Inhibition of contact-mediated activation of factor XI protects baboons against S aureus-induced organ damage and death.

Staphylococcus aureus infections can produce systemic bacteremia and inflammation in humans, which may progress to severe sepsis or septic shock, even with appropriate antibiotic treatment. Sepsis may be associated with disseminated intravascular coagulation and consumptive coagulopathy. In some types of mouse infection models, the plasma coagulation protein factor XI (FXI) contributes to the pathogenesis of sepsis. We hypothesize that FXI also contributes to the pathogenesis of sepsis in primates, and that pharmacological interference with FXI will alter the outcome of Staphylococcus aureus-induced lethality in a baboon model. Pretreatment of baboons with the anti-FXI antibody 3G3, a humanized variant of the murine monoclonal 14E11 that blocks FXI activation by FXIIa, substantially reduced the activation of coagulation, as reflected by clotting times and plasma complexes of coagulation proteases (FXIIa, FXIa, FIXa, FXa, FVIIa, and thrombin) with serpins (antithrombin or C1 inhibitor) following infusion of heat-inactivated S aureus 3G3 treatment reduced fibrinogen and platelet consumption, fibrin deposition in tissues, neutrophil activation and accumulation in tissues, cytokine production, kininogen cleavage, cell death, and complement activation. Overall, 3G3 infusion protected the structure and function of multiple vital organs, including lung, heart, liver, and kidney. All treated animals reached the end point survival (7 days), whereas all nontreated animals developed terminal organ failure within 28 hours. We conclude that FXI plays a role in the pathogenesis of S aureus-induced disseminated intravascular coagulation and lethality in baboons. The results provide proof of concept for future therapeutic interventions that may prevent sepsis-induced organ failure and save lives in certain forms of sepsis.

Comparing Pathways of Bradykinin Formation in Whole Blood From Healthy Volunteers and Patients With Hereditary Angioedema Due to C1 Inhibitor Deficiency.

Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood. We applied various forms of activation to fresh blood obtained from 10 healthy subjects or 10 patients with hereditary angioedema (HAE-1 or -2 only) to investigate kinin formation. An enzyme immunoassay for BK was applied to extracts of citrated blood incubated at 37°C under gentle agitation for 0-2 h in the presence of activators and/or inhibitory agents. Biologically active kinins in extracts were corroborated by c-Fos accumulation in HEK 293a cells that express either recombinant human B2 or B1 receptors (B2R, B1R). Biological evidence of HAE diagnostic and blood cell activation was also obtained. The angiotensin converting enzyme inhibitor enalaprilat, without any effect per se, increased immunoreactive BK (iBK) concentration under active stimulation of blood. Tissue kallikrein (KLK-1) and Kontact-APTT, a particulate material that activates the contact system, rapidly (5 min) and intensely (>100 ng/mL) induced similar iBK generation in the blood of control or HAE subjects. Tissue plasminogen activator (tPA) slowly (≥1 h) induced iBK generation in control blood, but more rapidly and intensely so in that of HAE patients. Effects of biotechnological inhibitors indicate that tPA recruits factor XIIa (FXIIa) and plasma kallikrein to generate iBK. KLK-1, independent of the contact system, is the only stimulus leading to an inconsistent B1R stimulation. Stimulating neutrophils or platelets did not generate iBK. In the HAE patients observed during remission, iBK formation capability coupled to B2R stimulation appears largely intact. However, a selective hypersensitivity to tPA in the blood of HAE patients suggests a role of plasmin-activated FXIIa in the development of attacks. Proposed pathways of kinin formation dependent on blood cell activation were not corroborated.

Hemostasis in Allergy.

The involvement of the hemostatic system in immune-mediated inflammation is widely reported. Many coagulation factors play a role in the pathogenesis of autoimmune diseases, such as systemic vasculitis and systemic lupus erythematosus. Hemostatic disorders are also involved in asthma and chronic spontaneous urticaria (CSU). Factor XIIa (FXIIa) was one of the first coagulation factors implicated in inducing both humoral and cellular responses and is therefore considered a prime new therapeutic target in immune-mediated inflammation. The involvement of coagulation factors, such as tissue factor and fibrinogen, in the pathogenesis of asthma has been reported. The finding of platelet activation in asthma also indicates a link between bronchial inflammation and hemostasis. The pathogenesis of mast cell degranulation and CSU was also shown to be associated with the activation of hemostatic factors such as fibrinogen and FXIIa. Increased plasma levels of D-dimer have been widely reported as a biological marker for the duration and severity of CSU. In addition, endothelial-induced cell activation by the kallikrein-high molecular weight complex and the release of heat shock protein 90 was shown to be involved in mast cell degranulation disorders. In this narrative review, the authors aim to summarize the role of hemostasis in inflammation, asthma, and CSU by focusing on the increasing information linking hemostatic factors and immune-mediated disorders.

New agents for thromboprotection. A role for factor XII and XIIa inhibition.

Blood coagulation is essential for hemostasis, however excessive coagulation can lead to thrombosis. Factor XII starts the intrinsic coagulation pathway and contact-induced factor XII activation provides the mechanistic basis for the diagnostic aPTT clotting assay. Despite its function for fibrin formation in test tubes, patients and animals lacking factor XII have a completely normal hemostasis. The lack of a bleeding tendency observed in factor XII deficiency states is in sharp contrast to deficiencies of other components of the coagulation cascade and factor XII has been considered to have no function for coagulation in vivo. Recently, experimental animal models showed that factor XII is activated by an inorganic polymer, polyphosphate, which is released from procoagulant platelets and that polyphosphate-driven factor XII activation has an essential role in pathologic thrombus formation. Cumulatively, the data suggest to target polyphosphate, factor XII, or its activated form factor XIIa for anticoagulation. As the factor XII pathway specifically contributes to thrombosis but not to hemostasis, interference with this pathway provides a unique opportunity for safe anticoagulation that is not associated with excess bleeding. The review summarizes current knowledge on factor XII functions, activators and inhibitors.

The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood.

BACKGROUND: Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. OBJECTIVE: In the present work the specificity of CTI for factor (F) XIIa is questioned. METHODS AND RESULTS: In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 µm CTI (assay concentration 2.4 µm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 µm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 µm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. CONCLUSION: To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 µm) and in plasma below 3 µm.

A new enzyme-linked immunosorbent assay recognizing both rat and human activated coagulation Factor XII (FXIIa).

We describe the first sandwich enzyme-linked immunosorbent assay (ELISA) capable of recognizing both rat and human activated coagulation Factor XII (FXIIa). Increased plasma concentrations of FXIIa have been associated with adverse outcomes in several cardiovascular disease states. In humans, the FXIIa antigen in plasma is quantified by a direct sandwich ELISA employing an antibody (mAb 2/215) raised against its ß-fragment (ß-FXIIa), but this assay is unable to detect rat ß-FXIIa antigen. Thus, experimental models are at present limited in their capacity to reveal mechanisms by which FXIIa might contribute to cardiovascular pathology. Consistent with overlap between human and rat FXIIa protein epitope sequences, Western blot analysis and ELISA demonstrate that another previously developed antibody against human FXIIa (mAb 201/9) detects ß-FXIIa in both human and rat plasma, with no evidence for cross-reactivity with the FXII zymogen or FXIIa complexed with its endogenous inhibitor. The mAb 201/9 based ELISA identified similar elevations in FXIIa in plasma from rats and humans with chronic renal failure. The capacity of this ELISA to identify rat plasma FXIIa has potential application to a wide range of experimental models of human cardiovascular disease.

Antiphospholipid antibodies may impair factor XIIa-dependent activation of fibrinolysis in pregnancy: in vitro evidence with human endothelial cells in culture and monoclonal anticardiolipin antibodies.

OBJECTIVE: The purpose of this study was to analyze the in vitro activation of the contact complex with the use of human umbilical vein endothelial cells (HUVEC) in culture and monoclonal anticardiolipin antibodies (MaCL). STUDY DESIGN: Cultured HUVECs were incubated with pooled plasma from third-trimester pregnant women with the addition (20 mg/mL) of MaCL with anti-beta-2 glycoprotein I activity that was obtained from patients with antiphospholipid syndrome (APS), MaCL from an individual without APS, or from a control patient with immunoglobulin M without aCL activity. Supernatants were evaluated. Activated factors XII and VII, prothrombin-fragment 1 + 2, urokinase-type plasminogen activator (UPA), and differentiating 2 chain UPA were determined. RESULTS: In the cultured HUVEC supernatants, the addition of MaCL increased activated factor VII and prothrombin-fragment 1 + 2, did not modify UPA, and decreased activated factor XII and differentiating 2 chain UPA, in comparison with samples with control immunoglobulin M added. The MaCL without APS activity did not change any parameter that was evaluated. CONCLUSION: MaCL with anti-beta-2 activity that was obtained from patients with APS may interfere in the activation of the contact complex during pregnancy.

Factor XIIIa+ dendritic cells and S-100 protein+ Langerhans' cells in adult periodontitis.

OBJECTIVE: The purpose of this study was to evaluate the presence of factor XIIIa+ dendritic cells and S-100 protein+ Langerhans' cells in the gingival epithelium and connective tissue of periodontal pockets, before and after non-surgical periodontal therapy. BACKGROUND: The microbial flora in periodontal pockets provokes complex immune reactions. Dendritic cells play a critical role in primary and secondary immune responses and are considered as antigen-presenting cells. Factor XIIIa positive dendritic cells and S-100 protein positive Langerhans' cells identified by immunoreactivity against factor XIIIa antigen and S-100 protein, respectively, are two distinct subpopulations of dendritic cells. METHODS: Fifty-four gingival tissue samples were obtained from periodontal pockets of initial depth 4-5 mm and > or = 6 mm. Each group was subdivided in to three subgroups. The first subgroup consisted of samples taken on baseline day and used as control. The second and third subgroups included those obtained 1 month after plaque and calculus removal, and 1 month after scaling and root planing, respectively, additionally to oral hygiene instructions. The tissues were removed from the palatal gingiva under local anaesthesia during routine periodontal surgery. Immunohistochemical staining with antibodies against factor XIIIa and S-100 protein was performed to identify dendritic cells positive and Langerhans' cells positive, respectively. RESULTS: Factor XIIIa+ dendritic cell numbers decreased compared to controls after plaque and calculus removal, oral hygiene instructions and scaling and root planing in periodontal pockets of 4-5 mm, but not in those of > or = 6 mm depth. S-100+ Langerhans' cell numbers decreased after periodontal treatment in the periodontal pockets > or = 6 mm. CONCLUSION: These results may reflect a tendency for reduction of these two distinctive subpopulations of dendritic cells after non-surgical periodontal therapy.

A monoclonal antibody raised against human beta-factor XIIa which also recognizes alpha-factor XIIa but not factor XII or complexes of factor XIIa with C1 esterase inhibitor.

A monoclonal antibody (mAb 2/215) against human beta-factor XIIa (beta-FXIIa), was shown by equilibrium binding studies to have a high affinity for alpha-factor XIIa (alpha-FXIIa) (Kd 1.8 nM) and beta-FXIIa (Kd 0.65 nM) but no detectable reaction with FXII zymogen or alpha-FXIIa:C1 esterase inhibitor (C1-INH) complex. Surface plasmon resonance studies showed that the mAb 2/215 bound to immobilized alpha-FXIIa with high affinity (KD 3.93 +/- 1.46 x 10(-11) M). Western blots employing mAb 2/215 indicated that human plasma contained small amounts of alpha-FXIIa but no beta-FXIIa. mAb 2/215 did not inhibit the amidolytic activity of beta-FXIIa and protected beta-FXIIa from inhibition by C1-INH. The recovery by ELISA,employing mAb 2/215 as the capture antibody, of alpha-FXIIa added to plasma was 11.3%, 42% after inhibition of alpha-FXIIa with 3:4dichloroisocoumarin, and 82% when 0.5% Triton-X100 was added to the assay. Gel filtration showed that the majority of plasma alpha-FXIIa existed as a complex (Mr approximately 170,000). This distinctive mAb increases the capacity to study the contact system in health and disease.

Multiple interactive residues of recognition: elucidation of discontinuous epitopes with linear peptides.

The discontinuous epitopes of beta-factor XIIa for three mAbs were mapped by a linear peptide-based immunoblotting technique, referred to as multiple interactive residues of recognition. The Abs were incubated with a set of overlapping synthetic peptides, deduced from the cDNA sequences of beta-factor XIIa, on a polymer membrane, and the signal was amplified by an ECL assay. Several discrete sequences of the protein were recognized by each Ab. The recognized peptides were further characterized using alanine substitution analogues and peptides of different lengths. The discontinuous epitopes found for each Ab were formed by several peptides and were composed of 20 to 31 residues. The sequence FLQEA was recognized by all three Abs and was the immunodominant peptide. Abs 201/9 and 2/15 bound to very similar discontinuous sequences, but with subtle differences. The sequences 76RLHEAFSP83, 88HDLALLRLQE97, 178GFLEG182, 146FLQEA150, and 237IRE239 formed the epitope for mAb 201/9, whereas 76RLHEAFSP83, 90LALLRLQE97, 146FLQEA150, and 235AWIREHT241 formed the epitope for Ab 2/15. The third Ab 202/7 recognized the sequences 79EAFSP83, 93LRLQE97, 133WGHQF137, and 146FLQEA150. We suggest that these sequences represent the sites to which the Abs bind. This procedure provides a sensitive and convenient tool to elucidate discontinuous epitopes for the binding of Abs, receptor ligand-binding sites, or enzyme inhibitor binding sites.