USE OF ALBUMIN AND TWEEN AS STABILIZERS TO PREVENT ACTIVITY LOSS DURING CLOTTING ASSAYS OF COAGULATION FACTOR IX AND X CONCENTRATES

1987 ◽  
Author(s):  
Shirley I Miekka
Keyword(s):  
Vitamin K ◽  
Surface Adsorption ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Test Tube ◽  
Factor Ix ◽  
Tween 20 ◽  
Factor X ◽  
Bovine Albumin ◽  
Activity Loss

Assays for clotting activities of Vitamin K-dependent coagulation factors in Factor IX complex concentrates are IcnovTn to give variable results depending on the composition of the sample diluent. Higher potency values are obtained when deficient plasma is used for sample pre-dilution compared with dilution in buffer. This discrepancy is more pronounced in assays of higher purity Factor IX (FIX) or Factor X (FX) concentrates. We have found that addition of a mixture of bovine albumin (0.1% w/v) and Tween 20 (0.01% v/v) (BAT) to the dilution buffer can eliminate the discrepancy, giving clotting times and plot slopes equal to chose obtained upon dilution in deficient plasma. Less protection was obtained with either albumin or Tween added separately. Polyethylene glycol 8000 (0.1% w/v), commonly used to stabilize thrombin solutions, gave variable results. BAT had no effect on clotting times of whole plasma or of FIX or FX samples pre-diluted in deficient plasma. Neither deficient plasma nor BAT had any effect when added after sample dilutions were prepared: activity of a FIX concentrate was 137 U/ml when pre-diluted in Factor IX-deficient plasma and 1312 U/ml diluted in BAT, compared with 49 U/ml diluted in buffer alcne; addition of deficient plasma or BAT to the dilutions of sample in buffer gave activities of only 36 a).id 34 U/ml, respectively. Similar results were obtained with FX samples. Furthermore, when a solution of FX (pre-diluted to 1 U/ml in buffer without stabilizer) was merely transferred from one test tube to another without further dilution, clotting times increased progressively and activity decreased by 85% after 8 transfers. By contrast, an identical sample diluted to 1 U/ml with BAT remained essentially unchanged after 8 serial transfers. These results indicate that Vitamin K-dependent coagulation factors are very susceptible to surface adsorption or inactivation after dilution of concentrates, and that either BAT or deficient plasma will prevent this loss. The use of albumin and Tween as stabilizers provides a simpler, .less expensive alternative to prevent nonspecific surface adsorption and achieve more accurate measurement of clotting activities.

Genetic Polymorphism of Factor IX Gene in Greek Patients with Thrombophilia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4121-4121
Author(s):  
Pantelis P.E. Makris ◽  
Michel M. Iskas ◽  
Rigini R. Papi ◽  
Dimitrios D.K. Kiriakidis
Keyword(s):  
Vitamin K ◽  
Polyacrylamide Gel Electrophoresis ◽  
Control Sample ◽  
Coagulation Factor ◽  
Factor Ix ◽  
University Hospital ◽  
Activate Factor ◽  
Factor X ◽  
Coagulation Factor Ix ◽  
Pcr Products

Abstract Introduction. Coagulation factor IX plays an important intermediate role in the activation of blood coagulation. It is located within the blood plasma as a zymogen, in its inactivated state. Factor IX is dependent on the presence of Vitamin K. The structure of factor IX closely resembles the structures of many other Vitamin K dependent plasma proteins, such as prothrombin, factor X and protein C. After being activated, Factor IX forms a complex with calcium ions, membrane phospholipids and coagulation factor VIIIa to activate factor X. The exact locus of the coagulation factor IX gene was found to exist in the Xq26-q27 region of the X chromosome. The FIX gene spans 34 kb and contains eight exons. Over 300 different mutations have been identified in the FIX gene, all of which result in the production of inactive FIX, causing hemophilia B. Aim. In this study we searched for mutations in the FIX gene which result in an increased activity of FIX thus being the cause of thrombophilia syndromes. Material: A total of 108 individuals from unrelated families were involved in this study, presenting thrombophilic syndromes. A control sample from a healthy non-thrombophilic individual was also used. Total DNA from the above individuals was supplied to us by the Haemostasis and Thrombosis Unit of AHEPA University Hospital, Thessaloniki, Greece. According to HAT (Heparin Antithrombin Test, Makris, Van Dreden 1998) method a mixture of human antithrombin and heparin is added in the plasma and partial thromboplastin time is estimated. 97% of normal individuals exhibit prolonged time values in this test, whereas in our patients the time was significantly reduced. However, after the addition of recombined human FIX (rhFIX) in the mixture, prolongation of PTT is noted. Methods: The promoter region and the eight exons of the FIX gene were amplified by PCR using seven labelled primer pairs specific for these regions, that were described previously in literature. The amplification reactions were performed in a MJ Research P200 thermal cycler while the Tm of each primer pair was optimised as shown in the table. PCR products were analyzed using LI-COR DNA analyzer which is based on fragment separation by polyacrylamide gel electrophoresis. With this method PCR products presenting up to a 1 bp difference in their molecular weight create distinct bands on the gel and thus an insertion, or deletion of a base can be detected. However, no such differentiation was present among the samples examined. Assuming that the potential mutations could involve point mutations and thus be undetectable by the above method, the samples were sequenced and compared with the control. Sequencing the promoter and the 8 exons sites of the FIX gene of the most high risk cases. A point mutation was detected in four of the samples. The mutation was a single base change (ACT →GCT) located at the 21975 bp of the FIX gene, in exon 6. This mutation causes a significant change, replacing the Thr194 residue with an Ala residue (T194A). The sequencing pattern of one of these patients and the control is shown in the figure. Figure Figure

Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1269-1277
Author(s):  
KJ Smith
Keyword(s):  
Replacement Therapy ◽  
Vitamin K ◽  
Disease Transmission ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Heat Treated ◽  
Coagulation Factor Concentrates ◽  
Risk Of Disease ◽  
Factor Ix Deficiency

Thrombosis and transmission of viral diseases are the principal adverse effects of current replacement therapy for factor IX deficiency when using heat-treated concentrates of vitamin K-dependent coagulation factors. More highly purified factor IX preparations could decrease the risk of disease transmission, reduce patient exposure to allogeneic proteins, and reduce the risk of thrombosis. In this study, two immunoaffinity-purified factor IX preparations from commercial vitamin K-dependent coagulation factor concentrates had specific activities of 134 and 155 U/mg. Crude concentrates and purified factor IX preparations were tested for thrombogenicity in rabbits. One of two crude concentrates tested in the stasis-thrombosis assay caused large thrombi at doses of 50 U/kg. Purified factor IX from this concentrate was not thrombogenic at 106 to 234 U/kg. A heparin-treated concentrate that was not active in the stasis model at 100 U/kg caused significant (P less than .05) delayed consumption of rabbit fibrinogen, platelets, antithrombin III antigen, and factor VIII activity at the same dose. Factor IX prepared from this concentrate caused no consumption of coagulation factors at 214 to 243 U/kg despite the presence of trace amounts of activated factor IX. These results indicate that more highly purified preparations could reduce the risk of thrombosis in replacement therapy for hemophilia B. Also, at least for the preparations tested, factor IX and factor IXa were not the thrombogenic components of the crude concentrates.

International Normalized Ratio Versus Plasma Levels of Coagulation Factors in Patients on Vitamin K Antagonist Therapy

2011 ◽  
Vol 135 (4) ◽  
pp. 490-494 ◽  
Author(s):  
Gene Gulati ◽  
Megan Hevelow ◽  
Melissa George ◽  
Eric Behling ◽  
Jamie Siegel
Keyword(s):  
Vitamin K ◽  
Warfarin Therapy ◽  
Clinical Laboratory ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
International Normalized Ratio ◽  
Activity Levels ◽  
Factor X ◽  
Life Threatening ◽  
Average Factor

Abstract Context.—The key question when managing patients on warfarin therapy who present with life-threatening bleeding is how to use the international normalized ratio (INR) to best direct corrective therapy. The corollary question for the clinical laboratory is at what level will the INR reflect a critical value that requires notifying the clinician. Objective.—To determine the levels of vitamin K–dependent factors over a range of INR values. Design.—Evaluation of the vitamin K–dependent coagulation factor levels on plasma remnants from patients in whom a prothrombin time and INR was ordered to monitor warfarin therapy. There were a total of 83 specimens evaluated with an INR range from 1.0 to 8.26. Results.—The mean activity levels of all 4 factors remained near or above 50% when the INR was less than 1.5. The average factor X level was 23% when the INR range was 1.6 to 2.5, but levels of factors II, VII, and IX did not drop below the hemostatic range until the INR was greater than 2.5. At an INR of 3.6 or more, the activity levels of all 4 factors were less than 30% in more than 90% of the specimens. Conclusion.—Levels of factors II, VII, IX, and X declined with increasing INR but not at the same rate and not to the same level at a given INR. However, most of the values were below the hemostatic value once the INR was 3.6 or more, the level that was also considered critical for physician notification.

scholarly journals Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1269-1277 ◽  
Author(s):  
KJ Smith
Keyword(s):  
Replacement Therapy ◽  
Vitamin K ◽  
Disease Transmission ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Heat Treated ◽  
Coagulation Factor Concentrates ◽  
Risk Of Disease ◽  
Factor Ix Deficiency

Abstract Thrombosis and transmission of viral diseases are the principal adverse effects of current replacement therapy for factor IX deficiency when using heat-treated concentrates of vitamin K-dependent coagulation factors. More highly purified factor IX preparations could decrease the risk of disease transmission, reduce patient exposure to allogeneic proteins, and reduce the risk of thrombosis. In this study, two immunoaffinity-purified factor IX preparations from commercial vitamin K-dependent coagulation factor concentrates had specific activities of 134 and 155 U/mg. Crude concentrates and purified factor IX preparations were tested for thrombogenicity in rabbits. One of two crude concentrates tested in the stasis-thrombosis assay caused large thrombi at doses of 50 U/kg. Purified factor IX from this concentrate was not thrombogenic at 106 to 234 U/kg. A heparin-treated concentrate that was not active in the stasis model at 100 U/kg caused significant (P less than .05) delayed consumption of rabbit fibrinogen, platelets, antithrombin III antigen, and factor VIII activity at the same dose. Factor IX prepared from this concentrate caused no consumption of coagulation factors at 214 to 243 U/kg despite the presence of trace amounts of activated factor IX. These results indicate that more highly purified preparations could reduce the risk of thrombosis in replacement therapy for hemophilia B. Also, at least for the preparations tested, factor IX and factor IXa were not the thrombogenic components of the crude concentrates.

Coagulation Induced by Dilute Tissue Factor Depends More Critically on Factor II and X Concentration Than on FVII or FIX

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4084-4084
Author(s):  
Brynja R. Gudmundsdottir ◽  
Alexia M Bjornsdottir ◽  
Pall T. Onundarson
Keyword(s):  
Prothrombin Time ◽  
Vitamin K ◽  
Platelet Rich Plasma ◽  
Coagulation Factor ◽  
Maximum Velocity ◽  
Coagulation Factors ◽  
Factor X ◽  
Platelet Poor Plasma ◽  
Stabilization Phase ◽  
Maximum Clot Firmness

Abstract Prothrombin time based tests used to monitor coumarin anticoagulation measure the initiation phase of fibrin formation (clotting time, CT) in platelet poor plasma. Additional information can be obtained using computerized rotational thromboelastometry (ROTEM) which also measures the consequent propagation phase (measured as maximum velocity, Vmax), and the stabilization phase (maximum clot firmness, MCF). We used ROTEM to study the effect of individual vitamin K dependent (VK) coagulation factor (F) concentration on clotting induced by dilute thromboplastin in platelet poor and platelet rich plasma (PPP and PRP). As expected, FII, FVII and FX in PPP equally affected the prothrombin time whereas FIX did not. In PPP the ROTEM CT, however, was affected more by the concentration of FII and FX than by FVII (data not shown). To imitate physiological clotting better, factor deficient platelet poor plasma was repleted by adding frozen platelets in an optimal concentration corresponding to 100 × 109/L.. In the PRP the ROTEM CT was more dependent on the concentration of FII than of FVII, more dependent on FX than on FII and not dependent on FIX at all (left figure). The Vmax (reflecting the propagation phase) was also more dependent on FII and factor X than on the concentration of FVII or FIX which both may demonstrate a threshold effect (right figure). The stabilization phase or maximum clot firmness (MCF, not shown) was influenced similarly by FII and FX but was not influenced by FVII or FIX. Figure Figure Conclusion: During deficiency of vitamin K dependent coagulation factors the concentration of FII and FX may be more critical for hemostasis than FVII or FIX concentration. This may have practical implications for the appropriate choice of monitoring assays during coumarin administration.

Factor X Levels, Polymorphisms in the Promoter Region of Factor X, and the Risk of Venous Thrombosis

2001 ◽  
Vol 85 (06) ◽  
pp. 1011-1017 ◽  
Author(s):  
Swibertus Poort ◽  
Hans Vos ◽  
Frits Rosendaal ◽  
Rogier Bertina ◽  
Marieke de Visser
Keyword(s):  
Risk Factor ◽  
Venous Thrombosis ◽  
Vitamin K ◽  
Promoter Region ◽  
Genotypic Variation ◽  
Coagulation Factor ◽  
Premenopausal Women ◽  
Factor Ix ◽  
Factor X ◽  
Increased Risk

SummaryElevated levels of procoagulant proteins factor II, factor VIII, factor IX, factor XI and fibrinogen are associated with an increased risk of venous thrombosis. In a population-based case-control study on venous thrombosis (Leiden Thrombophilia Study, LETS) we investigated whether elevated coagulation factor X (FX) levels are a risk factor for venous thrombosis and whether FX levels are determined by polymorphisms in the promoter region of the FX gene. We found that subjects with high FX levels (above the 90th percentile, ≥ 126 U/dl) had a 1.6-fold increased risk of venous thrombosis. The highest risk (OR = 4.3, 95% confidence interval: 1.5-12) was found in the subgroup of premenopausal women who are not using oral contraceptives. However, these estimated risks disappeared after adjustment for other vitamin K-dependent coagulation factors II, VII and IX. To study the influence of genotypic variation on plasma FX levels we assessed four polymorphisms in the promoter region of the FX gene: a TTGTGA insertion between position -343A and -342G, a C/T polymorphism at position -222, a C/A polymorphism at position -220 and a C/T polymorphism at position -40. No relationship between these investigated genotypes and FX levels was observed. We conclude that high FX levels predict risk of thrombosis, but are not a risk factor for venous thrombosis when the levels of other vitamin K-dependent proteins are taken into account.

DEVELOPMENT OF A COAGULATION FACTOR X CONCENTRATE AS A BY-PRODUCT OF COAGULATION FACTOR IX PRODUCTION

1987 ◽  
Author(s):  
Shirley I Miekka ◽  
David B Clark ◽  
Doris Menache
Keyword(s):  
Native Species ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Pilot Scale ◽  
Factor Ix ◽  
Red Cross ◽  
Factor X ◽  
Coagulation Factor Ix ◽  
The Usa

The American Red Cross is developing a Coagu.lation Factor X (FX) concentrate to provide a safer alternative for replacement therapy in Factor X deficient patients, who can experience thromboembolic complications with current treatments. Based on a survey of hemophilia treatment centers, we estimate the frequency of the homozygous disorder to be approximately 1/150th that of hemophilia A, or about 65 patients in the USA. We have devised a method for producing FX as a by-product of our Coagulation Factor IX concentrate (FIX). The method starts with adsorption of cryoprecipitate supernatant plasma with DEAE-Sephadex resin followed by elution of Vitamin K-dependent coagulation factors. This material is adsorbed to sulfated dejctran resin and Factors II and X are eluted by increasing the salt concentration. At 0.45 M NaCl, FII elutes quickly while FX is retarded and can be recovered essentially free of FIX by collecting the slower eluting material. FIX is then recovered at still higher ionic strength. The pooled FX is concentrated, diafiltered and treated to inactivate viruses. Approximately 30% of plasma FX was recovered in pilot scale experiments (600 liters plasma). Specific activity was > 51 FX units / mg protein corresponding to a purity of around 50% and 3000-fold purification over plasma. The ratios of Factors "X : II : IX : Protein C were 1.0 : <0.03 : <0.03 : 0.2. The major contaminant, conprising nearly 50% of the protein, was found to be inter-alpha trypsin inhibitor (IaI), a serine protease inhibitor whose function in plasma has not yet been determined. This inhibitor is also present in the DEAE-Sephadex eluate and. in the FIX concentrate. However, Western blot and HPLC analyses have shown that IaI is present in two different forms.In FX it behaves as expected for the IaI monomer (Mr = 160 kDa), while in the DEAE-eluate and in FIX it exists also in r higher molecular weight form (≥400 kDa) corresponding either to aggregates, complexes or larger native species not previously described. The nature of the possible interaction of Ial w.vdi these coagulation factors is unknown and is currently boinn evaluated.

scholarly journals Acquired Vitamin K Deficiency as Unusual Cause of Bleeding Tendency in Adults: A Case Report of a Nonhospitalized Student Presenting with Severe Menorrhagia

2017 ◽  
Vol 2017 ◽  
pp. 1-3 ◽  
Author(s):  
Omid Reza Zekavat ◽  
Gholamreza Fathpour ◽  
Sezaneh Haghpanah ◽  
Seyed Javad Dehghani ◽  
Maryam Zekavat ◽  
...  
Keyword(s):  
Vitamin K ◽  
Coagulation Factor ◽  
Energy Drinks ◽  
High Energy ◽  
Factor Ix ◽  
Factor Vii ◽  
Bleeding Tendency ◽  
Factor X ◽  
Vitamin K Deficiency ◽  
K Deficiency

We report a rare case of acquired vitamin K deficiency presenting with severe menorrhagia and without any gynecological problem. Partial thromboplastin time (59.2 seconds) and prothrombin time (33.1 seconds, INR: 5.97) were considerably prolonged in laboratory evaluations. A complete coagulation factor assay test was performed for the patient: factor IX, 24%; factor II, 41%; factor VII, 3%; and factor X, 52%. She had been taking many high-energy drinks and she had inadequate dietary intake for the past 6 months. Given that she had vitamin K deficiency (VKD), a course of vitamin K therapy was started for her in the hospital. This case showed the potential for menorrhagia due to VKD with use of high-energy drinks and the value of a complete and detailed history in early diagnosis.

Increased Activity of Vitamin K-Dependent Clotting Factors in Human Hyperlipoproteinaemia – Association with Cholesterol and Triglyceride Levels

1977 ◽  
Vol 38 (02) ◽  
pp. 0465-0474 ◽  
Author(s):  
M Constantino ◽  
C Merskey ◽  
D. J Kudzma ◽  
M. B Zucker
Keyword(s):  
Vitamin K ◽  
Plasma Lipids ◽  
Coagulation Factors ◽  
Clotting Factor ◽  
Factor X ◽  
Clotting Factors ◽  
Blood Coagulation Factors ◽  
Cholesterol Levels ◽  
Type V ◽  
Increased Activity

SummaryLevels of blood coagulation factors, cholesterol and triglyceride were measured in human plasma. Prothrombin was significantly elevated in type Ha hyperlipidaemia; prothrombin and factors VII, IX and X in type lib; and prothrombin and factors VII and IX in type V. Multiple regression analysis showed significant correlation between the levels of these plasma lipids and the vitamin K-dependent clotting factors (prothrombin, factors VII, IX and X). Higher cholesterol levels were associated with higher levels of prothrombin and factor X while higher triglyceride levels were associated with higher levels of these as well as factors VII and IX. Prothrombin showed a significant cholesterol-triglyceride interaction in that higher cholesterol levels were associated with higher prothrombin levels at all levels of triglyceride, with the most marked effects in subjects with higher triglyceride levels. Higher prothrombin levels were noted in subjects with high or moderately elevated (but not low) cholesterol levels. Ultracentrifugation of plasma in a density of 1.21 showed activity for prothrombin and factors VII and X only in the lipoprotein-free subnatant fraction. Thus, a true increase in clotting factor protein was probably present. The significance of the correlation between levels of vitamin K-dependent clotting factors and plasma lipids remains to be determined.

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