The hemostatic role of factor XI.

Coagulation factor (F)XI has been described as a component of the early phase of the contact pathway of blood coagulation, acting downstream of factor XII. However, patients deficient in upstream members of the contact pathway, including FXII and prekallikrein, do not exhibit bleeding complications, while FXI-deficient patients sometimes experience mild bleeding, suggesting FXI plays a role in hemostasis independent of the contact pathway. Further complicating the picture, bleeding risk in FXI-deficient patients is difficult to predict because bleeding symptoms have not been found to correlate with FXI antigen levels or activity. However, recent studies have emerged to expand our understanding of FXI, demonstrating that activated FXI is able to activate coagulation factors FX, FV, and FVIII, and inhibit the anti-coagulant tissue factor pathway inhibitor (TFPI). Understanding these activities of FXI may help to better diagnose which FXI-deficient patients are at risk for bleeding. In contrast to its mild hemostatic activities, FXI is known to play a significant role in thrombosis, as it is a demonstrated independent risk factor for deep vein thrombosis, ischemic stroke, and myocardial infarction. Recent translational approaches have begun testing FXI as an antithrombotic, with one promising clinical study showing that an anti-sense oligonucleotide against FXI prevented venous thrombosis in elective knee surgery. A better understanding of the varied and complex role of FXI in both thrombosis and hemostasis will help to allow better prediction of bleeding risk in FXI-deficient patients and also informing the development of targeted agents to inhibit the thrombotic activities of FXI while preserving hemostasis.

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice.

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1ß and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.

Novel identification of Factor XI deficiency in Indian Sahiwal (Bos indicus) cattle.

Factor-XI deficiency (FXID) is inherited as autosomal lethal recessive disorder of carrier Holstein-Friesian bulls. A 76 base pair segment insertion into exon 12 in Factor-XI gene causes FXID in cattle. Keeping this in view the present study was conducted to screen breeding bulls of both indigenous and exotic breeds for mutation in Factor-XI gene and to find out the frequency of FXID carrier animals in breeding bulls. A total of 120 bulls of different age group maintained at Frozen Semen Bull Station, India were randomly selected from different cattle breeds to screen presence of FXID syndrome in breeding sires. Genomic DNA was isolated from blood of the selected bulls. PCR parameters were standardized to obtain 244 and 320 bp amplicons. The results showed that 2 Sahiwal bulls out of 120 animals were carrier for FXID. Amplicons of the carrier animals were sequenced and annoted, which confirms a 76 bp insertion in the exon 12. Bleeding and clotting time showed considerable discrepancy in the carrier animals as compared to the normal animals. The findings of relative mRNA expression of Factor XI transcript revealed identical tendency in the carrier. The frequency of carrier animals and mutant allele was 2.5 % and 0.025 respectively. This study recommends for screening of breeding at AI bull centers in the country for FXID. The study also stands a merit for identification of FXID carrier in Bos indicus for the first time.

Factor XI Deficiency Protects Against Atherogenesis in Apolipoprotein E/Factor XI Double Knockout Mice.

OBJECTIVE: Atherosclerosis and atherothrombosis are still major causes of mortality in the Western world, even after the widespread use of cholesterol-lowering medications. Recently, an association between local thrombin generation and atherosclerotic burden has been reported. Here, we studied the role of factor XI (FXI) deficiency in the process of atherosclerosis in mice. APPROACH AND RESULTS: Apolipoprotein E/FXI double knockout mice, created for the first time in our laboratory. There was no difference in cholesterol levels or lipoprotein profiles between apolipoprotein E knockout and double knockout mice. Nevertheless, in 24-week-old double knockout mice, the atherosclerotic lesion area in the aortic sinus was reduced by 32% (P=0.004) in comparison with apolipoprotein E knockout mice. In 42-week-old double knockout mice, FXI deficiency inhibited atherosclerosis progression significantly in the aortic sinus (25% reduction, P=0.024) and in the aortic arch (49% reduction, P=0.028), with a prominent reduction of macrophage infiltration in the atherosclerotic lesions. CONCLUSIONS: FXI deprivation was shown to slow down atherogenesis in mice. The results suggest that the development of atherosclerosis can be prevented by targeting FXI.

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.

Sample conditions determine the ability of thrombin generation parameters to identify bleeding phenotype in FXI deficiency.

Individuals with Factor XI (FXI) deficiency have a variable bleeding tendency that does not correlate with FXI:C levels or genotype. Comparing a range of sample conditions, we tested whether the thrombin generation assay (TGA) could discriminate between control subjects (n = 50) and FXI-deficient individuals (n = 97), and between those with bleeding tendency (n = 50) and without (n = 24). The comparison used platelet-rich plasma (PRP) and platelet-poor plasma (PPP), either with or without corn trypsin inhibitor (CTI) to prevent contact activation, over a range of tissue factor (TF) concentrations. When contact activation was inhibited and platelets were absent, FXI:C levels did not correlate with thrombin generation parameters, and control and FXI-deficient individuals were not distinguished. In all other sample types, the best discrimination was obtained using TF 0.5 pM and assay measures: endogenous thrombin potential (ETP) and peak height. We showed that although a number of conditions could distinguish differences between the groups tested, TGA measured in PRP with CTI best differentiated between bleeders and nonbleeders. These measures provided high sensitivity and specificity (peak height receiver operating characteristic [ROC] area under the curve [AUC] = 0.9362; P < .0001) (ETP ROC AUC = 0.9362; P < .0001). We conclude that by using sample conditions directed to test specific pathways of FXI activation, the TGA can identify bleeding phenotype in FXI deficiency.

The dimeric structure of factor XI and zymogen activation.

Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit.

Thrombosis in rare bleeding disorders.

Inherited deficiencies of blood coagulation factors are usually associated with lifelong bleeding tendency. In addition to Haemophilias A and B and von Willebrand disease, congenital deficiencies of such factors as fibrinogen, prothrombin (FII)), FV, FVII, FX, FXI, FXIII, and combined deficiencies occur and can lead to a diversity of clinical conditions. Paradoxically, for some of these disorders associated with significant bleeding tendency there are reports of thrombotic events, both arterial and venous. Thrombosis in hemophilia patients has a multifactorial pathogenesis and the main conditions associated with this complication are the use of long-term central venous catheters, intensive replacement therapy usually in the setting of surgical procedures, the use of bypassing agents or the coexistence of acquired or inherited prothrombotic risk factors. Regarding other rare bleeding disorders, thrombotic phenomena has been described particularly in patients with afibrinogenemia, FXI and FVII deficiency and the events can occur even in young patients, in the presence of concomitant risk factors or spontaneously. Replacement therapy must be individualized and should take into account past history of haemostatic challenges, family history of bleeding and thrombosis, just like the level of factor. For mild deficiencies when patients are asymptomatic the use of antithrombotic prophylaxis must be considered with or without concomitant use of replacement therapy. In patients with history of thrombosis it may be helpful to perform a thrombophilia screening to exclude coexisting prothrombotic defects and for all patients it is recommended to control known cardiovascular disease risk factors.

A cluster of factor XI-deficient patients due to a new mutation (Ile 436 Lys) in northeastern Italy.

A new mutation (Ile 436 Lys) was found in a cluster of patients in northeastern Italy. The mutation was present in five patients at the homozygote level and in one patient as a compound heterozygote with an already known mutation namely Glu 117 stop. All these patients showed a mild bleeding tendency mainly associated with deliveries or surgery. The first two patients were two sisters, and their parents were consanguineous. The third patient was the only homozygote in the family, and parents apparently were not consanguineous. The fourth and fifth patients were a brother and a sister, and in this case too, parents were not consanguineous. The sixth patient, a compound heterozygote, negated also the existence of consanguinity between his parents. There were also seven heterozygotes among the family members of the patients homozygous for this new mutation (Ile 436 Lys). Finally, there were two heterozygotes for the Glu 117 stop mutation in the family of the sixth patient. The heterozygotes, regardless of the mutation, were asymptomatic. The Ile436Lys mutation is characterized by low factor XI activity and antigen, namely is a cross-reaction material negative form. Molecular modeling indicates that the Ile436Lys mutation causes a large conformational change within the 432-442 loop. No relation could be traced among the different families; however, all their ancestors were autochthonous of the same two small towns. Furthermore, no Jewish ancestry could be found. The close geographical area in which all these patients were found and the absence of the same mutation in the general population of the area strongly suggests a founder effect and that the mutation is responsible for the defect. The compound heterozygosis with the Glu 117 stop mutation, common among Jews, was not surprising because of the past strict ties of the Republic of Venice with the Middle East.

Factor XI and XII as antithrombotic targets.

PURPOSE OF REVIEW: Arterial and venous thrombosis are major causes of morbidity and mortality, and the incidence of thromboembolic diseases increases as a population ages. Thrombi are formed by activated platelets and fibrin. The latter is a product of the plasma coagulation system. Currently available anticoagulants such as heparins, vitamin K antagonists and inhibitors of thrombin or factor Xa target enzymes of the coagulation cascade that are critical for fibrin formation. However, fibrin is also necessary for terminating blood loss at sites of vascular injury. As a result, anticoagulants currently in clinical use increase the risk of bleeding, partially offsetting the benefits of reduced thrombosis. This review focuses on new targets for anticoagulation that are associated with minimal or no therapy-associated increased bleeding. RECENT FINDINGS: Data from experimental models using mice and clinical studies of patients with hereditary deficiencies of coagulation factors XI or XII have shown that both of these clotting factors are important for thrombosis, while having minor or no apparent roles in processes that terminate blood loss (hemostasis). SUMMARY: Hereditary deficiency of factor XII (Hageman factor) or factor XI, plasma proteases that initiate the intrinsic pathway of coagulation, impairs thrombus formation and provides protection from vascular occlusive events, while having a minimal impact on hemostasis. As the factor XII-factor XI pathway contributes to thrombus formation to a greater extent than to normal hemostasis, pharmacological inhibition of these coagulation factors may offer the exciting possibility of anticoagulation therapies with minimal or no bleeding risk.

Three dominant-negative mutations in factor XI-deficient patients.

Factor XI (FXI) deficiency results from genetic defects of the F11 gene and is generally considered to be inherited in an autosomal recessive manner. However, the homodimeric structure of FXI allows, in some cases, the dominant-negative transmission of the disease. The aim of this study was to characterize novel missense mutations in three unrelated patients and verify the dominant-negative effects of these mutations on the secretion of wild-type FXI protein by expression studies. The F11 gene was PCR amplified, from genomic DNA extracted from peripheral blood, and sequenced on an ABI 3100 Genetic Analyzer. Human wild-type FXI and FXI mutants were expressed in BHK570 cells using Lipofectamin transfection reagents. Conditioned media and cell lysates were collected for the measurement of luciferase activity, FXI antigen and Western blot analysis. DNA sequencing revealed three novel missense F11 mutations; c.127G>A in exon 3 (Ala43Thr), c.723C>G in exon 7 (Phe241Leu) and c.1207G>A in exon 11 (Val403Met). In vitro expression studies showed that the mutation Ala43Thr, Phe241Leu or Val403Met remarkably decreased the extracellular secretion of mutant FXI, rather than reducing synthesis of the mutant proteins. Cotransfection of wild-type FXI with mutant FXI constructs indicated that the mutation Ala43Thr, Phe241Leu or Val403Met reduced the secretion of wild-type FXI by 75.9%, 68.6% or 71.4%, respectively. Our study suggests that dominant-negative mutations in FXI-deficient patients of non-Ashkenazi Jewish origin may be more prevalent than thought, resulting from FXI's unique dimeric structure.

Molecular characterization of FXI deficiency.

Factor XI (FXI) deficiency is a rare autosomal bleeding disease associated with genetic defects in the FXI gene. It is a heterogeneous disorder with variable tendency in bleeding and variable causative FXI gene mutations. It is characterized as a cross-reacting material-negative (CRM-) FXI deficiency due to decreased FXI levels or cross-reacting material-positive (CRM+) FXI deficiency due to impaired FXI function. Increasing number of mutations has been reported in FXI mutation database, and most of the mutations are affecting serine protease (SP) domain of the protein. Functional characterization for the mutations helps to better understand the molecular basis of FXI deficiency. Prevalence of the disease is higher in certain populations such as Ashkenazi Jews. The purpose of this review is to give an overview of the molecular basis of congenital FXI deficiency.

Thrombin generation in patients with factor XI deficiency and clinical bleeding risk.

SUMMARY: Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty-four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non-bleeders (n = 15). All severe bleeders had a personal history of surgery-related severe bleeding. Thrombin generation (TG) was measured in platelet-rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl(-1), a clinically non-bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl(-1) had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

Molecular characterization of two novel mutations causing factor XI deficiency: A splicing defect and a missense mutation responsible for a CRM+ defect.

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency.

Update on the physiology and pathology of factor IX activation by factor XIa.

Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. Activation of factor IX by factor XIa is required in certain situations to prevent bleeding from premature clot degradation. Factor XIa is a coagulation protease comprised of two identical subunits. The biochemical and physiologic implications of this unusual structural feature are being actively investigated. Congenital factor XI deficiency causes a mild-to-moderate bleeding disorder, with hemorrhage typically involving the oral/nasal cavities and the urinary tract. Current treatment recommendations take this tissue-specific bleeding pattern into account and target factor replacement to certain types of procedures and clinical situations. Results from animal models and human population studies indicate that factor XI contributes to thromboembolic disease. This protease may therefore be a legitimate therapeutic target.

Characterization of seven novel mutations causing factor XI deficiency.

BACKGROUND AND OBJECTIVES: Factor XI (FXI) deficiency is a rare autosomal recessive disorder, the main manifestation of which is injury-related bleeding. The disorder is rare in most populations, but common among Jews in whom two mutations, E117X and F283L, account for 98% of cases. Other mutations, C38R and C128X, are prevalent in French Basques and Britons, respectively. Additional sporadic mutations have been described in most parts of the world. The objective of this study was to identify the mutations in 15 unrelated FXI-deficient patients and characterize missense mutations by expression in baby hamster kidney (BHK) cells. DESIGN AND METHODS: Clinical and laboratory information and DNA samples were obtained from the patients and mutations were identified by sequencing. Missense mutations were expressed in BHK cells and their effect on FXI secretion and dimerization was assessed using enzyme-linked immunosorbent assay and immunoblotting. RESULTS: Of 16 mutations detected, seven are novel including two deletions, one splice site and four missense mutations. Expression of the four novel missense mutations (C58Y, Y427C, C527Y and V20A) in cells revealed no secretion of FXI-C58Y, Y427C and C527Y and secretion of only 22% of normal in the medium for FXI-V20A. Secretion of FXI from BHK cells harboring a previously reported E297K substitution cells was also impaired (4.5% of wild-type). Homodimerization was normal for all five mutants. INTERPRETATION AND CONCLUSIONS: Defective homodimerization of FXI was previously recognized as a major mechanism for defective secretion of FXI from producing cells. In this study, five FXI missense mutations (four novel) were associated with impaired secretion albeit normal dimerization, underscoring the existence of other mechanisms for defective secretion.

A novel factor XI missense mutation (Val371Ile) in the activation loop is responsible for a case of mild type II factor XI deficiency.

Coagulation factor XI (FXI) is the zymogen of a serine protease that, when converted to its active form, contributes to blood coagulation through proteolytic activation of factor IX. FXI deficiency is typically an autosomal recessive disorder, characterized by bleeding symptoms mainly associated with injury or surgery. Of the more than 100 FXI gene mutations reported in FXI-deficient patients, most are associated with a proportional decrease in FXI functional and immunologic levels (type I defects), whereas only a few mutations leading to the presence of dysfunctional molecules in plasma have been molecularly analyzed to date (type II deficiencies). We report the functional and molecular characterization of a missense mutation (Val371Ile) identified, in the heterozygous state, in a 25-year-old Italian male with mild FXI deficiency. Laboratory analysis revealed reduced functional FXI levels (34%), but normal antigen levels (102%), distinctive of a type II defect. Given the proximity of Val371 to the FXI activation site, a possible interference with zymogen activation was postulated. Expression experiments of the FXI-Val371Ile recombinant protein, followed by activation assays, showed both a different time course in FXI activation and a slight delay in factor IX activation by thrombin-activated FXI.