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

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.

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

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

INTRODUCTION: ADAMTS-13 is a member of A Disintegrin 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 (CCl(4)) 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 CCl(4) 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 CCl(4) 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.

Microarray-based genetic analyses for studying susceptibility to arterial and venous thrombotic disorders.

We describe the potential of microarray technology for parallel, high-throughput approaches to molecular detection of DNA variations associated with progression to arterial and venous thrombotic diseases. The use of the newly commercialized NanoChip platform and a framework for genetic screening with a list of potential genetic targets useful to the evaluation of cardiovascular risk are presented. Implementation in clinical setting of analytical silicon and glass microarrays will facilitate early diagnosis, help direct specific advice to high-risk individuals and evaluate treatment strategies.

Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells.

The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.

Regulation of human coagulation factor X gene expression by GATA-4 and the Sp family of transcription factors.

Serine protease factor Xa plays a critical role in the coagulation cascade. Zymogen factor X is synthesized and modified in the liver. To understand the mechanisms governing the liver-specific expression of factor X, the proximal promoter of human factor X was previously characterized. Two crucial cis elements at -73 and -128 and their cognate binding proteins, HNF-4 and NF-Y, respectively, were identified. In this report, studies are extended to 3 additional cis elements within the factor X promoter. Using gel mobility shift assays, the liver-enriched protein GATA-4 was identified as the protein binding to the GATA element at -96. GATA-4 transactivates the factor X promoter 28-fold in transient transfection experiments. It was also determined that the Sp family of transcription factors binds 2 DNase I-footprinted sites at -165 and -195. Disruption of Sp protein binding at either site reduces the promoter activity by half. Simultaneous disruption of both sites reduces the promoter activity 8-fold. This is the first report indicating the involvement of GATA-4 in the regulation of clotting factor expression. These observations provide novel insight into mechanisms by which the vitamin K-dependent coagulation factors are regulated.

A new mutation in the HNF4 binding region of the factor VII promoter in a patient with severe factor VII deficiency.

Investigation of the molecular basis of a severe factor VII (fVII) deficiency revealed compound heterozygosity in the fVII gene. On the paternal allele the patient had 3 structural gene abnormalities frequently associated with fVII deficiency. A new mutation, a C to T transition at position -55 relative to the translational start site, was found on the maternal allele. The study demonstrates that this mutation partially impeded binding of the transcriptional activator, hepatic nuclear factor 4, to the fVII promoter while greatly reducing reporter gene expression in hepatic cells. (Blood. 2000;96:4370-4372)

Secretable human platelet-derived factor V originates from the plasma pool.

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into alpha-granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed.

Severe factor VII deficiency due to a mutation disrupting an Sp1 binding site in the factor VII promoter.

We have identified a point mutation in the promoter of the factor VII gene responsible for a severe bleeding disorder in a patient from a large French-Canadian family with known consanguinity. The proband has an extremely low plasma level of factor VII antigen and factor VII coagulant activity (<1 percent of normal) and suffers from hemarthroses and chronic arthropathy. Sequencing of the patient's factor VII 5' flanking region, intron/exon junctions, and coding regions showed a homozygous point mutation, a C to G transversion at position -94 relative to the translation start site. We show here that this mutation prevented binding of transcription factor Sp1 and of other nuclear proteins to this region of the factor VII promoter and resulted in a 20-fold reduction in reporter gene expression in HepG2 cells. These data underscore the importance of this region of the factor VII promoter for in vivo expression of the factor VII gene.

Severe factor VII deficiency due to a mutation disrupting a hepatocyte nuclear factor 4 binding site in the factor VII promoter.

Although small deletions, splice site abnormalities, missense, and nonsense mutations have been identified in patients with factor VII deficiency, there have been no reports of mutations in the factor VII promoter. We investigated a girl with factor VII levels that were less than 1% of normal in association with a severe bleeding diathesis. The patient is homozygous for a T to G transversion that occurs 61 bp before the translation start site. This nucleotide is in a sequence that is an hepatocyte nuclear factor 4 (HNF-4) binding site within the factor VII promoter (ACTTTG AE-->ACGTTG). Using gel mobility shift assays, we show that the mutation disrupts the binding of HNF-4 to its cognate binding site. In growth hormone reporter gene assays, the activity of a plasmid containing the mutant promoter was 6.7% of the wild-type promoter plasmid. Although HNF-4 was able to transactivate the wild-type factor VII promoter 5.4-fold in HeLa cells, no transactivation could be shown with the mutant promoter. These findings indicate that HNF-4 exerts a major positive regulatory effect on factor VII expression and provides in vivo evidence that binding of this transcription factor is critical for normal factor VII expression.

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, Spl, 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 non-hepatocyte 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.