Applications of a salt tolerant cation exchanger carrying sulfate groups

Difference between two strong cation-exchange resins, namely sulfonium type and sulfate type regarding both their salt tolerance and hydrophobicity were investigated. There is only tiny variation between sulfate and sulfonic group and at the first glance it seems unlikely that it could be the reason for changed selectivity and salt tolerance that was detected in our preliminary experiments. For that reason salt tolerance and hydrophobicity of both ligands was investigated by using two representative polymethacrylate-based ion exchangers as for the sulfonium type TOYOPEARL GigaCap S-650M and for the sulfate type TOYOPEARL Sulfate-650F. In addition some in-silico calculations were performed for model substances representing the sulfonium and sulfate group, and significant differences were calculated regarding their hydrophobicity. These experiments confirmed the working hypothesis that salt tolerance and higher affinity and selectivity for some human plasma derived vitamin K dependent clotting factors and inhibitors are interrelated and dependent from the presence of the sulfate group. The affinity for these proteins was experimentally verified by separation of clotting factor IX from the prothrombin complex concentrate. Presented results show that a simple and fast separation between clotting factor IX and other vitamin K dependent clotting factors II, VII and X is possible, only if the resin with the sulfate, and not with sulfonic acid ligand was applied. Consequently, an immediate application of undiluted feedstock or the eluate from previous isolation step to sulfate resin is possible, and a significant optimization of downstream process can be achieved.

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Emergency Oral Anticoagulant Reversal: The Relative Efficacy of Infusions of Fresh Frozen Plasma and Clotting Factor Concentrate on Correction of the Coagulopathy

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655992 ◽

1997 ◽

Vol 77 (03) ◽

pp. 477-480 ◽

Cited By ~ 277

Author(s):

Mike Makris ◽

Mike Greaves ◽

Wendy S Phillips ◽

Steve Kitchen ◽

Frits R Rosendaal ◽

...

Keyword(s):

Vitamin K ◽

Fresh Frozen Plasma ◽

Factor Ix ◽

Clotting Factor ◽

Clotting Factors ◽

Anticoagulant Reversal ◽

Fresh Frozen ◽

Pre Treatment ◽

Clotting Factor Concentrates ◽

Frozen Plasma

SummaryHaemorrhage, including intracranial bleeding, is a common, potentially lethal complication of warfarin therapy and rapid and complete reversal of anticoagulation may be life-saving. Fresh frozen plasma (FFP) and vitamin K are most frequently administered. Because of the variable content of vitamin K-dependent clotting factors in FFP, and the effects of dilution, the efficacy of this approach is open to doubt. We have therefore compared the effects of FFP and clotting factor concentrates on the INRs and clotting factor levels of orally anticoagulated subjects requiring rapid correction of their haemostatic defect. In many, the pre-treatment INR was considered to be dangerously above the target therapeutic range. In the 12 patients given FFP, the INR did not completely correct (range 1.6-3.8, mean 2.3) indicating an ongoing anticoagulated state in all. In contrast, the INR in 29 subjects given clotting factor concentrates was completely corrected in 28 (range 0.9-3.8, mean 1.3). Following treatment, marked differences were observed in clotting factor IX levels between the two groups. The median factor IX level was 19 u/dl (range 10-63) following FFP infusion and 68.5 u/dl (range 31-111) following concentrate. In FFP treated patients, poorer responses were also observed for each of the other vitamin K-depen- dent clotting factors but these were less marked than for factor IX, which was present in low concentrations in some batches of FFP. Thus, haemostatically effective levels of factor IX cannot be achieved, in most instances, by the conventional use of FFP in patients requiring reversal of their anticoagulant therapy. Clotting factor concentrates are the only effective option where complete and immediate correction of the coagulation defect is indicated in orally anticoagulated patients with life or limb-threatening haemorrhage.

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Hypercoagulable State in the Watanabe Heritable Hyperlipidemic Rabbit, an Animal Model for the Progression of Atherosclerosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646543 ◽

1989 ◽

Vol 61 (01) ◽

pp. 140-143 ◽

Cited By ~ 23

Author(s):

Yoshitaka Mori ◽

Hideo Wada ◽

Yutaka Nagano ◽

Katsumi Deguch ◽

Toru Kita ◽

...

Keyword(s):

Vitamin K ◽

Fibrinogen Level ◽

Plasma Cholesterol ◽

Age Groups ◽

Clotting Factor ◽

Clotting Factors ◽

Group A ◽

Hyperlipidemic Rabbit ◽

Group B ◽

Progression Of Atherosclerosis

SummaryBlood coagulation in a strain of rabbits designated as Watanabe heritable hyperlipidemic (WHHL) rabbits was examined. The activities of vitamin K-dependent clotting factors, contact factors and clotting factor VIII (F VIII) and the fibrinogen level were significantly higher in WHHL rabbits than in normolipidemic rabbits (all age groups). Values for vitamin Independent clotting factor were already higher at 2 months of age. Contact factors and fibrinogen levels increased age after 5 to 8 months. F VIII increased between 5 and 8 months and then decreased. At 2 months of age, WHHL rabbits were divided into two groups. Group A was fed standard rabbit chow and group B standard rabbit chow containing 1% probucol. Probucol prevented the progression of atherosclerosis in group B in the absence of a significant reduction in plasma cholesterol level. F VIII and fibrinogen levels were statistically decreased in all rabbits at all ages in group B (P<0.05). These differences in clotting factors between the two groups were most obvious at 8 months (P<0.02).We conclude that vitamin K-dependent clotting factors may increase with hyperlipemia and that increases in F VIII and fibrinogen may be closely related to the progression of throm- boatherosclerosis.

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Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650223 ◽

1996 ◽

Vol 75 (01) ◽

pp. 070-075 ◽

Cited By ~ 20

Author(s):

E G C Wojcik ◽

P Simioni ◽

M v d Berg ◽

A Girolami ◽

R M Bertina

Keyword(s):

Vitamin K ◽

Protein C ◽

Cysteine Residue ◽

Factor Ix ◽

Disulphide Bond ◽

Clotting Factors ◽

High Molecular Weight Complex ◽

Low Concentrations ◽

Gla Domain ◽

Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651852 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0465-0474 ◽

Cited By ~ 46

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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Coagulation Disturbances in Patients with Argininemia

Acta Haematologica ◽

10.1159/000493678 ◽

2018 ◽

Vol 140 (4) ◽

pp. 221-225 ◽

Cited By ~ 1

Author(s):

Ertugrul Kiykim ◽

Tanyel Zubarioglu ◽

Mehmet Serif Cansever ◽

Tiraje Celkan ◽

Johannes Häberle ◽

...

Keyword(s):

Autosomal Recessive ◽

Urea Cycle ◽

International Normalized Ratio ◽

Factor Ix ◽

Factor Vii ◽

Clotting Factor ◽

Clotting Factors ◽

Hepatic Involvement ◽

Prolonged Aptt ◽

Liver Transaminases

Background: Argininemia is an autosomal recessive urea cycle disorder (UCD). Unlike other UCD, hyperammonemia is rarely seen. Patients usually present in childhood with neurological symptoms. Uncommon presentations like neonatal cholestasis or cirrhosis have been reported. Although transient elevations of liver transaminases and coagulopathy have been reported during hyperammonemia episodes, a permanent coagulopathy has never been reported. Methods: In this retrospective study, coagulation disturbances are examined in 6 argininemia patients. All of the patients were routinely followed up for hepatic involvement due to argininemia. Laboratory results, including liver transaminases, albumin, prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), and clotting factor levels, were assessed in all of the patients. Results: All of the patients had a prolonged PT and an increased INR, while none of the patients had a prolonged aPTT. Five patients had slightly elevated liver transaminases. A liver biopsy was performed in 1 patient but neither cirrhosis nor cholestasis was documented. Five of the 6 patients had low factor VII and factor IX levels, while other clotting factors were normal. Conclusions: Argininemia patients should be investigated for coagulation disorders even if there is no apparent liver dysfunction or major bleeding symptoms.

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Reduction of salivary tissue factor (thromboplastin) activity by warfarin therapy

Blood ◽

10.1182/blood.v53.3.366.366 ◽

1979 ◽

Vol 53 (3) ◽

pp. 366-374 ◽

Cited By ~ 1

Author(s):

LR Zacharski ◽

R Rosenstein

Keyword(s):

Tissue Factor ◽

Vitamin K ◽

Coagulation Factors ◽

Human Saliva ◽

Factor Vii ◽

Clotting Factor ◽

Activate Factor ◽

Total Protein Content ◽

Factor X ◽

Clotting Factors

Abstract The coagulant of normal human saliva has been identified as tissue factor (thromboplastin, TF) by virtue of its ability to cause rapid coagulation in plasmas deficient in first-stage coagulation factors and to activate factor x in the presence of factor VII and by virtue of the fact that its activity is expressed only in the presence of factor VII and is inhibited by an antibody to TF. The TF is related to cells and cell fragments in saliva. Salivary TF activity has been found to be significantly reduced in patients taking warfarin. The decline in TF activity during induction of warfarin anticoagulation occurs during the warfarin-induced decline in vitamin-K-dependent clotting factor activity, as judged by the prothrombin time. The decrease in TF activity is not related to a reduction in salivary cell count or total protein content or to a direct effect of warfarin on the assay. It is hypothesized that the mechanism by which warfarin inhibits TF activity may be related to the mechanism by which it inhibits expression of the activity of the vitamin-K-dependent clotting factors. Inhibition of the TF activity may be involved in the antithrombotic effect of warfarin.

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EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

10.1055/s-0038-1643568 ◽

1987 ◽

Author(s):

K L Berkner ◽

S J Busby ◽

J Gambee ◽

A Kumar

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Factor Ix ◽

Biologically Active ◽

Factor Vii ◽

Clotting Factor ◽

Clotting Factors ◽

Mammalian Expression ◽

Plasma Factor ◽

Gla Domain

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

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Extravascular Clotting Factor Activity within Joint Tissues Protects Against Progression of Hemophilic Arthropathy.

Blood ◽

10.1182/blood.v110.11.496.496 ◽

2007 ◽

Vol 110 (11) ◽

pp. 496-496 ◽

Cited By ~ 2

Author(s):

Junjiang Sun ◽

Narine Hakobyan ◽

Leonard A. Valentino ◽

Paul E. Monahan

Keyword(s):

Factor Viii ◽

Coagulation Factor ◽

Joint Capsule ◽

Factor Ix ◽

Clotting Factor ◽

Needle Puncture ◽

Factor Activity ◽

Clotting Factors ◽

Hemophilic Arthropathy ◽

Human Factor Viii

Abstract Hemophilic arthropathy is the major morbidity of congenital factor VIII and IX deficiency. Therapies localized to hemophilic joints could provide adjunctive protection, in addition to that provided by systemic factor replacement. However, the ability of extravascular clotting factors to contribute to hemostatic protection within joint tissue is unknown. We hypothesized that replacing deficient factor VIII or IX within the injured joint capsule of mice with hemophilia A (FVIII −/ −) or hemophilia B (FIX −/ −), respectively, would decrease the progression of synovitis. We developed a bleeding model consisting of a unilateral knee joint capsule needle puncture to induce hemorrhage in hemophilic mice. Pathology of the joint at two weeks after the injury is graded 0 to 10 using a murine hemophilic synovitis grading system (Valentino, Hakobyan. Haemophilia, 2006). Hemostatically normal mice do not develop synovitis following this injury, but > 95% of FIX −/ − mice develop bleeding and synovitis with a mean grade of 3–4 or greater. Coincident with needle puncture, recombinant human coagulation factor doses ranging from 0 to 20 IU/kg body weight of factor IX or 0 to 25 IU/kg of factor VIII were instilled intraarticularly (I.A.). Comparison groups received the same injury and intravenous (I.V.) factor IX or VIII doses of 25 IU/kg to 100 IU/kg (n= 4–7 mice per study group). Joint bleeding phenotype of the two strains of mice was similar. Mice receiving only saline injection at the time of needle puncture developed mean synovitis scores of 5 ±0.5 in the FVIII −/ − mice and 6 ±0.5 in the FIX −/ − mice. Protection by human clotting factor in the mouse coagulation system was incomplete; mice receiving 100 IU/kg I.V. of factor VIII or factor IX developed synovitis scores of 2.6 ± 1.7 and 2.1 ± 0.2, respectively. In contrast, pathology grade of FVIII −/ − mice dosed with 25 IU/kg I.A. was 0.67 ± 0.3 (p = 0.05 for comparison of 25 IU/kg I.A. with 100 IU/kg IV); FIX−/ − mice receiving 20 IU/kg I.A. had synovitis scores of 0.45 ± 0.58 (p < 0.01 for comparison of 25 IU/kg I.A. with 100 IU/kg I.V.). We next ruled out the possibility that I.A. factor was entering the circulation, and via that route resulting in joint protection, either through technical error at the time of injection, or from a depot effect in the joint with late equilibration into the circulation. Additional groups of mice received factor VIII or IX intravenously at 100 IU/kg, or intraarticularly at 4 times the doses used in the hemarthrosis challenge (80 IU/kg FIX or 100 IU/kg FVIII), and factor activity assays were performed at 1, 4, 12, 24, and 48 hours. Expected circulation kinetics were seen following I.V. dosing; no increase in circulating factor VIII or IX activity were seen in the intraarticular dosing groups at any timepoint. In considering the potential immunogenicity of an intraarticular therapy approach for hemophilic joint therapy, factor VIII −/ − mice were treated with three doses of human factor VIII 100 IU/kg at five day intervals either I.V. or I.A. At two weeks after exposure, 5/5 I.V.-treated mice developed inhibitor antibodies with titers ranging 0.8–7.2 BU; 2/5 I.A.-treated mice had detectable low-titer antibodies (1.3 BU), indicating no greater immunogenicity in the I.A. model. Extravascular factor VIII and factor IX can contribute to protection against blood-induced joint deterioration; enhancing local tissue hemostasis with protein or gene therapy may prove a useful adjunct to systemic replacement.

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Clinical Application of Prothrombin Complex Concentrate in Blood Management in Patients

Critical Care Nurse ◽

10.4037/ccn2017333 ◽

2017 ◽

Vol 37 (2) ◽

pp. 49-56

Author(s):

Sherri Ozawa ◽

Tiffany Nelson

Keyword(s):

Vitamin K ◽

Prothrombin Complex Concentrate ◽

International Normalized Ratio ◽

Fresh Frozen Plasma ◽

Drug Discontinuation ◽

Clotting Factors ◽

Urgent Surgery ◽

Anticoagulant Reversal ◽

Fresh Frozen ◽

Frozen Plasma

Management of patients receiving anticoagulants is a major factor in achieving better outcomes. Anticoagulant therapy may need to be discontinued or rapidly reversed before urgent surgery or invasive procedures. In these situations, treatment with concentrated vitamin K, fresh frozen plasma, and/or clotting factors can achieve more rapid anticoagulant reversal than can drug discontinuation alone. Activated prothrombin complex concentrate is used to treat hemophiliac patients with acquired factor VIII inhibitors. Nonactivated prothrombin complex concentrates are used for anticoagulant reversal. The concentrates are effective within minutes of dosing, providing a nearly immediate decrease in the international normalized ratio. The concentrates are lyophilized powders that can be quickly reconstituted, do not require ABO blood typing before use, and contain 25 times the concentration of vitamin K–dependent clotting factors compared with fresh frozen plasma. Studies suggest that the concentrates are associated with better clinical end points than is fresh frozen plasma.

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Properties of Recombinant Human Thromboplastin that Determine Sensitivity to Vitamin K-Dependent Coagulation Factors.

Blood ◽

10.1182/blood.v104.11.533.533 ◽

2004 ◽

Vol 104 (11) ◽

pp. 533-533

Author(s):

Stephanie A. Smith ◽

James H. Morrissey

Keyword(s):

Ionic Strength ◽

Vitamin K ◽

Phospholipid Composition ◽

Coagulation Factors ◽

Factor Vii ◽

Clotting Factor ◽

Sensitivity Index ◽

Clotting Factors ◽

Individual Factor ◽

Factor Deficiency

Abstract Patients undergoing oral anticoagulant therapy (OAT) with coumarins have reduced plasma levels of vitamin K-dependent clotting factors. The primary laboratory test for monitoring OAT is the prothrombin time (PT), in which clotting is initiated by tissue factor (TF). Clotting factors that contribute to the PT, and whose levels respond to OAT, are factor VII (FVII), factor X (FX), and prothrombin, although they are not suppressed to the same extent. Thromboplastin reagents (the source of TF activity in PT tests) can vary dramatically in their sensitivities to the effects of OAT. A calibration system, the International Sensitivity Index (ISI), is widely used to correct the PT for variable thromboplastin sensitivity, but discrepant responses by reagents of similar ISI have been reported. We have undertaken studies aimed at understanding which factors control the sensitivity of thromboplastin reagents, with a goal of creating “designer thromboplastins” whose sensitivities to specific clotting factors can be individually tailored. Thromboplastin reagents were prepared by reconstituting recombinant human TF into phospholipid vesicles containing varying amounts of phosphatidylcholine, phosphatidylserine (PS), and phosphatidylethanolamine (PE). Thromboplastins containing low levels of PS and high ionic strength had the highest sensitivity to OAT (i.e., lowest ISI). PE shifted the dose-response such that lower levels of PS were required to obtain the same ISI value. These studies demonstrate that multiple combinations of phospholipid composition and ionic strength can be used to produce reagents of identical ISI. We hypothesized that reagents of identical ISI values but different composition could have very different responses to changes in the levels of individual coagulation factors. Accordingly, thromboplastin reagents of varying composition were evaluated for their responses to deficiencies of FVII, FX and prothrombin. PT tests were performed using pooled normal plasma mixed with individual factor-depleted plasmas to yield 10%, 3%, 1% or 0.3% of the normal level of the specific clotting factor. Responses of thromboplastin reagents to individual factors were compared by plotting the clotting times obtained with these plasmas on log-log scales versus the percent factor level and fitting lines to the data by linear regression. Interestingly, altering the composition of the thromboplastin reagents dramatically and independently altered their sensitivities to individual clotting factors. For example, increasing ionic strength had no impact on the response to FVII, but markedly enhanced the response to prothrombin deficiency. Furthermore, the effect of changes in ionic strength on specific factors levels differed depending upon the phospholipid composition. These studies demonstrate that thromboplastin reagents of dissimilar composition but nearly identical ISI values can have very different sensitivities to deficiencies in FVII, FX, or prothrombin, so reagents of identical ISI do not necessarily respond to the factor deficiencies induced by OAT in an identical fashion. These studies evaluated samples with isolated individual factor deficiency, whereas patients on OAT have combined factor deficiency and therefore have more potential for discrepancy in PT responses between reagents. Controlling the responsiveness of thromboplastin reagents to deficiencies in individual clotting factors may therefore be desirable for monitoring OAT and for the other clinical diagnostic uses to which PT tests are commonly applied.

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