Correction of factor XI deficiency by liver transplantation.

Orthotopic liver transplantation for other diseases typically results in a coincidental cure for hemophilia A and B; however, long-term outcomes of liver transplant in hemophilia C are not very well described. Herein, the authors report a patient of severe congenital factor XI (FXI) deficiency who received an orthotopic liver transplant. The authors discuss the perioperative management and long-term outcomes. The normalization of his FXI levels confirms that the liver is the most clinically relevant site of synthesis of FXI.

Quality assurance in hemostasis: the perspective from the College of American Pathologists proficiency testing program.

External quality assurance (EQA) is an important component of the total quality assurance program of a clinical hemostasis laboratory. The College of American Pathologists (CAP) helps meet this requirement by providing a proficiency testing program that evaluates a broad range of hemostasis methods and analytes. This article reviews the published experience of the CAP proficiency testing program in hemostasis. The purpose is to formulate general conclusions about the benefits of EQA. Between 1963 and 2006, the performance characteristics of a variety of tests have been evaluated, including the prothrombin time, activated partial thromboplastin time, coagulation factor activity assays (e.g., fibrinogen, factor [F] VIII, FIX, FXI), von Willebrand factor assays, unfractionated heparin monitoring, lupus anticoagulant testing, and platelet function. Based on the results of these evaluations, the major benefits of EQA are to (1) enhance patient care and safety through improved laboratory testing; (2) characterize test accuracy and precision across multiple methods; (3) correlate specific method variables with accuracy and precision; (4) identify interfering substances and quantify their effects across multiple methods; (5) identify clinical laboratories that are at risk for poor performance so that their performance can improve; and (6) satisfy accreditation and regulatory requirements.

Thrombophilic families with inheritably associated high levels of coagulation factors VIII, IX and XI.

We describe six families in which associated high levels of coagulation factors (F) XI, FIX and FVIII (each with a plasma concentration higher than the 95th percentile found in a control group of 500 asymptomatic individuals: respectively, 135, 145 and 155 IU dL-1) were inherited as a dominant autosomic genetic traits. In these six families, this syndrome is associated with venous thromboembolic events (Odds ratio 41 [4.9-353], P = 0.0006). It seems to predispose to idiopathic events and, as age increases, is often associated with recurrence. First thrombotic episodes occur in young patients (50% of the carriers are symptomatic at the age of 32 years) and in women, can be unmasked by hormonal treatments, mainly oral contraceptives. The association of high levels of coagulation FXI, FIX and FVIII is thus a new rare high-risk inherited thrombophilia syndrome.

Co-segregation of thrombophilic disorders in factor V Leiden carriers; the contributions of factor VIII, factor XI, thrombin activatable fibrinolysis inhibitor and lipoprotein(a) to the absolute risk of venous thromboembolism.

BACKGROUND AND OBJECTIVES: The clinical expression of factor V Leiden varies widely within and between families and only a minority of carriers will ever develop venous thromboembolism. Co-segregation of thrombophilic disorders is a possible explanation. Our aim was to assess the contributions of high levels of factor VIII:C, factor XI:C, thrombin activatable fibrinolysis inhibitor (TAFI) and lipoprotein (a) (Lp(a)) to the risk of venous thromboembolism in factor V Leiden carriers. DESIGN AND METHODS: Levels of the four proteins were measured, in addition to tests of deficiencies for antithrombin, protein C and protein S, and the prothrombin G20210A mutation, in 153 factor V Leiden carriers, derived from a family cohort study. The (adjusted) relative risk and absolute risk of venous thromboembolism for high levels of each protein were calculated. RESULTS: Of carriers, 60% had one or more concomitant thrombophilic disorders. Crude odds ratios (95% CI) of venous thromboembolism for high protein levels were: 3.2 (1.1-9.3) (factor VIII:C); 1.7 (0.6-4.9) (factor XI:C); 3.0 (1.1-8.2) (TAFI); and 1.9 (0.7-5.7) (Lp(a)). Adjusted for age, sex, other concomitant thrombophilic disorders and exogenous risk factors, the odds ratio for venous thromboembolism were 2.7 (0.8-8.7) for high factor VIII:C levels and 1.8 (0.6-5.3) for high TAFI levels. Annual incidences in subgroups of carriers were 0.35% (0.09-0.89), 0.44% (0.05-1.57) and 0.94% (0.35-2.05) for concomitance of high levels of factor VIII:C, TAFI and both, respectively, as compared to 0.09% (0.00-0.48) in single factor V Leiden carriers and 1.11% (0.30-2.82) for other concomitant disorders. INTERPRETATION AND CONCLUSIONS: High levels of factor VIII:C and TAFI, in contrast with factor XI:C and Lp(a), are mild risk factors for venous thromboembolism, and substantially contribute to the risk of venous thromboembolism in factor V Leiden carriers. Our data support the hypothesis that the clinical expression of factor V Leiden depends on co-segregation of thrombophilic disorders.

Contact system. New concepts on activation mechanisms and bioregulatory functions.

This review considers the data of recent years concerning the contact system initiating the activation of blood plasma proteolytic systems, such as hemocoagulation, fibrinolysis, kininogenesis, and also complement and angiotensinogenesis. The main proteins of the contact system are the factors XII and XI, prekallikrein, and high-molecular-weight kininogen. The data on the structure, functions, and biosynthesis of these proteins and on their genes are presented. Studies in detail on the protein-protein interactions during formation of the ensemble of the contact system components on the anionic surface resulted in the postulation of the mechanism of activation of this system associated with generation of the XIIa factor and of kallikrein. This mechanism is traditionally considered a trigger of processes for the internal pathway of the hemocoagulating cascade. However, the absence of direct confirmation of such activation in vivo and the absence of hemorrhagia in the deficiency of these components stimulated the studies designed to find another mechanism of their activation and physiological role outside of the hemostasis system. As a result, a new concept on the contact system activation on the endothelial cell membrane was proposed. This concept is based on the isolation of a complex of proteins, which in addition to the above-mentioned proteins includes cytokeratin 1 and the receptors of the urokinase-like plasminogen activator and of the complement q-component. The ideas on the role of this system in the biology of vessels are developed. Some of our findings on the effect of leukocytic elastase on the key components of the contact system are also presented.

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.

Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.

Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with approximately 2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an alpha-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of alpha-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0. 05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Defective vaccinia virus as a biologically safe tool for the overproduction of recombinant human secretory proteins.

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency.

Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

Characterization of a panel of monoclonal antibodies to human coagulation factor XI and detection of factor XI in Hep G2 cell conditioned medium.

We have produced a panel of ten monoclonal antibodies specific to coagulation factor XI. Western blot analysis demonstrates that 9 of these antibodies react with the heavy chain of factor XI and one with the light chain. Seven of these antibodies inhibit factor XI and factor XIa activity. We have used immobilised monoclonal antibody for the production of factor XI deficient plasma and to purify factor XI to homogeneity with high yield in a simple two-step procedure. These monoclonal antibodies were used to develop highly sensitive immunoassays capable of detecting less than 0.01 mu factor XI antigen ml-1. A strong correlation was found between antigen and activity levels in 11 patients with hereditary deficiency indicating that none was cross-reacting material positive. Cultured Hep G2 cells were found to synthesize small amounts of factor XI antigen and this could also be detected by functional assay and by western blot analysis.

Synthesis of procoagulant antihaemophilic factor in vitro.

The cells which produce the procoagulant part of antihaemophilic factor (factor VIII-C) in normal people have remained unknown. Normal blood leucocytes cultured in vitro with phytohaemagglutinin synthesised a procoagulant with the properties of factor VIII-C. The procoagulant was inhibited by human VIII-C inhibitor and itself absorbed human VIII-C inhibitor--properties which are regarded as specific for factor VIII-C. Unexpectedly, cultured leucocytes from haemophiliacs also synthesised the procoagulant in similar amounts. Possible explanations and implications of these findings are briefly considered.

Generation of coagulation factors V, XI, and XII by the isolated rat liver.

When isolated rat livers were perfused with platelet-free erythrocytes suspended in Tyrode's solution containing 6% bovine serum albumin, the generation of factors V, XI, and XII was clearly demonstrable. On average, after 5 h of perfusion of a single liver, the concentrations in the perfusate (as a percentage of normal rat plasma) were about 6% for factor V, 8% for factor XI, and 5% for factor XII, compared with 20% for factor VII, which was used as the reference standard. When two livers were perfused, approximately twice these concentrations were achieved. When the properties of these factors in perfusate and plasma were compared, they agreed well except for differences in the celite adsorbability of factors XI and XII.

Factors affecting the evolution of factor XIa during blood coagulation.

Certain conditions affecting the evolution of factor XI-a activity during blood coagulation have been examined. Earlier data had indicated that calcium ion was not required for the conversion of factor XI to its activated form, but very little XI-a could be isolated from citrated or EDTA plasma, whether or not the plasma had been clotted by recalcification. Conversely, factor XI-a activity was identified in resin-decalcified plasma, again with or without recalcification. This confirmed that calcium is not required for the evolution of factor XI-a. This observation also permitted us to perform experiments which indicate that there is no obligatory participation of cellular elements in the evolution of factor XI-a during blood coagulation.