
9.
Menopause ; 24(4): 360-370, 2017 04.
Article in English | MEDLINE | ID: mdl-27922933

ABSTRACT

OBJECTIVE: Vasomotor symptoms (VMS) may be a marker of cardiovascular risk. We aimed to evaluate the cross-sectional association of VMS presence and severity with hemostatic parameter levels measured at baseline among Women's Health Initiative (WHI) Hormone Therapy trial postmenopausal participants. METHODS: This cross-sectional analysis included 2,148 postmenopausal women with measures of VMS presence and severity reported in the 4 weeks before WHI baseline, who were not using warfarin or hormone therapy and for whom the following baseline hemostatic parameters were measured within the WHI Cardiovascular Disease Biomarker Case-Control Study: antithrombin, plasminogen activator inhibitor-1, protein C antigen, total and free protein S antigen, total and free tissue factor pathway inhibitor, D-dimer, normalized activated protein C sensitivity ratio, and thrombin generation. Using multiple linear regression, we estimated the adjusted average difference in each hemostatic parameter associated with VMS presence and severity. A multiple comparisons-corrected P value was computed using the P-min procedure to determine statistical significance of our smallest observed P value. RESULTS: Women were 67 years of age on average and 33% reported VMS presence at baseline. There was some suggestion that VMS presence may be associated with a -0.34 adjusted difference in normalized activated protein C sensitivity ratio compared with no VMS (95% CI, -0.60 to -0.087; P = 0.009), but this association was not significant after correction for multiple comparisons (P = 0.073). VMS presence or severity was not significantly associated with the other hemostatic parameters. CONCLUSIONS: We found no convincing evidence that VMS presence or severity was associated with levels of hemostatic parameters among postmenopausal women.


Subject(s)
Hot Flashes/blood , Postmenopause/blood , Sweating , Aged , Antigens/blood , Antithrombin Proteins/metabolism , Cross-Sectional Studies , Female , Fibrin Fibrinogen Degradation Products/metabolism , Hemostasis , Humans , Lipoproteins/blood , Middle Aged , Plasminogen Activator Inhibitor 1/blood , Protein C/immunology , Protein S/immunology , Severity of Illness Index , Symptom Assessment , Thrombin/biosynthesis
10.
Thromb Haemost ; 115(6): 1090-100, 2016 06 02.
Article in English | MEDLINE | ID: mdl-26819003

ABSTRACT

Thrombin generation (TG) is decreased in children. TG is determined by two underlying processes: the conversion of prothrombin to thrombin and the inactivation of thrombin. Therefore, lower TG capacity in children can either be caused by a reduction of prothrombin conversion, an increase of thrombin inactivation, or both. In 36 children and 8 adults, TG and the factors that determine thrombin inactivation (antithrombin, α2Macroglobulin (α2M) and fibrinogen) were measured. Prothrombin conversion, thrombin inhibitor complex formation, and the overall thrombin decay capacity were determined. In silico modelling was performed to determine the contribution prothrombin conversion and thrombin inactivation to deviant paediatric TG. Both the amount of prothrombin converted and the maximal prothrombin conversion rate are significantly reduced in children as compared to adults. This is partly due to the prothrombin levels being lower and partly to a lower prothrombin conversion rate. The overall thrombin decay capacity is not significantly different in children, but α2Macroglobulin plays a more important role than it does in adults. In silico experiments demonstrate that reduced prothrombin conversion and to a lesser extent elevated α2M levels provide an explanation for low TG in children. Young age has a dual effect on prothrombin conversion. Lower plasma prothrombin levels result in decreased prothrombin conversion but the rate of prothrombin conversion is also decreased, i. e. the development of prothrombinase is lower than in adults.


Subject(s)
Prothrombin/metabolism , Thrombin/biosynthesis , Adolescent , Adult , Age Factors , Antithrombin Proteins/metabolism , Blood Coagulation/physiology , Child , Child, Preschool , Female , Fibrinogen/metabolism , Humans , Infant , Male , Middle Aged , Models, Biological , Pregnancy-Associated alpha 2-Macroglobulins/metabolism , Thrombin/antagonists & inhibitors , Young Adult
11.
Glycobiology ; 26(5): 482-92, 2016 May.
Article in English | MEDLINE | ID: mdl-26747427

ABSTRACT

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the ß-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the ß-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The ß-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the ß-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The ß-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood.


Subject(s)
Antithrombin Proteins/metabolism , Heparin/metabolism , Mannose/metabolism , Oligosaccharides/metabolism , Antithrombin Proteins/chemistry , Glycosylation , Heparin/chemistry , Humans , Mannose/chemistry , Oligosaccharides/chemistry , Protein Binding
12.
J Thorac Cardiovasc Surg ; 151(2): 444-50, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26553458

ABSTRACT

BACKGROUND: Antithrombin is one of the main natural coagulation system inhibitors. It is potentiated by heparin, and may be a key component of heparin response, particularly in infants aged <1 year. We sought to determine the impact of baseline antithrombin activity on response to heparin and thrombin generation during cardiopulmonary bypass (CPB). METHODS: Secondary analysis was performed using linear regression analyses, which combined patients from a trial of individualized versus weight-based heparin management for 90 infants aged <1 year undergoing cardiac surgery. RESULTS: Mean baseline antithrombin activity was 0.69 ± 0.16 U/mL, and it was lower in neonates than in older infants (0.57 ± 0.15 vs 0.77 ± 0.12 U/mL; P < .001). Lower baseline antithrombin activity was associated with lower postheparin anti-Xa activity (EST [SE]: +0.47 (0.19) U/mL per 100 U/kg heparin; P = .01) and higher heparin doses during surgery (EST [SE]: +51 (17) U/kg per hour; P = .003). The administration of fresh frozen plasma attenuated the effect of low baseline antithrombin activity (interaction P value = .009). Patients with lower anti-Xa activity recorded during CPB had higher levels of thrombin-antithrombin complex (EST [SE]: +12.8 (4.7) ng/mL per -1 U/mL anti-Xa; P = .006); prothrombin activation fragment 1.2 (EST [SE]: +0.13 (0.07) log pg/mL per -1 U/mL anti-Xa; P = .06); and D-dimer (EST [SE]: -0.25 (0.09) log ng/mL per -1 U/mL anti-Xa; P = .009) in the postoperative period after adjustment for baseline antithrombin activity, duration of CPB, amount of fresh frozen plasma and heparin used throughout surgery in multivariable models. CONCLUSIONS: Low circulating antithrombin activity is associated with lower heparin efficacy, which ultimately leads to a lower ability to suppress thrombin generation during CPB. Determination of risk factors for heparin resistance, and potentially, antithrombin replacement therapy, may individualize and improve anticoagulation treatment.


Subject(s)
Anticoagulants/administration & dosage , Antithrombin Proteins/metabolism , Blood Coagulation/drug effects , Cardiac Surgical Procedures , Cardiopulmonary Bypass , Heparin/administration & dosage , Thrombin/metabolism , Age Factors , Anticoagulants/adverse effects , Blood Coagulation Tests , Blood Component Transfusion , Blood Loss, Surgical/prevention & control , Cardiac Surgical Procedures/adverse effects , Cardiopulmonary Bypass/adverse effects , Drug Resistance , Factor Xa/metabolism , Female , Fibrin Fibrinogen Degradation Products/metabolism , Heparin/adverse effects , Humans , Infant , Infant, Newborn , Linear Models , Male , Multivariate Analysis , Peptide Fragments/blood , Plasma , Postoperative Hemorrhage/prevention & control , Prothrombin , Randomized Controlled Trials as Topic , Risk Factors , Treatment Outcome
14.
J Thromb Haemost ; 14(3): 531-45, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26614707

ABSTRACT

UNLABELLED: ESSENTIALS: The lectin pathway's MASP-1/2 activates coagulation factors but the trigger of the activation is unknown. MASP-1/2 activation was assessed by quantifying complexes between MASPs and antithrombin/C1-inhibitor. Activated platelets and fibrin were demonstrated to activate MASP-1 and MASP-2 both in vitro and in vivo. These findings may represent a crossroad between the complement and the coagulation systems. BACKGROUND: The activated forms of the complement lectin pathway (LP) proteases MASP-1 and MASP-2 are able to cleave the coagulation factors prothrombin, fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor in vitro. In vivo studies also show that MASP-1 is involved in thrombogenesis. OBJECTIVES: To clarify the not yet identified mechanisms involved in triggering activation of the LP during thrombotic reactions. METHODS: Novel sandwich-ELISAs for detection of complexes between MASP-1 or MASP-2 and the serpins C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. RESULTS: Activated platelets were shown by flow cytometry to bind Ficolin-1, -2 and -3 but not MBL, which was associated with activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma, bind and activate MASP-1 and MASP-2. As demonstrated by the ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. CONCLUSIONS: MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation.


Subject(s)
Blood Coagulation , Blood Platelets/enzymology , Complement Pathway, Mannose-Binding Lectin , Inflammation/enzymology , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Platelet Activation , Thrombosis/enzymology , Adult , Aged , Aged, 80 and over , Antithrombin Proteins/metabolism , Blood Platelets/immunology , Case-Control Studies , Complement C1 Inhibitor Protein/metabolism , Enzyme Activation , Female , Fibrin/metabolism , Humans , Inflammation/blood , Inflammation/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/enzymology , Lupus Erythematosus, Systemic/immunology , Male , Mannose-Binding Protein-Associated Serine Proteases/immunology , Middle Aged , Multiple Trauma/blood , Multiple Trauma/enzymology , Multiple Trauma/immunology , Protein Binding , Signal Transduction , Thrombosis/blood , Thrombosis/immunology , Time Factors , Young Adult
15.
Int J Hematol ; 102(3): 253-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26134363

ABSTRACT

Venous thromboembolism (VTE) occurs frequently in pregnant women and is a significant cause of maternal death. Hemostatic abnormalities were examined in 18 pregnant women with thrombosis. We studied five families with congenital antithrombin (AT) deficiency, and two families with congenital protein C (PC) deficiency. One woman with PC deficiency showed protein S (PS) Tokushima. The AT activity levels were significantly lower at the onset of thrombosis in the pregnant women than during the stable state. The PS activity and antigen levels were also significantly lower at the onset of thrombosis. In the patients with congenital AT deficiency, AT activity was significantly low in the stable state and decreased further at the onset of thrombosis. Although AT levels were normal before pregnancy, they subsequently decreased and in two cases the patients required the administration of AT after pregnancy. Gene analysis revealed one family with AT Budapest, one family with AT Toyama, and three families with AT Glasgow. Additionally, there were one family with PC Tochigi and one family with combined heterozygous of PC deficiency and PS Tokushima. In conclusion, the deficiency of natural anticoagulants, especially AT, is an important cause of pregnancy-related VTE.


Subject(s)
Antithrombins/metabolism , Pregnancy Complications, Cardiovascular/blood , Protein C Deficiency/blood , Venous Thromboembolism/blood , Adult , Aged , Aged, 80 and over , Antithrombin Proteins/genetics , Antithrombin Proteins/metabolism , Female , Humans , Male , Pregnancy , Pregnancy Complications, Cardiovascular/genetics , Protein C Deficiency/genetics , Venous Thromboembolism/genetics
16.
Thromb Res ; 134(4): 914-7, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25149909

ABSTRACT

INTRODUCTION: Prothrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)-protein C (PC) anticoagulant system have not been previously elucidated. MATERIALS AND METHODS: We prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. RESULT AND CONCLUSIONS: rTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.


Subject(s)
Antithrombin Proteins/metabolism , Fibrinogen/metabolism , Mutation , Protein C/metabolism , Prothrombin/genetics , Thrombomodulin/metabolism , Blood Coagulation Tests , Enzyme Activation , Humans , Prothrombin/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
17.
Arch Pathol Lab Med ; 138(7): 967-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24979180

ABSTRACT

CONTEXT: The results of studies among patients with antithrombin deficiency have suggested that the use of warfarin will increase the level of antithrombin. OBJECTIVE: To reevaluate the effect of warfarin on antithrombin levels using an automated amidolytic method in current use. DESIGN: Antithrombin levels were measured in patients who were receiving warfarin for atrial fibrillation and were compared with antithrombin levels in preoperative patients who had not received warfarin. RESULTS: Patients receiving warfarin had a mean antithrombin level of 100.40% (range, 81%-153%). Patients not receiving warfarin had a mean antithrombin level of 99.97% (range, 79%-120%). The Student t test was not significant for a difference between the mean antithrombin levels of the 2 populations. CONCLUSIONS: The use of warfarin does not increase the level of antithrombin in patients receiving the drug.


Subject(s)
Anticoagulants/therapeutic use , Antithrombin Proteins/metabolism , Warfarin/therapeutic use , Adult , Aged , Aged, 80 and over , Antithrombin Proteins/analysis , Antithrombin Proteins/deficiency , Artifacts , Atrial Fibrillation/blood , Atrial Fibrillation/drug therapy , Blood Chemical Analysis/methods , Blood Chemical Analysis/statistics & numerical data , Case-Control Studies , Female , Humans , Male , Middle Aged
18.
Br J Haematol ; 166(1): 130-9, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24684277

ABSTRACT

In this study we report the largest descriptive cohort of congenital antithrombin (AT) deficiency in children, its clinical presentation, molecular basis and genotype-phenotype correlation. Paediatric patients diagnosed with AT deficiency at two tertiary care children's hospitals over a 10-year period were retrospectively reviewed. SERPINC1 gene sequencing was offered to subjects who did not already have the test performed. Molecular modelling and stability simulations were performed for the novel mutations identified. Twenty-nine subjects from 18 pedigrees were identified. Mean age (± standard deviation) at diagnosis and mean duration of follow-up were 8.4 (± 6.6 years and 6.6 (± 5.7 years respectively. Most recent mean AT activity and AT antigen levels (n = 20) were 0.5 (± 0.0) iu/ml and 0.6 (± 0.1) iu/ml respectively. Ten subjects were diagnosed secondary to low AT activity measured following venous thrombo-embolism (VTE). All 10 subjects had additional risk factors at the time of VTE. None of the 19 subjects diagnosed with AT deficiency in the setting of positive family history have had VTE with 7.4 (± 5.8) years follow-up. Mutation analysis has been completed on 19 subjects from 16 pedigrees. Nine unique mutations, including 4 novel mutations were identified.


Subject(s)
Antithrombin Proteins/deficiency , Thrombophilia/congenital , Adolescent , Antithrombin III/genetics , Antithrombin Proteins/metabolism , Child , Child, Preschool , Cohort Studies , DNA Mutational Analysis/methods , Female , Follow-Up Studies , Genetic Predisposition to Disease , Genotype , Humans , Infant , Infant, Newborn , Male , Mutation , Pedigree , Phenotype , Retrospective Studies , Survival Analysis , Thrombophilia/blood , Thrombophilia/genetics , Venous Thromboembolism/blood , Venous Thromboembolism/genetics
19.
Thromb Haemost ; 111(4): 583-9, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24477356

ABSTRACT

The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.


Subject(s)
Antithrombin Proteins/metabolism , Antithrombins/metabolism , Insect Proteins/metabolism , Serpins/metabolism , Thrombin/antagonists & inhibitors , Animals , Blood Coagulation , Crystallization , Humans , Protein Conformation
20.
Med Sci Monit ; 19: 1102-8, 2013 Dec 04.
Article in English | MEDLINE | ID: mdl-24300831

ABSTRACT

BACKGROUND: This study analyzes the influence the of kidney donor hemostasis on the risk of complications in the kidney recipient after transplantation. MATERIAL AND METHODS: We enrolled 38 deceased kidney donors, of whom 14 donors died from a physical injury and the others died from ischemic or bleeding central nervous system stroke. The donors were categorized into 2 subgroups. If the recipient's postoperative period proceeded smoothly, the kidney donor was assigned to the uncomplicated donors (UD) group. If the recipient's postoperative period was complicated, the donor was assigned to the complicated (CD) Group. The CD group of consisted of 9 donors who died from strokes or bleedings and 2 who died from physical injury. We examined the antithrombin (AT) protein C (PC), complexes of thrombin/antithrombin (TAT), fragments F1+2 of prothrombin (F1+2), plasminogen (Pl), complexes of plasmin/antiplasmin (PAP), and D-dimers (D-d). RESULTS: In the CD group had decreased activity of AT, PC, and Pl and increased activity of F1+2, TAT, and D-d. The UD group had a higher level of PAP. The CD group had evidence of intensive blood coagulation, but the UD group had evidence of fibrinolysis. Fisher's exact test revealed an increased risk in recipients who received a kidney from the CD group. CONCLUSIONS: The hemostasis of the kidney donors had a correlation with the occurrence of some complications in the kidney recipients, especially complications connected with activation of blood coagulation. It seems that the activation of fibrinolysis could be positive prognostic factor, but this requires further investigations.


Subject(s)
Hemostasis/physiology , Kidney Transplantation/adverse effects , Postoperative Complications/etiology , Renal Insufficiency/surgery , Tissue Donors , Antithrombin Proteins/metabolism , Cadaver , Female , Humans , Male , Plasminogen/metabolism , Poland , Protein C/metabolism , Prothrombin/metabolism , Statistics, Nonparametric
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