Eleanor S. Pollak

Functional Characterization of the Human Factor VII 5′-Flanking Region

Factor VII is a vitamin K-dependent coagulation protein essential for proper hemostasis. The human Factor VII gene spans 13 kilobase pairs and is located on chromosome 13 just 2.8 kilobase pairs 5′ to the Factor X gene. In this report, we show that Factor VII transcripts are restricted to the liver and that steady state levels of mRNA are much lower than those of Factor X. The major transcription start site is mapped at −51 by RNase protection assay and primer extension experiments. The first 185 base pairs 5′ of the translation start site are sufficient to confer maximal promoter activity in HepG2 cells. Protein binding sites are identified at nucleotides −51 to −32, −63 to −58, −108 to −84, and −233 to −215 by DNase I footprint analysis and gel mobility shift assays. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, are shown to bind within the first 108 base pairs of the promoter region at nucleotide sequences ACTTTG and CCCCTCCCCC, respectively. The importance of these binding sites in promoter activity is demonstrated through independent functional mutagenesis experiments, which show dramatically reduced promoter activity. Transactivation studies with an HNF-4 expression plasmid in HeLa cells also demonstrate the importance of HNF-4 in promoting transcription in nonhepatocyte derived cells. Additionally, the sequence of a naturally occurring allele containing a previously described decanucleotide insert polymorphism at −323 is shown to reduce promoter activity by 33% compared with the more common allelic sequence.

Increased ADAMTS-13 proteolytic activity in rat hepatic stellate cells upon activation in vitro and in vivo

Summary.  Introduction: ADAMTS‐13 is a member of ADisintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, primarily synthesized in hepatic stellate cells (HSCs), one of the major cell types transdifferentiating into myofibroblasts during liver fibrosis. However, the association between ADAMTS‐13 expression and HSC activation or liver fibrosis is not known. Methods: In this study, we determined the ADAMTS‐13 mRNA, protein, and activity in isolated primary HSCs upon activation on a plastic dish and in liver after administration of carbon tetrachloride (CCl4) in rats. Results: We showed that ADAMTS‐13 antigen and proteolytic activity in the activated rat HSCs were dramatically increased, whereas ADAMTS‐13 mRNA in these cells was only minimally altered. Similarly, the ADAMTS‐13 antigen and proteolytic activity in rat liver after CCl4 injury were also significantly increased, whereas the ADAMTS‐13 mRNAs in these liver tissues were only slightly increased compared with normal. Surprisingly, despite the dramatic up‐regulation of ADAMTS‐13 protein synthesis in the activated HSCs after CCl4 administration, the plasma levels of ADAMTS‐13 protease in rats did not increase concordantly. Conclusion: We conclude that the up‐regulation of ADAMTS‐13 protein expression in rat HSCs during activation in vitro and in vivo suggests the possibility of ADAMTS‐13 proteolysis, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries. The data also suggest the minimal contribution of the activated HSCs in regulation of plasma levels of ADAMTS‐13 protease.

Interlaboratory agreement in the monitoring of unfractionated heparin using the anti-factor Xa-correlated activated partial thromboplastin time

Summary.  Background: In an effort to improve interlaboratory agreement in the monitoring of unfractionated heparin (UFH), the College of American Pathologists (CAP) recommends that the therapeutic range of the activated partial thromboplastin time (APTT) be defined in each laboratory through correlation with a direct measure of heparin activity such as the factor Xa inhibition assay. Whether and to what extent this approach enhances the interlaboratory agreement of UFH monitoring has not been reported. Objectives: We conducted a cross‐validation study among four CAP‐accredited coagulation laboratories to compare the interlaboratory agreement of the anti‐FXa‐correlated APTT with that of the traditional 1.5–2.5 times the midpoint of normal (1.5–2.5:control) method for defining the therapeutic APTT range. Patients and methods: APTT and FXa inhibition assays were performed in each laboratory on plasma samples from 44 inpatients receiving UFH. Results: Using the anti‐FXa‐correlation method, there was agreement among all four laboratories as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in seven (16%) patient samples. In contrast, consensus was achieved in 23 (52%) samples when the 1.5–2.5:control method was employed. Conclusions: The anti‐FXa‐correlation method does not appear to enhance interlaboratory agreement in UFH monitoring as compared with the traditional 1.5–2.5:control method. Adoption of the anti‐FXa‐correlation method produces considerable disparity in UFH dosing decisions among different centers, although the clinical impact of this disparity is not known.

A novel missense mutation responsible for factor VII deficiency in research Beagle colonies

Summary.  Background: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Objective: Our aim was to identify and characterize the mutation causing cFVII deficiency. Methods: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII‐deficient Beagles and obligate carriers were utilized. Results: In all FVII‐deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor‐like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of ≤ 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII‐G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild‐type and mutant cFVII proteins demonstrated reduced activity of the mutant (< 2%) compared with wild‐type. Rotational thromboelastometry revealed a severe impairment of clotting activity in affected Beagles, and heterozygotes also exhibited changes in coagulation‐based assays. Using a mutation‐specific polymerase chain reaction/restriction digest that allows rapid identification of the G96E mutation, we surveyed a US research Beagle colony and identified a mutant allelic frequency of 31%. Conclusions: We have identified a single causative mutation for cFVII deficiency that may have implications for pharmacotoxicologic research, because reduced FVII coagulant activity may alter hemostatic and/or cardiovascular endpoints in this commonly used animal species.

D-dimer, Magnetic Resonance Imaging Diffusion-weighted Imaging, and ABCD2 Score for Transient Ischemic Attack Risk Stratification

BACKGROUND We sought to determine whether measurement of D-dimer (DD) would improve risk stratification after transient ischemic attack (TIA). METHODS We enrolled 167 patients with acute TIA in a prospective observational study. DD was measured using rapid enzyme-linked immunosorbent assay. The primary outcome measure was a composite end point consisting of stroke or death within 90 days or the identification of a high-risk stroke mechanism requiring specific early intervention (defined as > or =50% stenosis in a vessel referable to symptoms or a cardioembolic source warranting anticoagulation). RESULTS The composite end point occurred in 41 patients (25%). A 50% or greater stenosis was found in 25 patients (15%), a cardioembolic source in 14 (8%), and clinical events in 8 (5 strokes, 3 deaths), 6 of whom also had a high-risk cause of TIA. ABCD(2) score was associated with outcome (P for trend = .017, c-statistic 0.63). DD levels did not differ based on outcome status (geometric mean 0.75 v 0.82 microg fibrinogen equivalent unit/mL, P = .56), and DD had little use for predicting outcome (c-statistic 0.57), even when combined with ABCD(2) score. Of 96 patients with early magnetic resonance imaging (MRI), 23% had diffusion-weighted imaging (DWI) abnormalities, and MRI DWI was predictive of outcome (c-statistic 0.76). The addition of MRI DWI to ABCD(2) improved predictive accuracy (c-statistic 0.83) compared with either alone. CONCLUSIONS Many patients with TIA have a high-risk mechanism (large vessel stenosis or cardioembolism) or will experience stroke/death within 90 days. Increasing ABCD(2) scores were associated with this composite end point. Measurement of DD did not provide additional prognostic information.

Utility of D-dimer in the diagnosis of cerebral venous sinus thrombosis

(20 of 46 patients, 43.5%) compared with a D-dimer plasma level >6125 ng mL (11 of 46 patients, 23.9%). Up to 50% of the patients could be correctly classified as having DVT or not by the determination of theD-dimer and fibrinogen plasma levels. It is evident that the conclusions of the present report should be restricted to patient populations which are comparable to that investigated in this study and to the laboratory tests used here. The sample size of n 1⁄4 103 is small, and, although not uncommon in this type of study [4], a limitation and underscores the pilot character of the present study. The prevalence of DVT in our patient group was 44.7%, which is rather high, although in the range of what has been reported [4]. However, since accuracy indices depend on the prevalence of the disease, the conclusions of the present study should be generalized with caution.

Platelet glycoprotein Ibα polymorphisms modulate the risk for myocardial infarction

Platelet glycoprotein Iba (GPIba) gene polymorphisms have been reported to affect the risk of developing coronary heart disease. Here, within the GPIba gene, we determine the association between the variable number of tandem repeats (VNTR), the -5C/T Kozak sequence dimorphism, and the human platelet antigen (HPA)-2 polymorphisms with occurrence of myocardial infarction (MI). Patients (n=180) presenting survivors of MI were compared to 180 controls matched by age, gender, and race. Carriers of VNTR-CD genotype had a 2-fold higher risk for MI compared to controls.The prevalence of VNTR-BC was lower among patients than among controls (P=.007). These data are in agreement with recent reports of increased plug formation by human platelets containing VNTRCD but no other VNTR genotypes. Among patients, the number of vessels severely occluded was greater among carriers of the D-allele (P=.019) or VNTR-CD (P=.026) and lower among carriers of the C-allele (P=.003) or VNTR-CC (P=.0009) compared to non-carriers of these alleles. No influence was seen with the Kozak or HPA-2 polymorphisms. Determination of VNTR of the GPIba gene may prove useful for identifying high-risk individuals for MI.

Serologic results in >1000 patients with suspected heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

Asymptomatic Factor VII Deficiency in African Americans

African Americans with factor VII (FVII) deficiency, as defined by clinical laboratory values, are frequently asymptomatic. To date the genotypes underlying this FVII defect in asymptomatic African Americans have not been established. We show in 3 unrelated African-American patients that the defect is due to a G to A nucleotide change resulting in an arginine to glutamine mutation in Factor VII amino acid 304. This defect results in low FVII coagulant activity levels using rabbit brain thromboplastin but not using human thromboplastin. This report may aid transfusion and hematology specialists evaluate patient results and prevent unnecessary transfusions to treat patients with abnormal laboratory values.

Mutation in the factor VII hepatocyte nuclear factor 4α-binding site contributes to factor VII deficiency.

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at −60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the −60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4&agr; (HNF4&agr;) co-transfection and a chromatin immunoprecipitation assay indicate that the −60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4&agr;. The importance of interaction between the FVII gene and HNF4&agr; in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.