Preparation of Highly Purified Human Thrombin and Factor Xa in a Simultainous Procedure

1975 ◽  
Author(s):  
M. Miller-Andersson
Keyword(s):  
Specific Activity ◽  
Factor Xa ◽  
Fresh Frozen Plasma ◽  
Factor X ◽  
Serum Factors ◽  
Human Thrombin ◽  
Fresh Frozen ◽  
Brain Material ◽  
The Brain ◽  
Chromatography Step

A preparation procedure that purifies Thrombin and activated factor X in a simultainous process has been worked out. Prothrombin complex was first prepared from fresh frozen plasma by chromatography on DEAE-Sephadex. Prothrombin activator was prepared from human brain thromboplastin and bovine serum factors. The activation was performed at 20° C and followed by assaying thrombin activity. After activation the brain material was removed by centrifugation. The protein mixture was immediately adsorbed on to Amberlite IRC-50 and both enzymes were fully adsorbed to the resin. They were eluted in a CaCl2 gradient and the two enzymes eluted in two slightly owerlapping peaks.The thrombin obtained in this procedure was highly purified with a specific activity of 2100 U per mg protein. The activated factor X contained traces of thrombin. It was fully suitable for anti Xa assays of heparin. When higher degree of purity was needed the activated factor X was further purified in an iron-exchange or affinity chromatography step.

scholarly journals Inherited Moderate Factor X Deficiency Presenting as Cardiac Tamponade

2019 ◽  
Vol 2019 ◽  
pp. 1-4
Author(s):  
Tamer Othman ◽  
Ayman Abdelkarim ◽  
Karen Huynh ◽  
An Uche ◽  
Jennifer Lee
Keyword(s):  
Cardiac Tamponade ◽  
Clinical Manifestations ◽  
Missense Variant ◽  
Fresh Frozen Plasma ◽  
Factor X ◽  
Pericardial Window ◽  
Extensive Experience ◽  
Management Guidelines ◽  
Factor X Deficiency ◽  
Fresh Frozen

Factor X deficiency is a rare bleeding disorder that varies in the severity of its clinical manifestations. The symptoms of this disorder can occur at any age, although most severe cases appear in childhood. The rarity of this condition has not allowed for the establishment of evidence‐based management guidelines, and thus, individuals afflicted with factor X deficiency are treated based on limited literature and the opinions of clinicians with extensive experience. In this case report, we discuss a unique presentation of a 38-year-old male who was found to have cardiac tamponade as a result of his newly diagnosed inherited moderate factor X deficiency. This was discovered by obtaining a factor X activity assay and confirmed with genetic testing which demonstrated a missense variant on the factor X gene on chromosome 13. His management involved correction of his factor X deficiency with fresh frozen plasma, a pericardiocentesis, and placement of a pericardial window. He has been asymptomatic and without hemorrhagic episodes for the 10 months following his discharge.

Preparation of a Factor VII Concentrate

1979 ◽  
Author(s):  
A.J. MacLeod ◽  
I. Dickson
Keyword(s):  
Specific Activity ◽  
Fresh Frozen Plasma ◽  
Factor Vii ◽  
Batch Adsorption ◽  
Citrate Buffer ◽  
Linear Gradient ◽  
Fresh Frozen ◽  
Frozen Plasma ◽  
Phosphate Citrate Buffer

A factor VII concentrate has been prepared from pooled citrated fresh frozen plasma following removal of cryoprecipitate and factors II, IX and X. The method involved batch adsorption on DEAE-Sephadex A-50, fractionation of the subsequent batch eluate by PEG precipitation and passage through a column of DEAE-Sepharose CL-.6B. A phosphate-citrate buffer pH 6.9 was used throughout, this was made 0.2M with NaCl for the batch elution and a 0 - 0.2H NaCl linear gradient was used to elute the components from the column. Factor VII activity was clearly resolved from the bulk of the protein, including caeruloplasmin, and could be recovered as a concentrate at about 20 U FVII/ml with a specific activity of in excess of 1 U FVII/mg of protein and an overall recovery of 40% to 50%

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Treatment of Amyloidosis Associated Factor X Deficiency

1976 ◽  
Vol 35 (02) ◽  
pp. 377-381 ◽  
Author(s):  
Joel A. Spero ◽  
Jessica H. Lewis ◽  
Ute Hasiba ◽  
Lawrence D. Ellis
Keyword(s):  
Fresh Frozen Plasma ◽  
Factor Ix ◽  
Bleeding Complications ◽  
Primary Amyloidosis ◽  
Factor X ◽  
Associated Factor ◽  
Factor X Deficiency ◽  
Fresh Frozen ◽  
Frozen Plasma

SummaryThis is the tenth patient in thirteen years to be reported with the findings of an isolated factor X deficiency associated with primary amyloidosis. A favorable response to factor IX concentrate was manifested by temporary clinical and laboratory correction of her diathesis. This mode of treatment, therefore, provides an approach to therapy for bleeding complications in this group of patients who have previously failed to respond to fresh frozen plasma.

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1692-1692
Author(s):  
Jennifer Newell ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

Real-world management of oral factor Xa inhibitor-related bleeds with reversal or replacement agents including andexanet alfa and four-factor prothrombin complex concentrate: a multicenter study

2021 ◽  
Vol 17 (1) ◽  
pp. 127-135
Author(s):  
Craig I Coleman ◽  
Paul P Dobesh ◽  
Sherry Danese ◽  
Julie Ulloa ◽  
Belinda Lovelace
Keyword(s):  
Length Of Stay ◽  
Hospital Mortality ◽  
Real World ◽  
Prothrombin Complex Concentrate ◽  
Factor Xa ◽  
Fresh Frozen Plasma ◽  
Factor Xa Inhibitor ◽  
Fresh Frozen ◽  
Andexanet Alfa ◽  
Frozen Plasma

Aim: We describe the real-world utilization and outcomes associated with managing oral factor Xa inhibitor (FXai)-related major bleeds. Materials & methods: Electronic records from 45 US hospitals were queried (ICD-10-CM billing codes D68.32, T45.515x or T45.525x) to identify major bleed hospitalizations related to FXai use. Patient demographics, bleed type (intracranial hemorrhage, gastrointestinal, critical compartment, traumatic, other), FXai taken, reversal or replacement agents administered (including andexanet alfa, four-factor prothrombin complex concentrate, fresh frozen plasma, others), in-hospital mortality and length of stay were recorded. Results: Of 3030 FXai-related hospitalizations for major bleeds, patients averaged 68 years old and 47% were women. In-hospital mortality was highest for intracranial hemorrhage (23%, n = 507) and lowest for gastrointestinal bleeds (4%, n = 1453). In-hospital mortality was lowest (4%) for bleeds managed with andexanet alfa (n = 342), compared with 10% for four-factor prothrombin complex concentrate (n = 733), 11% for fresh frozen plasma (n = 925) and 8% for both other agents (n = 794) and no agents (n = 438). Median length of stay was 5 days across all agents, while ICU length of stay was shorter andexanet alfa (2 days) compared with other agents (3 days). Conclusion: In-hospital mortality differed by bleed type and agents administered. Andexanet alfa was associated with the lowest rate of in-hospital mortality across all bleed types.

Structural and Functional Characteristics of Proteolytically Modified Bovine Factor X and Xa (Des Light Chain Residues 1-41)

1979 ◽  
Author(s):  
T. Morita ◽  
C.M. Jackson
Keyword(s):  
Light Chain ◽  
Specific Activity ◽  
Factor Xa ◽  
Peptide Bond ◽  
Amino Acid Sequences ◽  
Structural Domain ◽  
Factor X ◽  
One Stage ◽  
The One ◽  
Bovine Factor

Incubation of bovine Factor X with α-chymotrypsin produces a peptide, residues 1-41 of the light chain and a modified Factor X designated “headless Factor X”. Clotting activity of “headless Factor X” is virtually zero by one stage assay. Composition, chromatographic elution characteristics and molecular weight estimates by SDS gel electrophoresis form the bases for the aforedesignated structure of “headless Factor X”. Activation of “headless Factor X” by the Russell’s viper venom activator requires Ca2+ as does normal Factor X, however, the rate is much slower. After full activation the specific activity of “headless Factor Xa” and normal Factor Xa differed by less than 20% using BOC-L-Val-L-Leu-Gly-L-Arg-pNA as substrate. However, clotting specific activity is less than 0.002% of normal Factor Xa in the one stage clotting assay. The activation peptides released from “headless Factor X” (residues 1-51 and 291-307) of the heavy chain were identical to those released from normal Factor X. Similar rapid, highly selective chymotryptic cleavage of Prothrombin Fragment 1, and the similarity in the amino acid sequences of the light chain of Factor X and Fragment 1 suggests that the region around the susceptible peptide bond must lie on the surface of both these molecules and perhaps exist in a “hinge” region connecting the Gla containing domain and the reamining structural domain of these portions of prothrombin and Factor X. (Supported by HL12820).

scholarly journals Successful use of BPL Factor X concentrate in a child with severe factor X deficiency

2013 ◽  
Vol 1 (2) ◽  
pp. 8-10 ◽  
Author(s):  
Kate Khair ◽  
Poornima Kumar ◽  
Mary Mathias ◽  
Jemma Efford ◽  
Ri Liesner
Keyword(s):  
Central Venous Access ◽  
Coagulation Factor ◽  
Venous Access ◽  
Fresh Frozen Plasma ◽  
Factor X ◽  
Post Dose ◽  
Thrombotic Risk ◽  
Factor X Deficiency ◽  
Fresh Frozen ◽  
The Right

Abstract Introduction: Severe factor X deficiency is a rare serious bleeding disorder historically treated with fresh frozen plasma (FFP) and more recently with prothrombin complex concentrate (PCC) which contains activated factors II, VII, IX and X. The infusion volume of PCC is smaller than FFP, but there is a risk of thromboembolic complications given the presence of activated forms of vitamin K-dependent factor concentrates when treating an isolated coagulation factor deficiency. Methods: We describe the case of a nine-year-old girl of consanguineous origin with co-existent congenital merosin deficient muscular dystrophy and severe factor X deficiency treated with twice-weekly PCC prophylaxis via an indwelling central venous access device (CVAD). Infusion occlusion of her fifth CVAD occurred 24-months post-insertion; thrombus within the right subclavian and brachiocephalic veins was seen on radiological imaging. She started peripheral treatment with BPL Factor X concentrate as infusion volumes were smaller and given her immobility further thrombotic risk was predicted to be reduced. A sixth CVAD was inserted seven months later and BPL Factor X prophylaxis was continued. Results:BPL Factor X concentrate was effective in maintaining trough levels of 13IU/ml 72-hours post-dose, with no intercurrent bleeding episodes or further problems in terms of occlusion of her portacath. Further radiological screening has not been undertaken. Conclusion: BPL Factor X has been shown to be a safe and effective alternative to PCC for treatment of severe factor X deficiency in this case.

Inhibitor Of Factor VII Activation In Multiple Myeloma

1981 ◽  
Author(s):  
R A McNutt ◽  
J A Penner
Keyword(s):  
Prothrombin Time ◽  
Fresh Frozen Plasma ◽  
Factor Vii ◽  
Affinity Column ◽  
Factor X ◽  
Inhibitor Activity ◽  
Myeloma Protein ◽  
Myeloma Proteins ◽  
Fresh Frozen ◽  
Recalcification Time

An investigation of a patient with IgG myeloma who developed a clotting abnormality is described. Factor VII activity was 4%. A prothrombin time of 20.4 seconds with control of 11.0 seconds was found in association with normal partial thromboplastin time, factor X level, and a failure to correct with VitaminK and fresh frozen plasma.Methods for characterization were as follows. After defibrination and ammonium sulfate fractionation, plasma globulin portion was further purified on a DEAE ion exchanger. To identify inhibitor, Ig, alpha and beta globulins, myeloma proteins as well as albumin and fibrinogen isolated from patient plasma were incubated with normal plasma for two hours. Inhibitor activity was found in monoclonal antibody fraction. SPA affinity column removed inhibitor activity.To elucidate whether inhibitor was directed against FVII, patient plasma, myeloma proteins, and monospecific xenogeneic antibodies to FVII were compared for their ability to bind 125I FVII. Free from bound forms were separated on a Sephacryl S200 column. Chromatograms indicated absence of binding of patient inhibitor to 125I tracer.Normal and patient plasma were incubated with cephalo- plastin for 2 hours at 37°C. Prolonged recalcification time from 50 to 170 seconds was seen with patient plasma. More significantly, when inhibitor plasma and cephaloplastin were incubated for 2 hours and added to FVII deficient plasma, correction of prothrombin time was evident. Patient plasma not incubated with cephaloplastin failed to correct FVII deficient plasma.We conclude that low activity obtained in the assay system was due to the myeloma protein which inhibited FVII/ thromboplastin interaction rather than an acquired inhibitor directly affecting FVII. It seems likely that the antibody is directed to the functional site of thromboplastin responsible for FVII activation.

Haemostatic alterations induced by treatment with asparaginases and clinical consequences

2015 ◽  
Vol 113 (02) ◽  
pp. 247-261 ◽  
Author(s):  
Tommaso Za ◽  
Angela Ciminello ◽  
Silvia Betti ◽  
Elena Rossi ◽  
Valerio De Stefano
Keyword(s):  
Risk Factors ◽  
Factor Xa ◽  
Central Venous Line ◽  
Fresh Frozen Plasma ◽  
Lymphoid Leukaemia ◽  
Inherited Thrombophilia ◽  
Clinical Consequences ◽  
Fresh Frozen ◽  
Fibrinolytic Proteins ◽  
Near Future

SummaryThe benefit of asparaginase for treating acute lymphoid leukaemia (ALL) has been well established. Native asparaginase derives from Escherichia coli (colaspase) or Erwinia chrysanthemi (crisantaspase); in a third preparation, colaspase is pegylated. Depletion of asparagine leads to decreased synthesis of procoagulant, anticoagulant, and fibrinolytic proteins, with resultant hypercoagulability and greater risk of venous thromboembolism (VTE). Colaspase and crisantaspase are not dose-equivalent, with crisantaspase displaying haemostatic toxicity only at dosages much higher and administered more frequently than those of colaspase. Cerebral venous thrombosis and pulmonary embolism are two life-endangering manifestations that occur during treatment with asparaginase particularly in children and in adults with ALL, respectively. Approximately one-third of VTEs are located in the upper extremities and are central venous line-related. Other risk factors are longer duration of asparaginase treatment and concomitant use of prednisone, anthracyclines, and oral contraceptives. The risk associated with inherited thrombophilia is uncertain but is clearly enhanced by other risk factors or by the use of prednisone. VTE prevention with fresh frozen plasma is not recommended; the efficacy of antithrombin (AT) concentrates has occasionally been reported, but these reports should be confirmed by proper studies, and AT should not be routinely employed. Therapeutic or prophylactic heparin doses are only partially effective, and direct thrombin or factor Xa inhibitors could play significant roles in the near future.

Sign in / Sign up

Export Citation Format

Share Document