A novel mutation in the coagulation factor 12 gene in subjects with hereditary angioedema and normal C1-inhibitor.

In hereditary angioedema with normal C1-inhibitor two different missense mutations of codon p.Thr328* in the coagulation factor 12 gene have been reported in some families. In this study a novel factor 12 gene mutation, the deletion of 72 base pairs (bp) (c.971_1018+24del72*), was identified in a family of Turkish origin, in two sisters with recurrent skin swellings and abdominal pain attacks and in their symptom-free father. This deletion caused a loss of 48 bp of exon 9 (coding amino acids 324* to 340*) in addition to 24 bp of intron 9, including the authentic donor splice site of exon 9. The large deletion of 72 bp was located in the same F12 gene region as the missense mutations p.Thr328Lys* and p.Thr328Arg* reported previously. Our findings confirm the association between F12 gene mutations modifying the proline-rich region of the FXII protein and hereditary angioedema with normal C1-inhibitor.

Isolation and properties of an abnormal Hageman factor (Factor XII) molecule in a cross-reacting material-positive hageman trait plasma.

We have previously described two unrelated individuals with homozygous Hageman trait (Factor XII deficiency) whose plasmas contained nonfunctional material immunologically indistinguishable from normal Hageman factor (HF). Abnormal HF from the plasma of one these subjects has now been purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, alkaline disc gel electrophoresis, and immunoelectrophoresis. Purified abnormal HF had no clot-promoting activity, but showed the same specific antigenicity as purified normal HF by an immunoassay. The abnormal HF was of a single chain polypeptide with the same molecular weight (80,000) as normal HF and was positively stained by periodic acid-Schiff reagent. Both normal and abnormal HF had similar amino acid compositions and isoelectric points (pI 6.5 approximately 7.1). When 125I-labeled abnormal HF and 131I-labeled normal HF were mixed with normal plasma and exposed to glass, both HF underwent an identical pattern of cleavage, yielding 52,000- and 30,000-mol wt fragments. Similarly, abnormal HF was fragmented by trypsin in the same way as normal HF, but no prekallikrein-activating activity was generated after cleavage. [3H]Diisopropyl phosphorofluoridate was incorporated into a 29,000-mol wt fragment of the trypsin-cleaved normal HF, but not into that of the trypsin-cleaved abnormal HF. These data suggest that the molecular defect in this abnormal HF resides at or near the active site serine residue in the 30,000-mol wt part of the molecule.

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12-/-Apoe-/-) mice. Compared to F12+/+Apoe-/- controls, atherosclerotic lesion formation was reduced in F12-/-Apoe-/- mice. This was associated with a decrease in serum interleukin (IL)-1ß and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12-/-Apoe-/- mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1ß, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4+ T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.

Functional characterization of a variant factor XII (F XII Locarno) in a cross reacting material positive F XII deficient plasma.

The plasma of a healthy woman was found to contain half normal factor XII (FXII) antigen level (0.46 U/ml) without any FXII clotting activity (less than 0.01 U/ml). The variant FXII in this plasma, denoted as FXII Locarno, was partially characterized by immunological and functional studies on the proposita's plasma. FXII Locarno is a single chain molecule with the same size (Mr = 80 kDa) as normal FXII. Isoelectric focusing suggested an excess of negative charge in the variant FXII as compared to normal FXII. In contrast to FXII in normal plasma, FXII Locarno was not proteolytically cleaved upon prolonged incubation of proposita's plasma with dextran sulfate. Adsorption to kaolin was similar for both, abnormal and normal FXII. Incubation of the proposita's plasma with dextran sulfate and exogenous plasma kallikrein showed normal cleavage of FXII Locarno outside of the tentative disulfide loop Cys340-Cys467, but only partial cleavage within this disulfide loop. Furthermore, plasma kallikrein-cleaved abnormal FXII showed neither amidolytic activity nor proteolytic activity against factor XI and plasma prekallikrein. These results suggest a structural alteration of FXII Locarno, affecting the plasma kallikrein cleavage site Arg353-Val354 and thus formation of activated FXII (alpha-FXIIa).

Autoantibodies to contact proteins in patients with recurrent pregnancy losses.

The kallikrein-kinin system or plasma contact system consists of three essential plasma proteins. These are coagulation factor XII, prekallikrein and high molecular weight kininogen. Deficiencies of these proteins are not associated with clinical bleeding despite marked prolongation of in vitro surface-activated coagulation time. Paradoxically, studies suggest that contact proteins have anticoagulant, profibrinolytic functions in a physiologic millieu, on endothelial cells. Recently, evidence has accumulated for the presence of the kallikrein-kinin system or plasma contact system in the fetoplacental unit. Kinins which are released within the placenta may play a role in regulating placental blood flow. This suggests that the plasma contact system may also have an important role in pregnancy. Several studies have reported the presence of autoantibodies to the contact proteins in patients with recurrent early pregnancy losses. Disruption of this system may be a risk factor for early gestational losses.

The "Hageman" connection: interrelationships of blood coagulation, fibrino(geno)lysis, kinin generation, and complement activation.

The activation pathways for the generation of enzymes involved in blood clotting, clot lysis, complement activation, and kinin generation are briefly reviewed. The interrelationship of the four systems is illustrated by the multiple functions of four key enzymes: Factor XIIa, kallikrein, plasmin, and C1 esterase. The pivotal role of Factor XIIa in establishing this connection is elucidated.

The influence of serum on random migration and chemotaxis of polymorphonuclear leucocytes: methodological evaluation using sera from infection-prone patients and normals.

The effects on normal polymorphonuclear leucocytes (PMN) of chemotactic and chemokinetic factors in sera from normal subjects and infection-prone patients were examined by means of the leading-front technique, using a modified Boyden chamber. The analysis scheme used made it possible to differentiate between chemotactic and chemokinetic factors and demonstrated that different factors account for the major chemotactic and chemokinetic activities of serum. The major chemotactic activity of unactivated serum was heat-labile, and the chemotactic activity of factor XII-deficient sera was normal, suggesting the major chemotactic activity to be distinct from C5a and factor XII-dependent pathways. The existence of both heat-stable and heat-labile chemokinetic factors was shown. The possibility that the reduced chemokinetic effect of several patient sera was caused by abnormal levels of the serum proteins alpha 1-antitrypsin, alpha 2-macroglobulin and albumin was excluded.

Severe Fletcher factor (plasma prekallikrein) deficiency with partial deficiency of Hageman factor (factor XII): report of a case with observation on in vivo and in vitro leukocyte chemotaxis.

A case of cross-reacting material-negative Fletcher trait with additional partial deficiency of Hageman factor (HF, Factor XII) is described. Although the patient presented with a recent history of frequent epistaxis, he had no other personal or family history of a tendency toward bleeding or infection. Similar to other cases of Fletcher trait, his plasma showed a markedly prolonged partial thromboplastin time which could be corrected by prolonged incubation with the surface-activator kaolin. Surface-induced fibrinolysis, amidolysis of alpha-N-benzoyl-proline-L-phenylalanine-L-arginine-p-nitroanilide, and cold-promoted enhancement of factor VII activity, reactions requiring the presence in the plasma of fletcher factor (prekallikrein), in addition to Hageman factor and Fitzgerald factor (high-molecular weight kininogen), were also defective. In vivo chemotaxis of polymorphonuclear leukocytes and monocytes (Rebuck's skin window technique) in response to skin abrasions was defective, but was normal when diphtheria-tetanus toxoid was also applied. In vitro leukocyte chemotaxis (Boyden chamber technique) in response to normal or patient's own serum activated with zymosan was normal. Together with previous observations that kallikrein generated chemotactic activity, possibly via activation of C5, the present observations suggest that prekallikrein activation may be important for in vivo leukocyte chemotactic response to skin abrasion. The inheritance of Fletcher trait in this patient is unclear.l Although the father was an apparent heterozygote, the mother was completely normal for Fletcher factor procoagulant activity and antigen. The mild Hageman factor deficiency in the patient did not contribute significantly to the plasma defects described and was likely inherited from the father who had a low HF procoagulant activity.

Significance of Hageman factor (coagulation factor XII) for neutrophil locomotion and chemotaxis.

Neutrophils separated from Hageman factor-deficient blood exhibit normal random and directional locomotion. No defect in cytotaxin formation could be demonstrated in Hageman factor-deficient plasma, nor did kallikrein inhibitor interfere with the chemotactic activity of normal immune complex-activated human plasma. The results suggest that the chemotactic activity which is detectable in unfractionated human plasma is not derived from Hageman factor-dependent pathways.

Tobacco, cocoa, coffee, and ragweed: cross-reacting allergens that activate factor-XII-dependent pathways.

A glycoprotein antigen has been isolated from cured tobacco leaves (TGP-L) Nicotiana tabacum) and from cigarette smoke condensate (TGP-CSC) to which approximately one-third of human volunteers, smokers and non-smokers, exhibit immediate cutaneous hypersensitivity. TGP-L and TGP-CSC contain polyphenol haptens that activate the factor XII (Hageman factor) dependent pathways of coagulation, fibrinolysis, and kinin generation in normal human plasma. The purpose of this communication is to describe the isolation antigens from cocoa powder (Theobroma cacao), ground coffee (Coffea arabica), and ragweed (Ambrosia eliator) pollen that are immunologically cross-reactive with TGP-L and TGP-CSC, contain similar polyphenol haptens, and are capable of activating factor-XII-dependent pathways in normal human plasma.

Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner.

beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.