Factor XII deficiency is common in domestic cats and associated with two high frequency F12 mutations.

Factor XII (FXII) is a coagulation protein that initiates surface-activation of the coagulation cascade in vitro. The protein's in vivo role, however, remains poorly defined. Factor XII deficiency, or Hageman trait, is a rare hereditary disorder that is not associated with bleeding, and wide variations in FXII activity (FXII:C) exist among healthy people. While FXII-deficient knockout mice appear to be resistant to arterial thrombosis, human F12 polymorphisms that influence FXII:C have not been associated with thrombotic risk in population surveys. Factor XII deficiency is a naturally occurring hereditary trait in domestic cats. We undertook phenotypic and genotypic analyses of FXII-deficient cats for comparative studies with the human disease counterpart. A retrospective review of feline submissions to our laboratory revealed that FXII deficiency is common in domestic cats, and also present in many different breeds. The trait has a geographic bias toward the Midwestern United States. Clinical history, coagulation assays, and samples for F12 sequencing were obtained from 26 FXII deficient cats. None of the cats had experienced abnormal bleeding and their residual FXII:C was related to F12 mutation number and mutation-type. We found 2 high frequency F12 mutations: an exon 13 missense mutation (c.1631G > C) and an exon 11 deletion mutation (c.1321delC), and additional sequence variants throughout the gene. Factor XII deficiency in pet cat populations provides an animal model system to help clarify the biologic actions and clinical relevance of FXII protein.

Diagnostic biologique des angioedèmes bradykiniques : les recommandations du CREAK.

Bradykinin mediated angioedema (BK-AE) can be associated either with C1Inhibitor deficiency (hereditary and acquired forms), either with normal C1Inh (hereditary form and drug induced AE as angiotensin converting enzyme inhibitors…). In case of high clinical suspicion of BK-AE, C1Inh exploration must be done at first: C1Inh function and antigenemy as well as C4 concentration. C1Inh deficiency is significant if the tests are below 50 % of the normal values and controlled a second time. In case of C1Inh deficiency, you have to identify hereditary from acquired forms. C1q and anti-C1Inh antibody tests are useful for acquired BK-AE. SERPING1 gene screening must be done if a hereditary angioedema is suspected, even if there is no family context (de novo mutation 15 %). If a hereditary BK-AE with normal C1Inh is suspected, F12 and PLG gene screening is suitable.

Advances in Antithrombotic Therapy.

Thrombosis remains a major cause of morbidity and mortality. Consequently, advances in antithrombotic therapy are needed to reduce the disease burden. This article focuses on 2 such advances. First, the prevention of atherothrombosis in patients with coronary or peripheral artery disease, which has been enhanced by the finding that the combination of low-dose rivaroxaban plus aspirin is superior to aspirin alone for prevention of recurrent ischemic events. However, this benefit comes at the cost of increased bleeding albeit not fatal bleeding. To overcome this problem, the second advance is the identification of factor XI as a target for new anticoagulants that are potentially safer than those currently available.

Factor XII in coagulation, inflammation and beyond.

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe-/- mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.

In vivo activation and functions of the protease factor XII.

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. The current review concentrates on activators and functions of the FXII-driven contact system in vivo. Elucidating its physiologic activities offers the exciting opportunity to develop strategies for the safe interference with both thrombotic and inflammatory diseases.

Antigen levels of coagulation factor XII, coagulation factor XI and prekallikrein, and the risk of myocardial infarction and ischemic stroke in young women.

BACKGROUND: High levels of activated protein­inhibitor complexes of the intrinsic coagulation proteins are associated with ischemic stroke (IS) but not with myocardial infarction (MI). This study was aimed at determining whether the antigen levels of coagulation factors(factor XII, FXII, and FXI and prekallikrein (PK)are associated with MI and IS, and whether this association is independent of levels of activated protein­inhibitor complexes. PATIENTS AND METHODS: The RATIO study included young women (< 50 years) with MI (N = 205)and IS (N = 175), and 638 healthy controls. Antigen levels of FXII, FXI and PK were measured and expressed as percentages of of those in pooled normal plasmas. Odds ratios (ORs) and corresponding 99% confidence intervals (CIs) were calculated for high levels (i.e. ≥ 90th percentile of controls) as measures of rate ratios. RESULTS: After adjustment for potential confounders, high levels of FXII antigen were not associated with MI risk or IS risk(OR(MI) 1.18, 99% CI 0.51­2.74; ORIS 1.03, 9% CI 0.41­2.55). High levels of FXI antigen were slightly associated with an increase in MI risk (OR(MI) 1.55, 9% CI 0.74­3.21), whereas there was a substantial association with IS risk (ORIS 2.65, 9% CI 1.27­5.56). PK antigen was slightly associated with MI risk but not with IS risk(ORMI 1.54, 9% CI 0.67­3.52; ORIS 0.90, 9% CI 0.35­2.33). All associations remained similar after adjustment for levels of protein­inhibitor complexes. CONCLUSION: Increased levels of FXI antigen were associated with an increase in IS risk, whereas they showed only a marginal association with MI risk. FXII antigen and PK antigen levels were not substantially associated with MI risk and IS risk.

Role of coagulation factor XII in unexplained recurrent abortions in the Greek population.

OBJECTIVE: To investigate the prevalence and clinical significance of congenital factor XII (FXII) deficiency in the South-European Caucasian (Greek) population in a cohort of women with recurrent spontaneous abortions (RSAs). STUDY DESIGN: One hundred women with a history of > or =2 RSAs of unexplained nature were compared to 100 age-matched, healthy controls with no history of thrombotic disease or adverse pregnancy outcomes, regarding FXII activity. Women were included in the RSA group if they had normal coagulation parameters and no congenital or acquired thrombophilia. RESULTS: Fifteen of 100 women with RSA had reduced FXII activity, whereas all controls had normal FXII activity. FXII activity was significantly lower in the RSA than in the control group (median 100.5, range 10-150 vs. median 104.2, range 58.3-143.2, p < 0.016 by Mann-Whitney test). FXII activity was positively correlated with age in both the RSA and the control groups (r = +0.1, p = 0.04 and r = +0.04, p = 0.2, respectively), but this correlation reached statistical significance in the RSA group only. A negative correlation between FXII activity and the number of abortions in the RSA group was found (r = -0.2, p = 0.03). CONCLUSION: Congenital FXII deficiency is strongly associated with RSA in the Greek population.

Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway.

Binding of negatively charged macromolecules to factor XII induces a conformational change such that it becomes a substrate for trace amounts of activated factor present in plasma (less than 0.01%). As activated factor XII (factor XIIa or factor XIIf) forms, it converts prekallikrein (PK) to kallikrein and kallikrein cleaves high molecular weight kininogen (HK) to release bradykinin. A far more rapid activation of the remaining unactivated factor XII occurs by enzymatic cleavage by kallikrein (kallikrein-feedback) and sequential cleavage yields two forms of activated factor XII; namely, factor XIIa followed by factor XII fragment (factor XIIf). PK circulates bound to HK and binding induces a conformational change in PK so that it acquires enzymatic activity and can stoichiometrically cleave HK to produce bradykinin. This reaction is prevented from occurring in plasma by the presence of C1 inhibitor (C1 INH). The same active site leads to autoactivation of the PK-HK complex to generate kallikrein if a phosphate containing buffer is used. Theoretically, formation of kallikrein by this factor XII-independent route can activate surface-bound factor XII to generate factor XIIa resulting in a marked increase in the rate of bradykinin formation as stoichiometric reactions are replaced by Michaelis-Menton, enzyme-substrate, kinetics. Zinc-dependent binding of the constituents of the bradykinin-forming cascade to the surface of endothelial cells is mediated by gC1qR and bimolecular complexes of gC1qR-cytokeratin 1 and cytokeratin 1-u-PAR (urokinase plasminogen activator receptor). Factor XII and HK compete for binding to free gC1qR (present in excess) while cytokeratin 1-u-PAR preferentially binds factor XII and gC1qR-cytokeratin 1 preferentially binds HK. Autoactivation of factor XII can be initiated as a result of binding to gC1qR but is prevented by C1 INH. Yet stoichiometric activation of PK-HK to yield kallikrein in the absence of factor XII can be initiated by heat shock protein 90 (HSP-90) which forms a zinc-dependent trimolecular complex by binding to HK. Thus, endothelial cell-dependent activation can be initiated by activation of factor XII or by activation of PK-HK. Hereditary angioedema (HAE), types I and II, are due to autosomal dominant mutations of the C1 INH gene. In type I disease, the level of C1 INH protein and function is proportionately low, while type II disease has a normal protein level but diminished function. There is trans-inhibition of the one normal gene so that functional levels are 30% or less and severe angioedema affecting peripheral structures, the gastrointestinal tract, and the larynx results. Prolonged incubation of plasma of HAE patients (but not normal controls) leads to bradykinin formation and conversion of PK to kallikrein which is reversed by reconstitution with C1 INH. The disorder can be treated by C1 INH replacement, inhibition of plasma kallikrein, or blockade at the bradykinin B-2 receptor. A recently described HAE with normal C1 INH (based on inhibition of activated C1s) presents similarly; the defect is not yet clear, however one-third of patients have a mutant factor XII gene. We have shown that this HAE has a defect in bradykinin overproduction whether the factor XII mutation is present or not, that patients' C1 INH is capable of inhibiting factor XIIa and kallikrein (and not just activated C1) but the functional level is approximately 40-60% of normal, and that α2 macroglobulin protein levels are normal. In vitro abnormalities can be suppressed by raising C1 INH to twice normal levels. Finally, aggregated proteins have been shown to activate the bradykinin-forming pathway by catalyzing factor XII autoactivation. Those include the amyloid ß protein of Alzheimer's disease and cryoglobulins. This may represent a new avenue for kinin-dependent research in human disease. In allergy (anaphylaxis; perhaps other mast cell-dependent reactions), the oversulfated proteoglycan of mast cells, liberated along with histamine, also catalyze factor XII autoactivation.

Factor XII inhibition reduces thrombus formation in a primate thrombosis model.

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

[Reference values for blood coagulation factor activity in the Mexican population].

INTRODUCTION: During the fluid phase of hemostasis, fibrinogen is converted into fibrin, but other hemostatic factors are required. Reference values of hemostatic factors are established by manufacturers producing reagents using individuals with a specific genetic background. OBJECTIVE: To establish reference values for hemostatic factors in the Mexican indigenous and Mestizo populations. MATERIAL AND METHODS: We carried out a cross-sectional, descriptive study of healthy adult Mexicans. Clotting activity was evaluated using coagulometric assays. Blood donors were informed about the nature of the study and informed consent was obtained prior to blood being drawn. The protocol was approved by the Ethics Committee of our institution. RESULTS: One hundred and twenty samples were assayed (60 females and 60 males). Fibrinogen was higher in mestizos and in females. Reference values for factor XII ranged from 40-170% in indigenous subjects and from 36-159% in mestizos. Factor VIII ranged from 57-160% in indigenous subjects and from 51-209% in mestizo subjects. Reference values for the other hemostatic factors were also clearly different from the commercial reference values. Reference values for hemostatic factors in the Mexican population are different from traditionally used commercial reference values. There were significant differences between indigenous and mestizo Mexicans in the concentration of hemostatic factors with a tendency among mestizos to have higher factor concentrations. Low levels of plasma factor XII are frequent and perhaps may represent a risk factor for thrombotic events. Using these reference values may individualize the reposition of factors in Mexican hemophiliac patients.

In vivo roles of factor XII.

Coagulation factor XII (FXII, Hageman factor, EC = 3.4.21.38) is the zymogen of the serine protease, factor XIIa (FXIIa). FXII is converted to FXIIa through autoactivation induced by "contact" to charged surfaces. FXIIa is of crucial importance for fibrin formation in vitro, but deficiency in the protease is not associated with excessive bleeding. For decades, FXII was considered to have no function for coagulation in vivo. Our laboratory developed the first murine knockout model of FXII. Consistent with their human counterparts, FXII(-/-) mice have a normal hemostatic capacity. However, thrombus formation in FXII(-/-) mice is largely defective, and the animals are protected from experimental cerebral ischemia and pulmonary embolism. This murine model has created new interest in FXII because it raises the possibility for safe anticoagulation, which targets thrombosis without influence on hemostasis. We recently have identified platelet polyphosphate (an inorganic polymer) and mast cell heparin as in vivo FXII activators with implications on the initiation of thrombosis and edema during hypersensitivity reactions. Independent of its protease activity, FXII exerts mitogenic activity with implications for angiogenesis. The goal of this review is to summarize the in vivo functions of FXII, with special focus to its functions in thrombosis and vascular biology.

The plasma contact system 2.0.

The contact system is a protease cascade that is initiated by factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood. However, activators of the system in vivo and their contributions to disease states have remained enigmatic. Recent experimental and clinical data have identified misfolded proteins, collagens, and polyphosphates as the long-sought activators of the contact system in vivo. Here we present an overview about contact system activators and their contributions to health and pathology.

Factor XII activation is essential to sustain the procoagulant effects of particulate matter.

BACKGROUND: Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. OBJECTIVES: To study the mechanisms of PM-driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs; < 0.1 µm) in genetically modified mice. METHODS: Thrombin generation in response to PM of different sizes was assessed in normal human platelet-poor plasma, as well as in plasmas deficient in the intrinsic pathway proteases factors XII (FXII) or XI (FXI). In addition, UFPs were intratracheally instilled in wild-type (WT) and FXII-deficient (FXII(-/-) ) mice and plasma thrombin generation was analyzed in plasma from treated mice at 4 and 20 h post-exposure. RESULTS: In normal human plasma, thrombin generation was enhanced in the presence of PM, whereas PM-driven thrombin formation was completely abolished in FXII- and FXI-deficient plasma. UFPs induced a transient increase in tissue factor (TF)-driven thrombin formation at 4 h post-instillation in WT mice compared with saline instillation. Intratracheal instillation of UFPs resulted in a procoagulant response in WT mice plasma at 20 h, whereas it was entirely suppressed in FXII(-/-) mice. CONCLUSIONS: Overall, the data suggest that PM promotes its early procoagulant actions mostly through the TF-driven extrinsic pathway of coagulation, whereas PM-driven long lasting thrombogenic effects are predominantly mediated via formation of activated FXII. Hence, FXII-driven thrombin formation may be relevant to an enhanced thrombotic susceptibility upon chronic exposure to PM in humans.

Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Intrinsic coagulation activation and the risk of arterial thrombosis in young women: results from the Risk of Arterial Thrombosis in relation to Oral contraceptives (RATIO) case-control study.

BACKGROUND: Classically, intrinsic coagulation proteins are thought to have a minor role in hemostasis. Recently, these proteins, especially FXII, were implicated as possible key players in the pathogenesis of arterial thrombosis. This study aims to determine the risks of arterial thrombosis conferred by increased activation of intrinsic coagulation proteins in young women and the effect of oral contraceptive use on this association. METHODS AND RESULTS: The Risk of Arterial Thrombosis In relation to Oral contraceptives (RATIO) study is a population-based case-control study including young women (age 18 to 50 years) with myocardial infarction (n=205) and ischemic stroke (n=175) and 638 healthy controls. Intrinsic coagulation protein activation was determined by measuring activated protein-inhibitor complexes. This complex is with C1 esterase inhibitor (FXIIa-C1-INH, FXIa-C1-INH, Kallikrein-C1-INH) or antitrypsin inhibitor (FXIa-AT-INH). Odds ratios (ORs) and corresponding confidence intervals (95% CIs) were calculated with logistic regression. High levels of protein activation (>90th percentile of controls) showed an increased risk of ischemic stroke: FXIIa-C1-INH (OR, 2.1; 95% CI, 1.3 to 3.5), FXIa-C1-INH (OR, 2.8; 95% CI, 1.6 to 4.7), FXIa-AT-INH (OR, 2.3; 95% CI, 1.4 to 4.0), and Kallikrein-C1 (OR, 4.3; 95% CI, 2.6 to 7.2). If anything, myocardial infarction risk was only increased by Kallikrein-C1-INH (OR, 1.5; 95% CI, 0.9 to 2.5). Oral contraceptive use further increased the risks. CONCLUSIONS: High levels of activated proteins of the intrinsic coagulation system are associated with arterial thrombosis, whereas the strength of these associations differs for myocardial infarction and ischemic stroke. This contradicts similar analyses among men in the Northwick Park Heart Study. Together with the finding that oral contraceptive use further increases the risks, the question of whether the role of intrinsic coagulation proteins in the pathogenesis of arterial thrombosis is sex-specific is raised.

Laminin promotes coagulation and thrombus formation in a factor XII-dependent manner.

BACKGROUND: Laminin is the most abundant non-collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis. OBJECTIVE: To investigate the ability of laminin to promote coagulation and support thrombus formation under shear. RESULTS AND METHODS: Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI- and FXII-dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin-coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX. CONCLUSION: This study identifies a role for laminin in the initiation of coagulation and the formation of platelet-rich thrombi under shear conditions in a FXII-dependent manner.