IntroductionHuman coagulation factor V (FV) is a single-chain glycoprotein that plays an important role in maintaining the hemostatic balance. It circulates in blood as an inactive procoagulant with a M r of 330 kd and a structure consisting of 3 homologous A-type domains and 2 homologous C-type domains connected by a heavily glycosylated B domain in the order A1-A2-B-A3-C1-C2. Proteolytic cleavage by thrombin at R709, R1018, and R1545 (single-letter amino acid codes) results in removal of the B domain and converts the procofactor into the fully active cofactor FVa, which consists of a M r 105-kd heavy chain (A1-A2) and a M r 74-or 71-kd light chain (A3-C1-C2), associated via a single Ca ϩϩ ion. [1][2][3] The difference in molecular weight of the light chain reflects the presence of 2 isoforms of FVa (FVa 1 and FVa 2 ) due to alternative glycosylation of the C2 domain, which leads to different affinities for biologic membranes and subsequent overall procoagulant activity. 4,5 In its active form, FVa forms an essential part of the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin by factor Xa in the presence of calcium and a phospholipid membrane. 1-3 Activated protein C (APC) inactivates FVa through cleavage of the active cofactor at R306, R506, and R679 and requires FV as a cofactor in the APC-mediated inactivation of factor VIIIa (FVIIIa). 6,7 Thus, FV plays an important role in the procoagulant pathway as well as in the protein C anticoagulant pathway. The structure of FV is similar to FVIII (both cofactors share approximately 40% homology in their heavy and light chains) and ceruloplasmin, the copper-binding protein in plasma. 8,9 Recently, the crystal structure of the C2 domain of FV has been established 10 and molecular models for the A and C domains of FV have been proposed. 11,12 The gene for coagulation FV has been mapped to chromosome 1q23 13 and spans more than 80 kilobases (kb). It consists of 25 exons and the messenger RNA (mRNA) encodes a leader peptide of 28 amino acids and a mature protein of 2196 amino acids. Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25. The entire B domain is encoded by exon 13, which contains 2 tandem repeats of 17 amino acids and 31 tandem repeats of 9 amino acids that are absent in the B domain of FVIII. 14,15 Deficiency of FV, or parahemophilia, was first described in 1947 by Owren. 16 It is a rare autosomal recessive bleeding disorder with an estimated frequency of one in one million. The phenotypic expression of FV deficiency is variable; heterozygotes are usually asymptomatic, whereas homozygous patients show mild, moderate, or severe bleeding symptoms. Identifying the molecular basis underlying this disease will help to obtain more insight into the mechanisms involved in this variable clinical expression. The recently published complete nucleotide sequence of the FV gene (GenBank accession number Z99572) has facilitated the molecular characterization underlying FV deficiency and reports have ide...

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Background: α1-Antitrypsin (α1AT) deficiency predisposes individuals to chronic obstructive pulmonary disease (COPD) and/or liver disease. Phenotyping of the protein by isoelectric focusing is often used to characterize α1AT deficiency, but this method may lead to misdiagnosis (e.g., by missing null alleles). We evaluated a workup that included direct sequencing of the relevant parts of the gene encoding α1AT, SERPINA1 [serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1], for patients with α1AT concentrations ≤1.0 g/L. Methods: During a 5-year period, we identified 66 patients with α1AT concentrations ≤1.0 g/L and amplified and sequenced exons 2, 3, and 5 of the α1AT gene in these patients. To ensure that no relevant genotypes were missed, we sequenced the same exons in 48 individuals with α1AT concentrations between 1.0 and 1.5 g/L. Results: Sequence analysis revealed 18 patients with combinations of disease-associated α1AT alleles: 8 homozygous for the deficient Z allele and 10 compound heterozygotes for various deficient or null alleles. We identified and named 2 new null alleles, Q0soest (Thr102→delA, which produces a TGA stop signal at codon 112) and Q0amersfoort (Tyr160→stop). No relevant disease-associated allele combinations were missed at a 1.0-g/L threshold. Conclusions: Up to 22% of the alleles in disease-associated α1AT allele combinations may be missed by conventional methods. Genotyping by direct sequencing of samples from patients with α1AT concentrations ≤1.0 g/L detected these alleles and identified 2 new null alleles.

We assessed whether large-scale expression profiling of leukocytes of patients with essential hypertension reflects characteristics of systemic disease and whether such changes are responsive to antihypertensive therapy. Total RNA from leukocytes were obtained from untreated (n=6) and treated (n=6) hypertensive patients without apparent end-organ damage and from normotensive controls (n=9). RNA was reverse-transcribed and labeled and gene expression analyzed using a 19-K oligonucleotide microarray using dye swaps. Samples of untreated and of treated patients were pooled for each sex and compared with age- and sex-matched controls. In untreated patients, 680 genes were differentially regulated (314 up and 366 down). In the treated patients, these changes were virtually absent (4 genes up, 3 genes down). A myriad of changes was observed in pathways involved in inflammation. Inflammation-dampening interleukin receptors were decreased in expression. Intriguingly, inhibitors of cytokine signaling (the PIAS family of proteins) were differentially expressed. The expression of several genes that are involved in regulation of blood pressure were also differentially expressed: angiotensin II type 1 receptor, ANP-A receptor, endothelin-2, and 3 of the serotonin receptors were increased, whereas endothelin-converting enzyme-1 was decreased. Strikingly, virtually no changes in gene expression could be detected in hypertensive patients who had become normotensive with treatment. This observation substantiates the long-standing idea that hypertension is associated with a complex systemic response involving inflammation-related genes. Furthermore, leukocytes display differential gene expression that is of importance in blood pressure control. Importantly, treatment of blood pressure to normal values can virtually correct such disturbances.