Edward G. D. Tuddenham

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational γ-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.

The Hemophilias — From Royal Genes to Gene Therapy

Of the various types of hemophilia, the most common of these lifelong bleeding disorders are due to an inherited deficiency of factor VIII or factor IX (Table 1). The genes for these blood coagulat...

Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex.

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But ∼30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.

Factor VII deficiency and the FVII mutation database.

Factor VII (FVII) is a zymogen for a vitamin K‐dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 μg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N‐terminal domain (Gla) post‐translationally modified by γ‐carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C‐terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152‐Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc.mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild‐moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels. Hum Mutat 17:3–17, 2001.

Complete Inhibition of Acute Humoral Rejection Using Regulated Expression of Membrane-tethered Anticoagulants on Xenograft Endothelium

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane‐tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non‐transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T‐cell‐mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.

Bleeding symptoms in 27 Iranian patients with the combined deficiency of factor V and factor VIII

Inherited deficiency of factors V and VIII is the most frequent combined coagulation defect. The cases reported so far are mostly single cases or small series from different centres, making it difficult to evaluate the overall pattern of clinical manifestations of the combined defect. We examined at a single institution 27 Iranian patients. Mucocutaneous and post‐surgical bleeding were the most frequent clinical manifestations. The presence of two defects did not make the severity of bleeding greater than that expected in patients with single coagulation defects of similar degrees.

Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts

Summary.u2002 Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. Objectives:u2002To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein‐1 (MCP‐1), interleukin‐8 (IL‐8), interleukin‐6 (IL‐6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. Methods:u2002Steady‐state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse‐transcription–polymerase chain reaction. Results:u2002FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP‐1 mRNA and protein secretion strongly, IL‐8 moderately, and IL‐6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR‐1 and PAR‐3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR‐2. Conclusions:u2002FXa and thrombin induce expression of MCP‐1, IL‐8 and IL‐6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.

Factor VIII – novel insights into form and function

Factor VIII (FVIII) is a key cofactor in blood coagulation, deficiency of which results in the serious bleeding disorder haemophilia A. The traditional treatment for haemophilia is by replacement therapy using plasma-derived or recombinant FVIII products. Recent developments in FVIII biochemistry have improved our understanding of how FVIII interacts with other macromolecules in the performance of its normal procoagulant function and how naturally occurring mutations result in the spectrum of clinical phenotypes found in haemophilia A. This review describes elements of recent studies on the structure of the FVIII molecule (as determined by X-ray crystallography, electron crystallography and modelling studies), its interaction with plasma von Willebrand factor and how this relates to the activation state of FVIII, interactions with procoagulant phospholipid surfaces and factor IXa in the formation of the tenase complex and with the low-density lipoprotein receptor-related protein (LRP), a hepatic cell-surface receptor now known to be at least partly responsible for FVIII clearance from the circulation. Our improving understanding of the life cycle of the FVIII molecule carries with it the potential for better treatment for haemophilia, either by augmentation of its functional characteristics or by suppression of the natural clearance mechanisms.

The locus for combined factor V-factor VIII deficiency (F5F8D) maps to 18q21 , between D18S849 and D18S1103

Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factor V and coagulation factor VIII are diminished. In order to map and subsequently clone the gene responsible for this phenotype, DNAs from 19 families (16 from Iran, 2 from Pakistan, and 1 from Algeria) with a total of 32 affected individuals were collected for a genomewide linkage search using genotypes of highly informative DNA polymorphisms. All pedigrees except two contained at least one consanguineous marriage. A maximum LOD score (Zmax) of 14.82 for theta = .02 was generated with marker D18S1129 in 18q21; LOD scores > 9 were obtained for several other markers-D18S849, D18S1103, D18S64, and D18S862. Multipoint analysis resulted in Zmax = 18.91 for the interval between D18S1129 and D18S64. Informative recombinants placed the locus for F5F8D between D18S849 and D18S1103, in an interval of approximately 1 cM. These results are similar to the recently reported linkage of this disease to chromosome 18q in Jewish families (Nichols et al. 1997) and provide evidence that the same gene is responsible for all F5F8D among human populations. The difference in clinical severity of the phenotype in unrelated families, as well as the failure to detect a specific haplotype of DNA polymorphisms in the consanguineous Iranian families, suggests the existence of different molecular defects in the F5F8D gene. There exists an apparently gap-free contig with CEPH YACs linking the two markers on either side of the critical region. Positional cloning efforts are now in progress to clone the F5F8D gene.

Contact activation in shock caused by invasive group A Streptococcus pyogenes

ObjectivesThe aim of this study was to characterize abnormalities of coagulation in mice with experimental, invasive group A, streptococcal shock, in an attempt to explain the prolongation of the activated partial thromboplastin time identified in patients with streptococcal toxic shock syndrome. DesignA longitudinal descriptive animal model study of coagulation times and single coagulation factors in mice infected with Streptococcus pyogenes. This was followed by an experimental study to determine whether streptococci or streptococcal products could activate the human contact system in vitro. SettingUniversity infectious diseases and hemostasias molecular biology laboratories. SubjectsCD1 outbred mice. InterventionsNone. Measurements and Main ResultsCoagulation times, single factor assays, and bradykinin assays were conducted on murine plasma at different times after streptococcal infection and compared with uninfected mice. In experiments in which streptococcal products were co-incubated with human plasma, we compared coagulation times, single factor assays, and activities against a range of chromogenic substrates with control plasma. In a murine model of streptococcal necrotizing fasciitis, the activated partial thromboplastin times were significantly prolonged in infected mice compared with controls, whereas prothrombin times were normal, suggesting an isolated abnormality of the intrinsic pathway. Bleeding was not seen. Prolongation of activated partial thromboplastin time was associated with reduced factor XII and prekallikrein, whereas levels of factors VIII, IX, XI, and high molecular weight kininogen were elevated. In vitro studies suggested that streptococcal supernatants can activate prekallikrein, in addition to causing plasminogen activation through the action of streptokinase. ConclusionsProlongation of activated partial thromboplastin time in streptococcal toxic shock syndrome is associated with activation of the contact system, possibly contributing to the profound shock associated with streptococcal toxic shock syndrome.