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

Blood ◽  
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.

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

Blood ◽  
1979 ◽  
Vol 53 (3) ◽  
pp. 366-374 ◽  
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.

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

Blood ◽  
1979 ◽  
Vol 53 (3) ◽  
pp. 366-374 ◽  
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

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.

VKCFD2 – from clinical phenotype to molecular mechanism

2016 ◽  
Vol 36 (S 02) ◽  
pp. S13-S20 ◽  
Author(s):  
K. J. Czogalla ◽  
M. Watzka ◽  
J. Oldenburg
Keyword(s):  
Vitamin K ◽  
Physiological Activity ◽  
Coagulation Factors ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Vitamin K Cycle ◽  
Factor Deficiency ◽  
Er Retention ◽  
Deficiency Type ◽  
Diagnosis Therapy

SummaryVitamin K 2,3-epoxide reductase complex, subunit 1 (VKORC1) is an enzyme essential for the vitamin K cycle. VKORC1 catalyses the reduction of vitamin K 2,3-epoxide to the quinone form of vitamin K and further to vitamin K hydroquinone. The generated vitamin K hydroquinone serves as substrate for the enzyme γ-glutamyl-carboxylase which modifies all vitamin K-dependent proteins, allowing them to bind calcium ions necessary for physiological activity. Vitamin K-dependent proteins include the coagulation factors FII, FVII, FIX, FX, and proteins C, S und Z. Insufficient VKORC1 enzyme activity results in deficiency of the vitamin K-dependent clotting factors leading to haemorrhagic disorders. This phenotype is known as vitamin K clotting factor deficiency type 2 (VKCFD2). Worldwide, only four families of independent origin have been reported with this rare bleeding disorder. Affected family members carry the mutation VKORC1:p.Arg98Trp in homozygous form, the only mutation found so far to be associated with VKCFD2. Now, ten years after the identification of the VKORC1 gene, the molecular pathomechanism of VKCFD2 has been clarified. The Arg98Trp mutation disrupts an ER retention motif of VKORC1 leading to mislocalisation of the protein to outside the endoplasmatic reticulum. In this review, we summarize the clinical data, diagnosis, therapy and molecular patho -mechanism of VKCFD2.

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.

scholarly journals Intracellular maturation of the γ-carboxyglutamic acid (Gla) region in prothrombin coincides with release of the propeptide

1993 ◽  
Vol 291 (3) ◽  
pp. 723-727 ◽  
Author(s):  
R Wallin ◽  
C Stanton ◽  
S M Hutson
Keyword(s):  
Conformational Change ◽  
Vitamin K ◽  
Secretory Pathway ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Coagulation System ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Post Translational Modifications ◽  
Carboxyglutamic Acid

Vitamin K-dependent coagulation factors undergo several post-translational modifications before the proteins are secreted into the blood as functional zymogens of the coagulation system. The modifications include Asn-linked glycosylation, Asn/Asp hydroxylation, removal of a signal peptide for translocation of the polypeptide into the endoplasmic reticulum and removal of a propeptide which, when attached to the intracellular coagulation factor precursor, directs the protein for vitamin K-dependent gamma-carboxylation. gamma-Carboxylation of targeted Glu residues results in formation of Ca(2+)-binding gamma-carboxyglutamic acid (Gla) residues. Ca2+ binding by these residues induces a conformational change in the protein which is a necessary event for optimal activation or activity of the clotting factor in blood. In the present study we have monitored the intracellular prothrombin precursor in the secretory pathway of liver cells to determine the effect that the propeptide has on Ca(2+)-dependent folding of the protein. The data provide evidence that the Ca(2+)-induced conformational change required for activation of prothrombin coincides with release of the propeptide in the trans-Golgi apparatus of the liver cell and elucidates an important function for the endoproteinase furin in biosynthesis of vitamin K-dependent clotting factors.

scholarly journals Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

2020 ◽  
Author(s):  
Melissa A. Chiasson ◽  
Nathan J. Rollins ◽  
Jason J. Stephany ◽  
Katherine A. Sitko ◽  
Kenneth A. Matreyek ◽  
...  
Keyword(s):  
Vitamin K ◽  
Active Site ◽  
Large Scale ◽  
Transmembrane Domain ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Missense Variants ◽  
Vitamin K Cycle ◽  
Factor Deficiency ◽  
Anticoagulant Drug

ABSTRACTVitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency 2 or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

scholarly journals Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Melissa A Chiasson ◽  
Nathan J Rollins ◽  
Jason J Stephany ◽  
Katherine A Sitko ◽  
Kenneth A Matreyek ◽  
...  
Keyword(s):  
Vitamin K ◽  
Active Site ◽  
Large Scale ◽  
Transmembrane Domain ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Missense Variants ◽  
Vitamin K Cycle ◽  
Factor Deficiency ◽  
Anticoagulant Drug

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

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

1989 ◽  
Vol 61 (01) ◽  
pp. 140-143 ◽  
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.

Recombinant Tissue Factor as Substitute for Conventional Thromboplastin in the Prothrombin Time Test

1992 ◽  
Vol 67 (01) ◽  
pp. 042-045 ◽  
Author(s):  
Armando Tripodi ◽  
Arnaldo Arbini ◽  
Veena Chantarangkul ◽  
Pier Mannuccio Mannucci
Keyword(s):  
Prothrombin Time ◽  
Tissue Factor ◽  
Oral Anticoagulant Therapy ◽  
Clotting Factor ◽  
Rabbit Brain ◽  
Sensitivity Index ◽  
Clotting Factors ◽  
Wide Range ◽  
Reference Preparation ◽  
Time Test

SummaryRelipidated recombinant tissue factor (r-TF) has been assessed in comparison with conventional rabbit brain thromboplastin (Manchester Reagent) for its suitability for measurement of prothrombin time (PT). The International Sensitivity Index (ISI) of r-TF calibrated against the International Reference Preparation BCT/253 (human plain) was found to be 0.96 and 1.12 with instrumental and manual techniques. Our study of plasmas from patients with congenital deficiencies of clotting factors covering a wide range of severity demonstrates that r-TF is able to detect even minor deficiencies of factors involved in the extrinsic and common coagulation pathways. Patients with liver diseases were correctly diagnosed with a prevalence of abnormal results comparable for both reagents. Between-assay reproducibility expressed as coefficient of variation was 2.3 % and 3.9 % at normal and abnormal PT levels.In conclusion, our evaluation shows that relipidated r-TF possesses the necessary requisites of sensitivity, diagnostic accuracy and reproducibility which make it a suitable candidate for PT determination both for monitoring oral anticoagulant therapy and diagnosing congenital and acquired clotting factor deficiencies. Moreover, being a highly defined reagent it may constitute a step forward in the standardization of PT testing.

scholarly journals Coagulation Disturbances in Patients with Argininemia

2018 ◽  
Vol 140 (4) ◽  
pp. 221-225 ◽  
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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