Kirk Chu

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Activation-dependent Exposure of the Inter-EGF Sequence Leu83-Leu88 in Factor Xa Mediates Ligand Binding to Effector Cell Protease Receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88-(Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 ~ 20–40 μM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83–88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.

A viable mouse model of factor X deficiency provides evidence for maternal transfer of factor X

Summary.  Background: Activated factor X (FXa) is a vitamin K‐dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal. Objective: We sought to generate a viable mouse model of FX deficiency. Methods: We used a socket‐targeting construct to generate F10‐knockout mice by eliminating F10 exon 8 (knockout allele termed F10tm1Ccmt, abbreviated as ‘−’; wild‐type ‘+’), and a plug‐targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4–9% normal in humans carrying the defect, Pro343→Ser, termed FX Friuli (mutant allele termed F10tm2Ccmt, abbreviated as F)]. Results: F10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of ∼5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age‐dependent iron deposition and cardiac fibrosis. Interestingly, F10 (−/F) mice with FX activity levels of 1–3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival. Conclusions: We demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1–3% is sufficient to rescue the lethal phenotype. This viable low‐FX mouse model will facilitate the development of FX‐directed therapies as well as investigation of the FX role in embryonic development.

Severe factor X deficiency in a pair of siblings: clinical presentation, phenotypic and genotypic features, prenatal diagnosis and treatment.

Severe factor X deficiency is a rare bleeding disorder with a reported incidence of around 1:10 inhabitants [1]. Factor X plays a key role in the intersection between initiation and propagation of the coagulation processes and in the absence of FX, the functions of the TF-FVIIa complex and the prothrombinase complex are compromised with the net result of a seriously reduced thrombin generation. In consequence, patients with the severest forms of FX deficiency suffer from serious and potentially disabling bleeds. The present report deals with a pair of siblings suffering from severe functional FX deficiency. The male, born in 1996 was admitted to our paediatric department due to a swollen knee-joint. The initial investigation showed a prolonged APTT of 137 s FX:C was 0.01 U mL, factor VIII:C 2.00 U mL, factor IX:C 1.83 U mL, the relative PT was <0.15 U mL and factors II, V, VII, and XIII were all normal. The platelet count was 601 · 10 L. The female born in 1999 was diagnosed in utero following request of the parents who feared risk of intracranial bleeding during labour. As shown in Table 1, these children displayed a baseline level of FX:C at <0.01 IU mL. Both were CRM with intermediate FX:Ag levels as assessed by an inhouse ELISA method for FX:Ag. Parents were of Caucasian origin, asymptomatic and unrelated, and both had FX:C levels at close to 0.50 U mL, the mother being CRM with a normal value of FX:Ag, while the father had comparable levels of FX:C and FX:Ag. Mutational analysis revealed a missense mutation in exon 8, producing a p.Val298Met mutation in the mother and an exon 2 Gla-domain change causing a p.Glu16Lys amino acid shift in the father. The latter is novel as no previous reporting has been identified in the ISTH database [2] or in a publication appearing postupdate [3]. Both offsprings were doubly heterozygous for these mutations, and the genotype of the girl was diagnosed before birth utilizing a chorion villus tissue sample. Factor X deficiency was diagnosed in the boy at 17 months of age, when presenting with haemarthrosis of a knee joint requiring FFP treatment. The bleeding phenotype was severe in both children. The boy suffered a total of 30 bleeds during a 12-month period from the time of diagnosis (13 joint bleeds of ankle, knee, hip, elbow and wrist). Additionally four muscle bleeds and several mucosal bleeds were treated with FFP. After 17 treatment episodes he developed increasing signs of intolerance to FFP manifesting as urticaria requiring use of corticosteroid and antihistamine. Subsequently, treatment was shifted to a pasteurized FIX concentrate (factor IX P, ZLB Behring, Marburg, Germany). This concentrate labels 600 IU of factor IX and most often, the FX:C activity is around 900 IU per bottle. The clinical response was excellent and during the following 4 months he was treated on demand for five joint bleeds. At the age of 4 years he received a Port-àCath device and started prophylactic treatment with FIX P 600 IU twice weekly. Initially, the boy had been infused 900 IU FX once weekly, and monitoring disclosed a detectable FX:C trough level. During the first year of prophylactic treatment, the number of bleeds diminished to three in total, and during subsequent 2 years there were no bleeds. The sister had been diagnosed in utero and coagulation studies in the neonatal period confirmed the expected Correspondence: Jørgen Ingerslev, Centre for Haemophilia and Thrombosis, Department of Clinical Biochemistry, University Hospital Skejby, Aarhus, Denmark. Tel.: +45 89495182; fax: +45 89495192; e-mail: ingerslev@ki.au.dk

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2

Background: PRRG proteins were cloned more than a decade ago, but their function is still not known. Results: Several novel protein-protein interactions for PRRG are identified by array screening and pulldown analysis. Conclusion: PRRG-initiated signaling events most likely depend on proteins with WW domains. Significance: The protein-protein interactions identified here may help to elucidate the roles of PRRG proteins in different physiological settings. The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins.