Vitamin K-dependent Factors Research Articles

Unbalanced protein S deficiency due to warfarin treatment as a possible cause for thrombosis

The prothrombin time/International Normalized Ratio is an accurate parameter for following most warfarin-treated patients. However, it reflects mostly factor VII levels, and significant disproportionate alterations in other vitamin K-dependent factors may not be reflected properly. We report a rare case of a woman who developed arterial thrombosis whilst receiving therapeutic doses of warfarin, and was found to have a significant and unbalanced protein S deficiency. Discontinuation of warfarin resulted in complete normalization of coagulation factors, and no further thrombotic events under treatment with enoxaparin, with a follow-up of 2.5 years.

VKORC1: molecular target of coumarins.

The genetic diagnosis of a single family with combined vitamin K-dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra- and inter-individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin-resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose-influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low-dose vitamin K (80-100 microg day(-1)) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co-expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate-limiting step in the synthesis of biologically active vitamin K-dependent factors. Thus, co-expression of VKORC1 leads to a more efficient production of recombinant vitamin K-dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.

Warfarin Cessation Before Cardiopulmonary Bypass: Lessons Learned from a Randomized Controlled Trial of Oral Vitamin K

No standard protocol of warfarin cessation and bridging for cardiac surgery exists. This study examined a single institution's protocol with respect to timing of cessation and low molecular weight heparin bridging. The recovery of vitamin K-dependent factors and the effects of cardiopulmonary bypass on coagulation factors were explored. Administration of preoperative oral vitamin K was investigated in a randomized, placebo-controlled trial. A post hoc analysis examined residual anti-Xa activity of enoxaparin bridging. Forty patients on warfarin undergoing cardiopulmonary bypass were randomized to receive 5 mg of oral vitamin K or placebo 6 days before surgery. Blood samples were acquired at six times and assayed for prothrombin time, anti-Xa activity, and functional levels of factors II, V, VII, and IX and of protein C. Measures of bleeding and transfusion were also collected. No difference in bleeding or transfusion was observed between the treatment groups. Appropriate recovery of coagulation factors was observed with warfarin cessation irrespective of treatment group. The coagulation factors decreased by an average of 0.36 units/mL during the period of surgery. Enoxaparin 1 mg/kg until the evening before surgery resulted in 70% of patients entering the operating room with therapeutic anti-Xa activity (0.6 +/- 0.3 units/mL). The cessation of warfarin 6 days preoperatively is sufficient for functional recovery of vitamin K-dependent factors, which undergo significant changes during the operative course. A 5-mg dose of vitamin K with warfarin discontinuation did not enhance recovery of vitamin K-dependent factors and is unnecessary. With the observation that enoxaparin up until the night before surgery resulted in high residual anti-Xa levels in the operating room, our center now administers the last dose of enoxaparin 24 hours before surgery.

Pseudoxanthoma Elasticum-Like Phenotype with Cutis Laxa and Multiple Coagulation Factor Deficiency Represents a Separate Genetic Entity

Data on six patients with a Pseudoxanthoma Elasticum (PXE)-like phenotype, characterized by excessive skin folding (resembling cutis laxa) and a deficiency of the vitamin K-dependent clotting factors (II, VII, IX, and X) are presented. A comparison is made between the clinical, ultrastructural, and molecular findings in these patients and those seen in classic PXE and cutis laxa, respectively. Clinical overlap with PXE is obvious from the skin manifestations of yellowish papules or leathery plaques with dot-like depressions at presentation, angioid streaks and/or ocular peau d'orange, and fragmentation and calcification of elastic fibers in the dermis. Important phenotypic differences with PXE include much more severe skin laxity with spreading toward the trunk and limbs with thick, leathery skin folds rather than confinement to flexural areas, and no decrease in visual acuity. Moreover, detailed electron microscopic analyses revealed that alterations of elastic fibers as well as their mineralization were slightly different from those in classic PXE. Molecular analysis revealed neither causal mutations in the ABCC6 gene (ATP-binding cassette subfamily C member 6), which is responsible for PXE, nor in VKORC1 (vitamin K 2,3 epoxide reductase), known to be involved in vitamin K-dependent factor deficiency. However, the GGCX gene (gamma-glutamyl carboxylase), encoding an enzyme important for gamma-carboxylation of gla-proteins, harbored mutations in six out of seven patients analyzed. These findings all support the hypothesis that the disorder indeed represents a separate clinical and genetic entity, the molecular background of which remains to be unraveled.

Monitoring direct thrombin inhibitors with a plasma diluted thrombin time

Activated partial thromboplastin time (aPTT) monitoring of direct thrombin inhibitors (DTIs) is vulnerable to interference from many sources. If the baseline aPTT is prolonged, as occurs with lupus inhibitors, alternative methods are required to monitor DTI levels. We compared the plasma diluted thrombin time (1:4 dilution of patient plasma with normal plasma) and the aPTT in patient samples spiked with argatroban, bivalirudin, or lepirudin at three concentration levels. Each drug was spiked into five samples with lupus inhibitors, five samples with deficient vitamin K-dependent factors, three samples with elevated D-dimers, and eight samples with normal baseline aPTT values. The aPTT overestimated the spiked DTI concentration in all samples with lupus inhibitors, low levels of vitamin K-dependent factors, and elevated D-dimers. In samples with normal baseline aPTTs, the aPTT failed to correctly estimate the spiked drug concentration in four of 24 samples spiked with argatroban, seven of 24 spiked with lepirudin, and three of 24 spiked with bivalirudin. The plasma diluted thrombin time was not affected by lupus inhibitors, low vitamin K-dependent factor levels or elevated D-dimer levels and correctly estimated the spiked drug level in 63 of 63 samples spiked with argatroban, 63 of 63 samples spiked with bivalirudin, and 62 of 63 samples spiked with lepirudin. In conclusion, the plasma diluted thrombin time appears to be a viable alternative to the aPTT for monitoring DTI levels, especially in patients with lupus inhibitors or low levels of vitamin K-dependent factors.

The influence on INRs and coagulation factors of the time span between blood sample collection and intake of phenprocoumon or acenocoumarol: Consequences for the assessment of the dose

Managing treatment with vitamin K antagonists, the prothrombin time (PT), expressed as international normalized ratio (INR), may not represent the INR during the entire 24 hour (h) period, and this variation may be different between long-acting phenprocoumon and short-acting acenocoumarol. For both drugs we investigated the variation in 24 h of the PT/INR, the consequencesfor the assessment of the doses and which vitamin K-dependent factor causes the daily variation. Patients on self-management took their medication at 6 p.m. and determined their INRs for eight weeks, once a week and three times daily (8.30 a.m., 6 p.m. and 11 p.m., thus 14.5 h, 24 h and 29 h after taking the medication, respectively). Acenocoumarol showed a significant variation in INRs over the 24 h period, with 22 out of 80 INRs >20% lower at 11 p.m. versus 8.30 a.m. Phenprocoumon showed only few variations. Patients managed by the anticoagulation clinic took their medication at 6 p.m. for four weeks and then at 8 a.m. for four weeks, 15 h and 25 h, respectively, before the weekly blood collection. PT/INR and coagulation factors VII, X and II were determined. With acenocoumarol, taken 25 h before blood collection, the INRs were significantly different compared to 15 h, especially attributed to plasma levels of factor VII. Those on phenprocoumon were equal. These variations of INRs during 24 h may have major effects on the prescribed dose of short-acting vitamin K antagonists, such as acenocoumarol, especially for INRs at the limits of the therapeutic ranges.

Efficacy of Prothrombin Complex Concentrate (PCC) for Reversal of Warfarin Effect.

In the United States supratherapeutic INRs are typically reversed with fresh frozen plasma (FFP). This requires transfusion of large volumes (10–15 ml/kg) with therapeutic delay, volume overload, risk for TRALI (transfusion related acute lung injury) and multiple donor exposures. Our Blood Utilization Review Committee recommends PCC as the preferred product for warfarin reversal. Profilnine (Alpha Therapeutic Corp, Los Angeles, CA) is PCC containing vitamin-K dependent clotting factors and dosed by FIX content. FII, VII and X content is no more than 150%, 35% and 100% of the FIX amount, respectively. The manufacturer's suggested dose for warfarin reversal is 15–50 U/kg. The dose of PCC and need for vitamin K or FFP is determined by the clinician after consultation with the transfusion service. Sixty-five patients received PCC; 25 for reduction of INR prior to surgery or other interventions and 40 for treatment of a supratherapeutic INR (>5.0) with bleeding or risk of bleeding. Twenty-three patients with supratherapeutic INRs received low dose PCC (mean 25 U/kg) and 17 received high dose (mean 50 U/kg). The average admission INR was 9.0 (5.2–15.0) for the low-dose group and 8.6 (5.3–15) for the high-dose group. Repeat INR measurements occurred after PCC and again if FFP was transfused. Twenty patients had samples frozen for analysis of FII, VII, IX and X with a mean time between PCC infusion and sample draw of 2 hours (0.5–8.1). Ten of these were stable at the time of PCC infusion and were used to calculate relative factor recovery. The INR corrected (decreased to ≤3) in 55%(6/11) of cases following treatment with low-dose PCC, while high-dose PCC corrected the INR in 43%(3/7). Combination of PCC and FFP (mean 2 units, ~5–7 mL/kg) corrected the INR in 89% (16/18) and 87%(13/15) of cases for low and high-dose PCC, respectively. PCC dose, with or without FFP, had no effect on the likelihood of INR correction (Chi-square, p> 0.05). The addition of low dose FFP, however, significantly increased the response rate (Chi-square, p<0.05) for both doses. The average factor increment is summarized in Table 1. Differences in factor levels between the PCC and PCC plus FFP groups were not significant(ANOVA, p>0.05). Based on PCC dose the median recovery of factor IX was 100% with a median recovery for FII, VII and X of 168%, 32%, and 100% of the factor IX increment. When FFP was added to the treatment regimen the recovery of factor IX was 106% of the PCC dose. FII, VII, and X were 182%, 46%, and 119% of the FIX increment. Only FVII recovery was significantly higher in the PCC plus FFP group (Mann-Whitney, p< 0.05). In conclusion, treatment with Profilnine increases factor levels compatible with the manufacturer's insert, but PCC plus FFP causes a larger FVII increment. Treatment with low or high dose PCC results in similar rates of INR correction. However, addition of FFP increases the rate of INR correction for both doses. Profilnine alone is, therefore, unable to reliably correct a supratherapeutic INR, and supplementation with small amounts of FFP is necessary to provide additional FVII. A PCC product with a higher content of FVII is a better therapeutic option but is not readily available in the United States.Factor increments following PCC infusionINRF II(%)F VII(%)F IX(%)F X(%)PCC onlyPre8.3±4.511±107±710±106±3Post2.6±1.453±2114±1028±1931±12PCC plus FFPPre12.2±5.05±11±112±1912±19Post1.9±0.876±2820±1245±2253±19mean±SD

Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes

AbstractUpon local delivery, adenovirus (Ad) serotype 5 viruses use the coxsackie and Ad receptor (CAR) for cell binding and αv integrins for internalization. When administered systemically, however, their role in liver tropism is limited because CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor (F) IX or complement protein C4BP binding to the adenovirus fiber and “bridging” to either low-density lipoprotein receptor-related protein or heparan sulfate proteoglycans. Here, we show that hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C, and FVII in addition to FIX but not by prothrombin (FII), FXI, and FXII. This phenomenon was not dependent on proteolytic activation or cell signaling activity and for FX was mediated by direct virus-factor binding. Human FX substantially enhanced hepatocyte transduction by CAR-permissive and mutated viruses in an ex vivo liver perfusion model. In vivo, global down-regulation of vitamin K-dependent zymogens by warfarin significantly diminished liver uptake of CAR-deleted Ads; however, this phenomenon was fully rescued by acute infusion of human FX. Our results indicate a common and pivotal role for distinct vitamin K-dependent coagulation factors in mediating hepatocyte transduction by adenoviruses in vitro and in vivo.

Compound heterozygosity of novel missense mutations in the gamma-glutamyl-carboxylase gene causes hereditary combined vitamin K–dependent coagulation factor deficiency

AbstractHereditary combined vitamin K–dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the γ-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wild-type carboxylase, and their compound heterozygosity can therefore account for functional VKD factor deficiency. The implications for carboxylase mechanism are discussed.

Defective γ-Glutamyl Carboxylase Activity and Bleeding in Rambouillet Sheep

A flock of Rambouillet sheep was examined because of increased lamb mortality caused by ineffective hemostasis at parturition. Neonatal-affected lambs presented with inadequate hemostasis at the umbilicus, pale mucus membranes, and markedly prolonged activated clotting time. Affected lambs had consistently prolonged 1-stage prothrombin times and activated partial thromboplastin times that supported a defect in the common pathway or defects in both the intrinsic and extrinsic pathway of the coagulation cascade. Decreased activity of vitamin K-dependent procoagulant factors II, VII, IX, and X in male and female lambs suggested either a defect of the hepatic enzyme gamma-glutamyl carboxylase, or vitamin K(1) 2,3 epoxide reductase. Affected lamb hepatic gamma-glutamyl carboxylase activity was markedly decreased compared with that of age- and sex-matched control lambs, while vitamin K(1) 2,3 epoxide reductase and glucose-6-phosphatase activities were similar between an affected and normal lamb. Subcutaneous vitamin K(1) supplementation did not increase vitamin K-dependent procoagulant factor activities in 3 lambs administered vitamin K(1) daily. These data confirm defective gamma-glutamyl carboxylase activity as the cause of impaired coagulation of sheep in this flock. This flock represents the only viable animal model of hereditarily defective gamma-glutamyl carboxylase activity.

Poor correlation of supratherapeutic international normalised ratio and vitamin K‐dependent procoagulant factor levels during warfarin therapy

Patients with a supratherapeutic international normalised ratio (ST-INR) are at risk for bleeding. ST-INR is corrected by withholding warfarin therapy and often by supplementing vitamin K or providing vitamin K-dependent factors; the exact therapeutic decision is based on the extent of the prolonged INR. Currently, ST-INRs are frequently observed in clinical practice due to the use of sensitive recombinant tissue thromboplastin reagents and automation. However, there are scant data correlating an ST-INR with various vitamin K-dependent factors. This prospective cohort study, set in a large tertiary care teaching hospital for the University of Texas Southwestern Medical Center at Dallas, defined the relationship between ST-INR (>5.0) and measured vitamin K-dependent procoagulant factors. Prothrombin time, INR and vitamin K-dependent factors II, VII, IX and X were measured in 78 patients with an INR > 5.0 (ST-INR) who were on warfarin therapy for more than 2 months. There was no significant relationship between the ST-INR and levels of important vitamin K-dependent factors II and X. These data support the recent guidelines that the management of an INR > 5.0 should be driven by the clinical determinants rather than specific INR values per se.

Warfarin Initiation and the Potential Role of Genomic-Guided Dosing

Warfarin Initiation and the Potential Role of Genomic-Guided Dosing

Congenital vitamin K-dependent coagulation factor deficiency: a case report

Congenital vitamin K-dependent coagulation factor deficiency is a very rare bleeding disorder, which usually presents with episodes of intracerebral bleed in the first few weeks of life, sometimes leading to a fatal outcome. We report a case of combined factor deficiency of vitamin K-dependent factors in which the patient presented with both intracerebral bleeding, and possibly also thrombosis, and responded to a vitamin K supplement along with fresh frozen plasma.

Inherited Coagulation Factor VII and X Deficiencies Associated with Severe Bleeding Diathesis: Molecular Genetics and Pathophysiology

The rare inherited coagulation disorders are a fascinating group of diseases that have provided us with important insights into the structure and functions of their respective deficient proteins. Factor (F)VII deficiency is the commonest of these inherited disorders of coagulation, whereas FX deficiency is one of the rarest. Genes encoding the two proteins are located on the same chromosome at 13q34 and are separated by 2.8 kb only. Both proteins are vitamin K-dependent coagulations factors with a very strong structural homology. This review summarizes current knowledge on the prevalence, diagnosis and molecular pathology of FVII and FX deficiencies, and focuses on the genetic abnormalities associated with severe deficiencies and bleeding diathesis

Increased Production of Functional Recombinant Human Clotting Factor IX by Baby Hamster Kidney Cells Engineered to Overexpress VKORC1, the Vitamin K 2,3-Epoxide-reducing Enzyme of the Vitamin K Cycle

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

Battery of Tests for Profiling Abnormalities of Vitamin K-Dependent Coagulation Factors in Drug-Toxicity Studies in Rats

A battery of simple tests for profiling abnormalities of vitamin K-dependent coagulation factors encountered in drug-toxicity studies was verified in rats treated with warfarin (3 and 10 mg/kg, p.o). The thrombotest, or hepaplastin-test, is useful as a follow-up test after routine screening tests for coagulation abnormalities based on PT and APTT, to rule out other coagulation-factor abnormalities. Measurement of coagulation factor activities (factors II, VII, IX and X) using factor-deficient human plasmas provides direct evidence of decreased activities of vitamin K-dependent factors. Furthermore, Echis carinatus venom coagulation time, together with factor II activity, allows us to confirm the generation of PIVKA-II.

Consensus protocol for the emergency reversal of coumarin-induced anticoagulation

The management of haemorrhage or risk of haemorrhage in anticoagulated patients increasingly involves those providing services for haemophilia. This is particularly so in the use of prothrombin complex concentrates where experience of the use of these products exists almost entirely within haemophilia centres. As the majority of bleeding anticoagulated patients present to hospitals that do not have haemophila centres, and where there is little experience in the use of PCCs, there is clearly a need for practical guidelines to guide use of these products. Clinicians must be made aware of the potential benefits and risks of using concentrated coagulation factors prepared from large plasma pools. A key aspects of the safe use of PCCs is the careful selection of the clinical indications for their use. Only those patients with or at risk from life-threatening haemorrhage should be treated. And where a licensed product is available, this should be used in preference to those that are not. Data are lacking on the comparative efficacy of products containing factors II, VII, IX (FIX) and X against those lacking factor VII. There is evidence to show that factor VII levels may be extremely low in patients with high INRs, suggesting that overanticoagulated patients may benefit more from PCCs containing all four vitamin K-dependent factors. The optimal dose of PCC required to reverse anticoagulation is not fully established but does depend on the degree of the coagulation defect. The experience of this group suggests that doses between 25 IU FIX/kg and 50 IU FIX/kg are effective. We present a practical protocol for the use of PCC in reversal of coumarin-induced anticoagulation.

Gas6, warfarin, and kidney diseases

The product of growth arrest-specific gene 6 (Gas6) is a unique vitamin K-dependent autocrine growth factor for mesangial cells, and warfarin inhibits mesangial cell proliferation by interfering with the activation process of Gas6. A recent series of studies has revealed the in vivo roles of Gas6 and its receptor Axl in the progression of acute and chronic glomerulonephritis, diabetic nephropathy, chronic allograft rejection, and human kidney diseases. This review summarizes these studies and discusses the possible interventions targeting the Gas6/Axl pathway to prevent the progression of kidney diseases.

A Quantitative Trait Locus for Cholesterol/Low Density Lipoprotein within the Promoter of the Factor IX Gene.

The correlation between vitamin K-dependent coagulation factors (CF) and lipids has been previously established, with Factor VII notably receiving attention in the investigation of atherosclerosis. Current evidence suggests that plasma phospholipids (PL) may enhance the procoagulant reactions and may increase the availability of fat soluble vitamin K. PL may also affect the clearance of CF. There appears to be a concomitant decrease in CF as the result of lipid lowering therapies. While investigating single nucleotide polymorphisms (SNP) of the Factor IX (fIX) gene as potential quantitative trait loci (QTL) for fIX activity level (fIX:C) in the GAIT study, we observed an apparent relationship between two SNPs, G-793A and C-698T, with plasma cholesterol concentration. As expected, a similar relationship existed with low density lipoprotein (LDL) concentrations. In the current study, we examine these fIX promoter SNPs, which exhibit near perfect linkage disequilibrium (ρ=0.998, p<0.0001), as a possible QTL for cholesterol and LDL. We performed variance components analyses using Solar. We observed significant (p<0.05) marginal associations between LDL and factor VIII activity level (fVIII:C), fIX:C, cholesterol, VLDL, triglycerides, and BMI. We observed significant marginal associations between cholesterol and fVIII:C, fIX:C, LDL, VLDL, triglycerides, lipoprotein A (Lp(a)), and BMI. There was no association for smoking status or contraceptive use. For LDL, the most parsimonious model included age, age2, sex, age2 by sex, fIX:C, and the SNP covariate (coded −1,0,1). C(−698)T was significantly associated with LDL levels (nominal p=0.0262) and these covariates accounted for 33% of the total variance of LDL, with a similar result for G(−793)A. For cholesterol, the most parsimonious model included age, age2, fIX:C, Lp(a), and the SNP covariate. C(−698)T was significantly associated with LDL levels (nominal p=9.54 X 10−3) and these covariates accounted for 40% of the total variance of cholesterol, with a similar result for G(−793)A. Interestingly, these SNPs showed no association with fIX:C. Even though an increase in fIX:C results in an increase in cholesterol in this study, it is the same unit increase regardless of which allele is present, i.e. there is no interaction. The results of a linkage analysis for markers on the X chromosome were insignificant. Though this finding is intriguing and interesting with regard to the correlation between the vitamin K-dependent coagulations factors and lipids, it is entirely possible that it is by chance alone. These findings and the correlation between lipids and the vitamin K-dependent coagulation factors merit verification in an independent study population.

Expression of Blood Coagulation Factor X Is Not Liver-Restricted.

Factor X (FX) is a vitamin K-dependent clotting factor that plays a critical role in blood coagulation by catalyzing the conversion of prothrombin to thrombin. Although predominantly found in liver, expression of FX is not liver-restricted as shown in previous studies (JBC 271:2323–2331, 1996; EMBO J. 11:467–472, 1992). Expression of FX was also detected in whole mouse embryo extracts by RT-PCR as early as E7.5, prior to the formation of a liver bud (TH 84:1023–1030, 2000). Other studies have suggested additional biological functions for FX that are independent of its role in blood coagulation, including stimulation of mitogenic activity in endothelial cells, enhancement of platelet-derived growth factor (PDGF) release from vascular smooth muscle cells, induction of cytokine production, and up-regulation of the early growth response-1 (egr-1) gene transcription. FX-knockout [FX (−/−)] mice generated by our group as well as by Dewerchin et al. showed partial embryonic lethality beginning as early as E10.5 and fatal perinatal bleeding in the remaining FX (−/−) mice surviving to term. However, as for several other coagulation-related knock-out mice, the exact cause of embryonic lethality observed in some FX (−/−) mice remains to be deciphered.In light of these observations, we sought to determine the spatial and temporal patterns of FX expression in both developing and adult mice. Our preliminary studies, utilizing the combined techniques of Northern blot analysis, immuno-histochemistry, and in situ hybridization revealed the following. Northern blot analysis of mRNA isolated from different tissues of wild-type (+/+) adult mice showed FX transcript in multiple tissues including liver, stomach, spleen, lung, colon, ovaries, placenta, and heart (in decreasing levels of FX expression). Results of immuno-histochemistry on selective adult mouse tissues were similar to the results of Northern blot analysis with the exception of kidney, in which we found FX protein in the cortical, but not in the medullary region. Specifically, we noted expression of FX protein in the bronchi/bronchioles of the lung, and selective cells in the myocardium and in pancreas. However, despite detection of FX transcript in spleen and placenta, we failed to detect FX protein in either of these tissues. In developing embryos, immuno-histochemistry revealed expression of FX protein in liver, small intestines, and thymus for wild-type E14.5 paraffin-embedded sagittal sections, and expression of FX protein in liver and selective cells within the brain for E15.5 sagittal sections. Additionally, we carried out in situ hybridization of paraffin-embedded sagittal sections, using digoxigenin-labeled Factor X antisense riboprobe constructed from a 1 kb fragment of the 5′ end of the murine FX cDNA (identical to the probe used in Northern blot). For E12.5 embryos, FX transcript was found predominantly in the liver. For E14.5 embryos, we detected FX transcript not only in the liver, but also in the kidney (specifically in the primitive glomeruli). For E15.5 embryos, we noted FX transcripts in liver, lung, and selective cells in the brain. Already in progress, additional studies including both FX (+/+) and FX (−/−) embryos at earlier stages of development (for example E9.5 to E13.5) and additional adult tissues should provide a more complete delineation of the spatial and temporal patterns of FX expression in mouse development and adulthood. In conclusion, expression of murine FX is not restricted to liver during embryonic development or during adulthood.