Factor XII deficiency evaluated by thrombin generation assay.

INTRODUCTION: Coagulation factor XII (FXII) plays a role in thrombin generation, fibrinolysis, inflammation, angiogenesis, chemotaxis and diapedesis. FXII deficiency is not associated with bleeding risk unlike other coagulation factors. MATERIALS/METHODS: We investigated thrombin generation assay (TGA) profile modification in FXII deficiency and the correlation with TGA and deficiency severity. TGA was performed in platelet poor plasma (PPP) with tissue factor (1 pmol/L) and phospholipid (4 µmol/L) standardized concentration. Thrombin generation profiles were compared in 54 patients with FXII deficiency, 25 healthy controls and 23 patients with hemophilia A (factor VIII (FVIII) deficiency. Patients with FXII deficiency were classified in three groups based on FXII activity (30-50%, 10-29%, <10%). FVIII deficiency was included as a bleeding control group. RESULTS: As expected, we found a correlation between FXII deficiency and activated partial thromboplastin time (aPTT). A decrease of thrombin generation was observed in healthy controls and all FXII deficiency groups. A decrease of endogenous thrombin potential (ETP), peak and velocity was observed in patients with FVIII deficiency compared to FXII deficiency. A decrease of thrombin generation was noted in patients with FXII deficiency and bleeding history compared to patients with FXII deficiency and thrombosis history. CONCLUSION: In this study, thrombin generation profiles were not sensitive to FXII deficiency. TGA could distinguish bleeding and thrombotic tendency in FXII deficiency. Our results should therefore be considered as exploratory and deserve confirmation.

Acquired factor XII deficiency following transanal excision of rectal lesion by transanal minimally invasive surgery (TAMIS): a case report and literature review.

BACKGROUND: Local excision (LE) is currently one of the most effective methods used in cases of large benign polyps, not suitable for endoscopic treatment, or early-stage neoplasms. LE is also alternative to anterior rectal resection in selected patients suffering from major comorbidities and limits for major abdominal procedure. Furthermore, LE results in less pain, reduced impact on bowel function, shorter duration of hospital stay, and lower rates of morbidity, mortality and stoma creation. In particular, early data on transanal minimally invasive surgery (TAMIS) are promising, but they come from single centre case series related to small groups of patients and more data are needed to draw a final conclusion on the safety of this novel approach for transanal resection. CASE PRESENTATION: A 62-year-old woman, following a positive faecal occult blood test and with unremarkable medical history, was admitted to hospital for excision of a large flat neoplastic lesion. Endoscopic biopsy demonstrated a tubular adenoma with high-grade dysplasia and was decided to proceed with surgical excision by TAMIS. After surgery, short-term outcomes revealed prolonged activated partial thromboplastin time, undetectable factor XII activity, fever, and partial dehiscence of rectal wall defect suture. Cross-mixing studies of patient plasma show no correction in either the immediate or incubated activated partial thromboplastin time, indicating the presence of an acquired factor XII inhibitor. Activated partial thromboplastin time and factor XII improved in the following weeks without any specific therapy in addition to antibiotic therapy. CONCLUSION: This is the first report in which acquired inhibitor of coagulation factor XII is associated with a specific surgical procedure. This case has shown how trans-anal excision of rectal lesions, even when performed by minimally invasive means such as in case of TAMIS, is not free of complications. We consider the acute infection, resulting from early dehiscence of the suture, the trigger in an abnormal immune response, and inhibitor development.

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.

[Are we to pay attention to factor XII deficiency?]. / Máme venovat' pozornost' defektu koagulacného faktora XII?

Severe coagulation factor XII (FXII) deficiency is a very rare, mysterious and not well known inherited condition. Unlike other coagulation factor deficiencies, it is totally asymptomatic. Surprisingly, it does not lead to abnormal bleeding, even with major surgical procedures. The explanation for the lack of bleeding manifestations is unknown. It is suggested, but unproven, that patients are not sufficiently protected from thrombosis. FXII deficiency is usually discovered by accident through a routine coagulation testing done prior to surgery. Since FXII plays an important role in clot formation during in vitro measurements, its deficiency causes a marked prolongation of the activated partial thromboplastin time in the laboratory examination. The main concern related to FXII deficiency is the unnecessary testing, delay in health care and worry of surgical interventions that may be prompted by the abnormal laboratory result.

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.

Coagulation on endothelial cells: the underexposed part of Virchow's Triad.

The process of thrombin generation involves numerous plasma proteases and cofactors. Interaction with the vessel wall, in particular endothelial cells (ECs), influences this process but data on this interaction is limited. We evaluated thrombin generation on EA.hy926, human coronary arterial ECs (HCAECs) and patient-derived human venous ECs (HVECs) by means of a modified calibrated automated thrombogram (CAT) method and especially looked into contribution of the intrinsic and extrinsic pathways. Thrombin generation was measured in presence of confluent ECs with normal pooled and factor XII-deficient (FXII-deficient) platelet-poor plasma, with/without active site inhibited factor VIIa (ASIS) to block the extrinsic pathway and corn trypsin inhibitor for blocking contact activation (intrinsic pathway). Fetal bovine serum (FBS) was removed from culture conditions as FXIIa from the serum retained on ECs apparently, thereby inducing strong contact activation. In serum-free conditions, EA.hy926 and patient-derived HVECs induced thrombin generation mainly via the contact activation pathway with minor influence of ASIS on peak height and very low thrombin generation curves in FXII-deficient plasma. HVECs derived from coronary arterial bypass graft (CABG) patients showed increased thrombin generation compared to control patients, which could be ascribed to increased contact activation. Contribution of the extrinsic pathway on patient-derived ECs was limited. We conclude that the CAT method in combination with serum-free cultured ECs offers a valuable high-throughput method to evaluate endothelial influences on thrombin generation, which appears to involve predominantly contact activation on ECs. Contact activation-mediated thrombin generation was increased on ECs from CABG patients compared to controls.

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography.

BACKGROUND: Thrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells. DESIGN AND METHODS: Malignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombo-gram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression. RESULTS: In normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation - by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma. CONCLUSIONS: This study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global pro-coagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.

Perioperative management of cardiac surgery patients with factor XII deficiency - two case reports.

This paper presents two patients with factor XII deficiency, a rare coagulation disorder, who successfully underwent surgery with cardiopulmonary bypass (CPB) in Cardiac Surgery Clinic of the 4th Military Clinical Hospital in Wroclaw. Diagnosis, intra- and postoperative course as well as proposed management strategy are described.

Implications for cardiac surgery in patients with factor XII deficiency.

Factor XII deficiency is associated with a prolonged activated partial thromboplastin time and activated clotting time used for monitoring during cardiopulmonary bypass. It does not predispose to an increased risk of bleeding. We present the strategy used for a case of coronary artery bypass grafting in a patient with factor XII deficiency, followed by a brief discussion of the important clinical considerations when patients with factor XII deficiency undergo cardiac surgery. Monitoring of heparin and the avoidance of anti-fibrinolytic agents are the main intraoperative issues. Postoperative care must include careful thromboembolic prophylaxis and vigilance against infection.

Adult cystic fibrosis patients with and without infective exacerbations and their factor XII levels.

We investigated haemostatic and inflammatory parameters in patients with cystic fibrosis in an attempt to understand a previous finding of low factor XII levels in this patient population. We selected two groups of patients, adults attending outpatient annual review clinic who were well, chronically inflammed and adult patients with an infective exacerbation requiring antibiotics or admission to hospital, acutely inflammed. We measured known positive acute phase haemostatic factors, fibrinogen and factor VIII. Antithrombin and factor XII were also measured as both these factors have been proposed to be negative acute phase proteins in in-vitro cell models. Interleukin-6 was also measured as the proposed modulator of these factors during inflammation. Activated factor XII was measured to exclude XII activation as a cause of the low XII activity levels. Cystic fibrosis patients admitted to hospital with infective exacerbations, showed significantly more evidence of inflammation than the annual review patients. Fibrinogen and factor VIII were higher and factor XII was lower in these patients. This work suggests that factor XII behaves as a negative acute phase protein with no signs of elevated activated XII levels in either group. This supports similar findings from in-vitro cell culture. This study also shows low antithrombin levels in both patient populations, although there was no statistical difference between groups, which is probably related to their liver disorder.

Contact factor deficiencies and cardiopulmonary bypass surgery: detection of the defect and monitoring of heparin.

Contact factor pathway deficiencies do not cause surgical bleeding but make heparin monitoring by the activated partial thromboplastin time (APTT) and activated clotting time (ACT) unreliable. Heparin monitoring during cardiopulmonary bypass (CPB) surgery in these patients is particularly challenging. Here we describe heparin monitoring during CPB using the chromogenic anti Xa assay in two patients with severe factor XII deficiency (FXII < 0.01 U/mL) and one patient with severe prekallikrein (PK) deficiency (PK < 0.01 U/mL). Anti Xa levels of the three patients during CPB varied between 3.8 and 4.8 U/mL in keeping with a control group (mean anti Xa 4.5 U/mL and ACT > 480 s). There were no bleeding or thrombotic complications. We also found that detection of severe PK deficiency by the APTT in the PK deficient patient was dependent on the reagent used and discuss the sensitivity of different APTT reagents for contact factor deficiencies. We conclude that the sensitivity of APTT methods for contact pathway deficiencies is highly variable and although insensitivity is not a clinical problem in terms of bleeding, it can be a cause of discrepancy between different APTT reagents and the ACT. This can lead to confusion about a possible haemorrhagic tendency and delays in surgery. If these patients need to undergo cardiac surgery requiring high dose heparin treatment, monitoring by chromogenic anti Xa assay is a good alternative.

Qualitative thrombelastographic detection of tissue factor in human plasma.

BACKGROUND: Tissue factor (TF) is the principal in vivo initiator of coagulation, with normal circulating TF concentrations reported to be approximately 23-158 pg/mL. However, patients with atherosclerosis or cancer have been reported to have TF concentrations ranging between 800 and 9000 pg/mL. Of interest, thrombelastographic (TEG)-based measures of clot initiation and propagation have demonstrated hypercoagulability in such patients at risk for thromboembolic events. Thus, our goal in the present investigation was to establish a concentration-response relationship of the effect of TF on TEG variables, and determine specificity of TF-mediated events with a monoclonal TF antibody. METHODS: Thrombelastography was performed on normal human plasma exposed to 0, 500, 1000, or 2000 pg/mL TF. Additional experiments with plasma exposed to 0 or 750 pg/mL TF in the presence or absence of a monoclonal TF antibody (1:360 dilution, 10 min incubation) were also performed. Clot initiation time (R) and the speed of clot propagation (MRTG, maximum rate of thrombus generation) were determined. RESULTS: The addition of TF to normal plasma resulted in a significant, concentration-dependent decrease in R and increase MRTG values. The addition of TF antibody to samples with TF significantly increased R and decreased MRTG values compared to samples with TF addition. CONCLUSIONS: In conclusion, changes in TEG variables in conjunction with use of a TF antibody can detect pathological concentrations of TF in human plasma in vitro. Further investigation is warranted to determine if TEG(R)-based monitoring could assist in the detection and prevention of TF-initiated thromboembolic events.

Myocardial infarction and arterial thrombosis in severe (homozygous) FXII deficiency: no apparent causative relation.

Twenty-one patients (12 female and 9 male) with severe (homozygous) factor XII (FXII) deficiency and 58 (32 female and 26 male) with heterozygous FXII deficiency were observed for an average 16.2 years. No patient with homozygous FXII deficiency experienced myocardial infarction or any other arterial thrombosis. The same was true for heterozygotes. The cases of FXII deficiency and arterial thrombosis reported in the literature were evaluated. In every instance, associated risk factors were present that could justify the arterial thrombosis. Dyslipidemia, hypertension, smoking, and diabetes mellitus were the most frequent findings. The examination of the few papers that dealt with the prevalence of arterial thrombosis in patients with severe FXII deficiency showed that only 1 patient of 61 experienced myocardial infarction. In conclusion, it seems that the role of FXII deficiency in the pathogenesis of arterial thrombosis is minor.

Low factor XII level in an individual with Sotos syndrome.

Sotos syndrome is an overgrowth disorder that manifests characteristic dysmorphic features, neurological problems, and an increased risk for cancers and heart defects. Alterations of NSD1 are responsible for this disease. A subset of cases arise from deletions, which is of interest as the factor XII locus lies in close proximity to NSD1. This case report describes an individual with Sotos syndrome and factor XII deficiency, providing a potential link between these two genes and, consequently, expanding the clinical phenotype of Sotos syndrome.

Assessment of Actin FS and Actin FSL sensitivity to specific clotting factor deficiencies.

We present a two centre study designed to assess the sensitivity of Actin FS and Actin FSL to deficiencies of factor VIII, IX, XI or XII. The study was undertaken at two centres to avoid bias due to the investigations being undertaken on one analyser. Samples from patients with a factor VIII (n = 36, F VIII = < 1.0-50 iu/dl), factor IX (n = 22, F IX = 2-48 iu/dl), factor XI (n = 23, F XI = 5-50 u/dl) or a factor XII (n = 18, F XII = 1-50 u/dl) deficient state were studied. Activated partial thromboplastin times (APTT) were determined using two batches of Actin FS and of Actin FSL; comparison of APTT results between centres was facilitated by the conversion of clotting times to ratios (test divided by geometric mean normal clotting time). APTT ratios were considered to be elevated if greater than two standard deviations above the mean normal. The factor deficient status of each sample was verified by assaying all samples for factors VIII, IX, XI and XII. Clotting factor assays were performed on a Sysmex CA-1000 fitted with research software, which permitted the auto-dilution and testing of three serial dilution of both a reference preparation and each patient's sample. Assay results were calculated using parallel-line Bioassay principles. This procedure allowed for variation in clotting times due to the effect of temporal drift of any of the reagents within the assay system. Actin FS and Actin FSL demonstrate acceptable sensitivity to factor VIII deficiency, however, both reagents failed to detect a large proportion of factor XI (17.4% and 30.4% of samples, respectively) and factor XII (66.7% and 72.2%, respectively) deficiencies. The detection rate with Actin FSL for factor IX deficiency was also poor (36.4% not detected). As factor IX and XI deficiencies are both associated with haemorrhagic disorders, the inability of these reagents to detect such abnormalities gave cause for concern.

Amidolytic assay of factor XI in human plasma--significance of kallikrein for the activity measured.

Factor XI (FXI) deficiency is associated with an abnormal bleeding state. The extent of bleeding does not correlate well with the plasma concentration of FXI, and it has been suggested that also unknown factors interfere with the bleeding tendency. In a recent paper (Thromb. Res. 74, 477-485, 1994) we found that FXIa activated in human plasma was present in association with part of factor XIIa (FXIIa) and part of kallikrein, influencing their functional activities. Should the activity level of FXIa also be altered by the other contact factors this might provide one approach to the problem of the failure of assays of FXIa to correlate with bleeding tendency. In the present study we have developed an assay procedure for FXIa based on its amidolytic (S-2366) activity, and allowing at the same time a quantification of the amount of FXIa associated to kallikrein. The total amidase activity obtained was separated into two main fractions by use of soybean trypsin inhibitor (STI), corn inhibitor (CI) and lima bean trypsin inhibitor (LTI). One fraction contained free FXIa which could be specifically blocked by LTI. An inhibitor resistant fraction was found to contain FXIa inactive in association with kallikrein. The content of FXIa could be assessed in experiments with mixtures of normal plasma and plasma deficient in prekallikrein, and was taken into account in the calculations. This fraction increased during storage of plasma at -70 degrees C. To obtain stable and comparable assay conditions the method was based on plasma stored for at least four weeks. The specificity of the method was verified by parallel radial immunodiffusion tests. The results imply that the activity level of FXIa is dependent on kallikrein present. If the experimental results has relevance to the situation under physiological conditions, they indicate one possible cause of the failure of assays of FXI to correlate with bleeding tendency.

Role of factor XII in thrombin generation and fibrinolysis during cardiopulmonary bypass.

During cardiopulmonary bypass, thrombin is generated, which is thought to be initiated by activation of factor XII on the surface of the bypass equipment. We present a patient with severe factor XII deficiency who underwent cardiac surgery. As much thrombin was formed during cardiopulmonary bypass (measured by the prothrombin activation fragment F1 + 2 and thrombin-antithrombin complexes) as in normal patients, showing that factor XII was not necessary for thrombin generation. Factor X, but not factor IX, was activated (as measured by their activation peptides), and this activation correlated with F1 + 2 and thrombin-antithrombin complexes, suggesting that the tissue-factor/factor-VIIa pathway is the trigger for thrombin formation.