The carboxylation efficiency of the vitamin K-dependent clotting factors: studies with factor IX

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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Intrauterine Subdural Hemorrhage Associated with Profound Deficiency of the Vitamin K Dependent Clotting Factors in an Infant Homozygous for a Common Polymorphism in the Vitamin K Epoxide Reductase Complex 1 (VKORC1) Promoter.

Blood ◽

10.1182/blood.v110.11.2152.2152 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2152-2152

Author(s):

Nadav Schwartz ◽

Johannes Oldenburg ◽

David Hart ◽

Michael Nardi ◽

Ori Langer Most ◽

...

Keyword(s):

Vitamin K ◽

Gestational Age ◽

Subdural Hematoma ◽

Factor Ix ◽

Factor Vii ◽

Dietary Vitamin ◽

Subdural Hemorrhage ◽

Factor X ◽

Clotting Factors ◽

Complete Sequencing

Abstract Hemorrhage in newborn infants who have not received vitamin K supplementation is a well recognized entity, but hemorrhage occurring prenatally due to deficiency of the vitamin K dependent factors is not. We report a subdural hemorrhage in a fetus at 29 weeks of gestation associated with very low levels of vitamin K dependent factors. The pregnancy was monitored by periodic ultrasound because of a previous near term stillbirth to this mother for which no etiology was identified. At 29 weeks a 4.6 × 7.7 × 8.8 cm left subdural hematoma was detected. There is no history of bleeding in this Hispanic family and no known consanguinity. His mother was not taking coumadin or any other medication, and her coagulation studies were normal. The baby was delivered by cesarean section, blood drawn for coagulation studies and 1mg Vitamin K given intramuscularly. The baby oozed from the injection and venipuncture sites. The hematocrit was 19% (mean for gestational age = 40.88) and packed red blood cells were given followed by fresh frozen plasma (10ml/kg). Platelets were 284,000/cmm. At six hours of life coagulation studies were greatly improved, the factor levels now being in the normal range for 29 weeks gestational age. Results at birth, 6 hours and 6 months are shown in the table. At 6 hours the baby no longer oozed from venipuncture sites. He received 1mg vitamin K daily for 3 days and no further supplementation thereafter other than that contained in infant formula. The subdural hematoma was drained on day 1 of life. Growth and development at 20 months are normal. Complete sequencing of the VKORC1 gene showed a homozygous functional promoter polymorphism: VKORC1:c.[1–1639>A]+[1–1639>A] (VKORC1*2/*2). This A allele is associated with 25% less VKORC1 expression and protein (50% less for homozygotes) compared to the G allele. Complete sequencing of the gamma-glutamyl carboxylase gene revealed no functional abnormalities. Individuals homozygous for this VKORC1 polymorphism have a relatively low capacity to regenerate reduced vitamin K and should require more vitamin K intake than others. They are known to require less warfarin. We hypothesize that insufficient vitamin K was available in utero to this fetus to compensate for the relatively low reductase level, and he became severely factor deficient. In the extra uterine environment sufficient dietary vitamin K was available to compensate for his relatively low reductase level. It is possible that the previous unexplained stillbirth was due to hemorrhage because of a similar factor deficiency. A reason for an intrauterine paucity of vitamin K has not been determined but must be rare as 17% of Europeans and a larger number of Chinese are homozygous VKORC1*2/*2, and intrauterine hemorrhage due to this cause is not reported. Birth 6 hours 6 months Values in parentheses are ELISA assays. Other assays are functional. nd = not done PT (sec) >120 17.6 13.7 PTT (sec) 176 50.3 35.2 Fibrinogen (mg/dL) 194 196 287 % Factor II 1 39 96 % Factor V 81 nd 116 % Factor VII 3 61 99 % Factor IX 1 nd 108 % Factor X 2 (33) nd 107 % Protein C <1 (18) 27 nd

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Applications of a salt tolerant cation exchanger carrying sulfate groups

10.22541/au.161420234.42544291/v1 ◽

2021 ◽

Author(s):

Egbert Müller ◽

Djuro Josic ◽

Marija Begic ◽

Suzana Pecenkovic ◽

Uros Andjelkovic

Keyword(s):

Salt Tolerance ◽

Vitamin K ◽

Prothrombin Complex Concentrate ◽

Factor Ix ◽

Sulfate Group ◽

Clotting Factor ◽

Clotting Factors ◽

Cation Exchange Resins ◽

Model Substances ◽

Exchange Resins

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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Haemophilia B Leyden in Greece

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661679 ◽

1986 ◽

Vol 56 (03) ◽

pp. 340-342 ◽

Cited By ~ 2

Author(s):

T Mandalaki ◽

C Louizou ◽

C Dimitriadou ◽

E Briët

Keyword(s):

Prothrombin Time ◽

Vitamin K ◽

Older Patients ◽

Factor Ix ◽

Severe Haemophilia ◽

Clotting Factors ◽

Haemophilia B ◽

Dutch Family ◽

Proportional Reduction ◽

Negative Form

SummaryIn this paper, a five generation Greek family is described with haemophilia B. The disease is characterized by a normal ox-brain prothrombin time, normal levels of the vitamin-K dependent clotting factors VII and X and a proportional reduction of factor IX acttivity and antigen levels all of which is consistent with the cross-reacting material negative form of haemophilia B. However, in this family the factor IX levels in the three patients of generation V are around 1 U/dl while the three older patients in generation III have factor IX levels ranging from 28 to 44 U/dl. In the oldest patient of generation V we observed a rise of the factor IX level from 1 U/dl up to the age of 13 to 10 U/dl at age 14. In addition, the older patients have very mild bleeding symptoms or none at all, while the young ones have occasional spontaneous haemorrhages in muscles and joints, compatible with severe or moderately severe haemophilia. The disease appears to be similar to haemophilia B Leyden which has been described in a Dutch family.

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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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Kinetic Aspects of the Interaction of Blood-Clotting Enzymes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651250 ◽

1968 ◽

Vol 20 (01/02) ◽

pp. 078-087 ◽

Cited By ~ 34

Author(s):

H. C Hemker ◽

A. D Muller

Keyword(s):

Vitamin K ◽

Blood Clotting ◽

Experimental Results ◽

Factor X ◽

Clotting Factors ◽

Vitamin K Deficiency ◽

K Deficiency

SummaryPIVKA, the circulating anticoagulant protein found in vitamin K deficiency can, on kinetical grounds, be recognized as an analogue of factor X. The existence of analogues of other vitamin K-dependent clotting factors cannot be ruled out, but need not be assumed to explain the experimental results.

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The Reaction of Thiol Compounds with the Vitamin-K Dependent Clotting Factors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653570 ◽

1969 ◽

Vol 21 (03) ◽

pp. 573-579 ◽

Cited By ~ 1

Author(s):

P Fantl

Keyword(s):

Prothrombin Time ◽

Vitamin K ◽

Anaerobic Conditions ◽

Clotting Factors ◽

Thiol Compounds ◽

Aerobic Conditions ◽

One Stage ◽

Factor Ii ◽

Ph Dependent ◽

The One

SummaryTreatment of human and dog oxalated plasma with 0.2 to 1.0 × 10−1 M 2.3-dithiopropanol (BAL) or dithiothreitol (DTT) at 2–4° C for 30 min results in the reduction of the vitamin-K dependent clotting factors II, VII, IX and X to the respective-SH derivatives. The reaction is pH dependent. Under aerobic conditions the delayed one stage prothrombin time can be partly reversed. Under anaerobic conditions a gradual prolongation of the one stage prothrombin time occurs without reversal.In very diluted plasma treated with the dithiols, prothrombin can be converted into thrombin if serum as source of active factors VII and X is added. In contrast SH factors VII, IX and X are inactive in the specific tests. Reoxidation to active factors II, VII, IX and X takes place during adsorption and elution of the SH derivatives. The experiments have indicated that not only factor II but also factors VII, IX and X have active-S-S-centres.

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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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Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656082 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0944-0948 ◽

Cited By ~ 20

Author(s):

Darla Liles ◽

Charles N Landen ◽

Dougald M Monroe ◽

Celeste M Lindley ◽

Marjorie s Read ◽

...

Keyword(s):

Gene Therapy ◽

Vitamin K ◽

Absorption Rate ◽

Venous Access ◽

Factor Ix ◽

Hemophilia B ◽

Current Therapy ◽

Home Therapy ◽

Routes Of Administration ◽

Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

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