Bone metastases are associated with worse prognosis in patients affected by metastatic colorectal cancer treated with doublet or triplet chemotherapy plus bevacizumab: a subanalysis of the TRIBE and TRIBE2 trials.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers; ∼20% of patients have metastases at diagnosis, and 50%-60% subsequently develop metachronous metastases. Bone involvement, despite being rare, is usually associated with higher disease burden, worse prognosis, impaired quality of life, and significant health-related cost. In the last few years, following the positive results of the TRIBE and TRIBE2 trials, the association of FOLFOXIRI plus bevacizumab has become the new standard of care for metastatic CRC. Despite being highly efficacious in all subgroups, little is known about the activity of this regimen in patients with bone metastases. PATIENTS AND METHODS: We carried out a pooled analysis of TRIBE and TRIBE2 studies focusing on patients with skeletal deposits. RESULTS: Our analyses on the whole population showed that patients with baseline bone involvement reported shorter overall survival [OS; 14.0 versus 26.2 months; hazard ratio (HR) 2.04, 95% confidence interval (CI) 1.46-2.87; P < 0.001] and progression-free survival (PFS; 6.2 versus 11.1 months; HR 1.96, 95% CI 1.42-2.69; P < 0.001) compared with those without bone metastases; no significant interaction with the treatment was reported for PFS (P = 0.094) and OS (P = 0.38). Bone metastases had a negative prognostic implication in the multivariate analysis (HR 2.24, 95% CI 1.54-3.26; P < 0.001). Furthermore, patients with bone lesions at first radiological progression (including those with baseline bone metastases) had a shorter OS compared with those who progressed in other sites (10.4 versus 13.2 months; HR 1.48, 95% CI 1.15-1.91; P = 0.002). A trend toward inferior OS (7.5 versus 11 months, HR 1.50, 95% CI 0.92-2.45; P = 0.10) appeared in patients with basal skeletal deposits compared with those with bone involvement at first radiological progression. CONCLUSIONS: Our study confirmed the negative prognostic impact of bone metastases in CRC. Furthermore, we demonstrated for the first time that the survival advantage of triplet chemotherapy plus bevacizumab is maintained even in this prognostically unfavorable subgroup.

Evaluation of COVID-19 impact on DELAYing diagnostic-therapeutic pathways of lung cancer patients in Italy (COVID-DELAY study): fewer cases and higher stages from a real-world scenario.

INTRODUCTION: COVID-19 has disrupted the global health care system since March 2020. Lung cancer (LC) patients (pts) represent a vulnerable population highly affected by the pandemic. This multicenter Italian study aimed to evaluate whether the COVID-19 outbreak had an impact on access to cancer diagnosis and treatment of LC pts compared with pre-pandemic time. METHODS: Consecutive newly diagnosed LC pts referred to 25 Italian Oncology Departments between March and December 2020 were included. Access rate and temporal intervals between date of symptoms onset and diagnostic and therapeutic services were compared with the same period in 2019. Differences between the 2 years were analyzed using the chi-square test for categorical variables and the Mann-Whitney U test for continuous variables. RESULTS: A slight reduction (-6.9%) in newly diagnosed LC cases was observed in 2020 compared with 2019 (1523 versus 1637, P = 0.09). Newly diagnosed LC pts in 2020 were more likely to be diagnosed with stage IV disease (P < 0.01) and to be current smokers (someone who has smoked more than 100 cigarettes, including hand-rolled cigarettes, cigars, cigarillos, in their lifetime and has smoked in the last 28 days) (P < 0.01). The drop in terms of new diagnoses was greater in the lockdown period (percentage drop -12% versus -3.2%) compared with the other months included. More LC pts were referred to a low/medium volume hospital in 2020 compared with 2019 (P = 0.01). No differences emerged in terms of interval between symptoms onset and radiological diagnosis (P = 0.94), symptoms onset and cytohistological diagnosis (P = 0.92), symptoms onset and treatment start (P = 0.40), and treatment start and first radiological revaluation (P = 0.36). CONCLUSIONS: Our study pointed out a reduction of new diagnoses with a shift towards higher stage at diagnosis for LC pts in 2020. Despite this, the measures adopted by Italian Oncology Departments ensured the maintenance of the diagnostic-therapeutic pathways of LC pts.

The lung immuno-oncology prognostic score (LIPS-3): a prognostic classification of patients receiving first-line pembrolizumab for PD-L1 ≥ 50% advanced non-small-cell lung cancer.

BACKGROUND: To stratify the prognosis of patients with programmed cell death-ligand 1 (PD-L1) ≥ 50% advanced non-small-cell lung cancer (aNSCLC) treated with first-line immunotherapy. METHODS: Baseline clinical prognostic factors, the neutrophil-to-lymphocyte ratio (NLR), PD-L1 tumour cell expression level, lactate dehydrogenase (LDH) and their combination were investigated by a retrospective analysis of 784 patients divided between statistically powered training (n = 201) and validation (n = 583) cohorts. Cut-offs were explored by receiver operating characteristic (ROC) curves and a risk model built with validated independent factors by multivariate analysis. RESULTS: NLR < 4 was a significant prognostic factor in both cohorts (P < 0.001). It represented 53% of patients in the validation cohort, with 1-year overall survival (OS) of 76.6% versus 44.8% with NLR > 4, in the validation series. The addition of PD-L1 ≥ 80% (21% of patients) or LDH < 252 U/l (25%) to NLR < 4 did not result in better 1-year OS (of 72.6% and 74.1%, respectively, in the validation cohort). Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 2 [P < 0.001, hazard ratio (HR) 2.04], pretreatment steroids (P < 0.001, HR 1.67) and NLR < 4 (P < 0.001, HR 2.29) resulted in independent prognostic factors. A risk model with these three factors, namely, the lung immuno-oncology prognostic score (LIPS)-3, accurately stratified three OS risk-validated categories of patients: favourable (0 risk factors, 40%, 1-year OS of 78.2% in the whole series), intermediate (1 or 2 risk factors, 54%, 1-year OS 53.8%) and poor (>2 risk factors, 5%, 1-year OS 10.7%) prognosis. CONCLUSIONS: We advocate the use of LIPS-3 as an easy-to-assess and inexpensive adjuvant prognostic tool for patients with PD-L1 ≥ 50% aNSCLC.

Osimertinib beyond disease progression in T790M EGFR-positive NSCLC patients: a multicenter study of clinicians' attitudes.

BACKGROUND: In most cases, T790M EGFR-positive NSCLC patients receiving osimertinib developed "non-drugable" progression, as the patients with common EGFR-sensitizing mutations were treated with first-line osimertinib. In both settings, chemotherapy represents the standard treatment and local ablative treatments (LATs) are potential useful options in the case of oligo-progression. METHODS: We conducted a study on "post-progression" (pp) outcomes of T790M EGFR-positive NSCLC patients treated with osimertinib, according to the therapeutic strategy applied: osimertinib beyond progression (± LATs), "switched therapies" or best supportive care only (BSC). RESULTS: 144 consecutive patients were evaluated: 53 (36.8%) did not received post-progression treatments (BSC), while 91 (63.2%) patients received at least 1 subsequent treatment; 50 patients (54.9%) received osimertinib beyond disease progression [19 (20.9%) of them with adjunctive LATs] and 41 (45.1%) a switched therapy. Median ppPFS (progression-free survival) and median ppOS (overall survival) of patients who received osimertinib beyond progression vs. switched therapies were 6.4 months vs. 4.7 months, respectively [HR 0.57 (95% CI 0.35-0.92), p = 0.0239] and 11.3 months vs 7.8 months, respectively [HR 0.57 (95% CI 0.33-0.98), p = 0.0446]. Among patients who received osimertinib beyond progression with and without LATs median ppPFS was 6.4 months and 5.7 months, respectively [HR 0.90 (95% CI 0.68-1.18), p = 0.4560], while median ppOS was 20.2 months and 9.9 months, respectively [HR 0.73 (95% CI 0.52-1.03), p = 0.0748]. At the univariate analysis, the only factor significantly related to the ppPFS was the therapeutic strategy in favor of osimertinib beyond progression (± LATs). Moreover, the only variable which was significantly related to ppOS at the multivariate analysis was osimertinib beyond progression (± LATs). CONCLUSION: Our study confirmed that in clinical practice, in case of "non-druggable" disease progression, maintaining osimertinib beyond progression (with adjunctive LATs) is an effective option.

Nutritional intervention in a hemodialysis pregnant woman: a case report.

Pregnancy in dialysis patients is a rare occurrence. When pregnancy does occur, the risk of spontaneous abortion, stillbirth and neonatal complications, such as prematurity and growth retardation, are fairly high. The authors describe their experience in the follow-up of a patient with chronic renal failure who became pregnant during regular dialysis treatment and followed nutritional care. The outcomes were successful and she gave birth to a healthy baby. It is emphasized that special dedication to the nutritional control enabled a good outcome of the pregnancy. The importance of the nutritionist intervention in the follow-up of dialysis patients with the integration of a multidisciplinary staff is stressed.

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001). Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.

[Hydatidiform mole with coexistent fetus. Cytogenetic features, diagnosis, and management]. / Mola idatiforme con feto coesistente. Aspetti cito-genetici, diagnosi e management.

Hydatidiform mole with coexistent fetus is an unusual entity caused by two distinct types of pregnancy: the first one is a partial hydatidiform mole, while the second is a twin pregnancy in which a mole coexists with a normal fetus. In these two separate genetic entities, the counseling and the mother-fetus prognosis are different. Two cases of mole with coexistent fetus are reported: a partial hydatidiform mole typically tripliod and a partial mole with unusual diploid karyotype. Prenatal diagnosis is remarkable for the evaluation of fetus development related with his karyotype. Triplody excludes all hope of a non-malformed surviving child and termination of pregnancy is desirable, while normal karyotype the possibility of a continuation of pregnancy may be considered.

Identification of a putative binding site for negatively charged surfaces in the fibronectin type II domain of human factor XII--an immunochemical and homology modeling approach.

Monoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (FXII), which inhibits kaolin-induced clotting activity by preventing the binding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK5 likely is directed against the binding site of FXII for negatively charged surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino acid consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contains two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ), the second one with residues 57-60 (TPNF); both amino acid stretches belonging to the fibronectin type II domain of FXII. Sequence B also reveals homology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40-47 and 57-60 are close to each other and accessible for the solvent, i.e. in a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type III domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37) or 39-47 (FXII39-47), were synthesized and tested for the capability to inhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhibited the binding of labeled FXII to kaolin and effectively prevented both dextran sulfate- and kaolin-induced activation of the contact system in plasma. Hence, we suggest that the fibronectin type II domain of FXII, in particular residues 39 to 47, contribute to the binding site of FXII for negatively charged surfaces.

The second exon-encoded factor XII region is involved in the interaction of factor XII with factor XI and does not contribute to the binding site for negatively charged surfaces.

Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-triangle up19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-triangle up19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-triangle up19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-triangle up19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-triangle up19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-triangle up19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-triangle up19 bound to kaolin, which binding was equally well inhibited by either, rFXII-triangle up19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-triangle up19 generated a similar amount of FXIIa- and kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1-inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-triangle up19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.

Initiation of contact system activation in plasma is dependent on factor XII autoactivation and not on enhanced susceptibility of factor XII for kallikrein cleavage.

Various mechanisms have been hypothesized to explain the initiation of contact system activation in plasma. We investigated the capability of dextran sulphate (DS) of different molecular weights to initiate contact system activation in normal human plasma, and compared this with their capability to support factor XII autoactivation and to enhance factor XII susceptibility for cleavage by kallikrein. Dextran sulphate of Mr 500,000 (DS500) and 50,000 (DS50) was able to initiate contact system activation in plasma (determined by measuring the amount of factor XIIa-C1-inhibitor, kallikrein-C1-inhibitor and factor XIa-C1-inhibitor complexes generated) as well as to support factor XII autoactivation and to enhance factor XII susceptibility for cleavage by kallikrein (as measured with amidolytic assays using purified proteins). In contrast, dextran sulphate of Mr 15,000 (DS15) and 5000 (DS5) neither induced contact system activation in plasma, nor supported autoactivation of factor XII, although both of these DS species enhanced the rate of activation of factor XII by kallikrein in the purified system. Based on these properties (i.e. binding of factor XII without inducing autoactivation), DS15 and DS5 were predicted to be inhibitors of contact system activation induced in plasma by DS500, which indeed was observed. We conclude that enhanced factor XII susceptibility for kallikrein activation and factor XII autoactivation are distinct phenomena, the latter being necessary to support activation of the contact system in plasma.

Interleukin-6 downregulates factor XII production by human hepatoma cell line (HepG2).

Involvement of the contact system of coagulation in the pathogenesis of various inflammatory diseases is suggested by reduced plasma levels of factor XII (Hageman factor) and prekallikrein generally considered to result from activation of the contact system. However, in many of these diseases patients develop an acute-phase response and, therefore, an alternative explanation for the decreased levels of factor XII could be the downregulation of factor XII gene expression in the liver as described for negative acute-phase proteins. We report here that interleukin-6 (IL-6), the principal cytokine mediating the synthesis of most acute-phase proteins in the liver, downregulates the production of factor XII by the human hepatoma cell line HepG2 by up to 75%. The decrease in protein secretion correlated with an equivalent decrease of factor XII mRNA likely indicating a pretranslational control of factor XII gene expression by IL-6. Downregulation of factor XII production by IL-6 in vitro parallelled that of transthyretin, a known negative acute-phase protein. Moreover, we show that, in patients developing an acute-phase response after immunotherapy with IL-2, plasma levels of factor XII correlate (r = .76, P < .0001) with those of transthyretin. Taken together, these results suggest that factor XII behaves as a negative acute-phase protein.

Modulation of contact system proteases by glycosaminoglycans. Selective enhancement of the inhibition of factor XIa.

We investigated the influence of dextran sulfate, heparin, heparan sulfate, and dermatan sulfate on the inhibition of FXIa (where FXIa is activated factor XI, for example), FXIIa, and kallikrein by C1 inhibitor, alpha1-antitrypsin, alpha2-antiplasmin, and antithrombin III. The second-order rate constants for the inhibition of FXIa by C1 inhibitor, alpha1-antitrypsin, alpha2-antiplasmin, and antithrombin III, in the absence of glycosaminoglycans, were 1.8, 0.1, 0.43, and 0.32 x 10(3) M-1 s-1, respectively. The rate constants of the inactivation of FXIa by C1 inhibitor and by antithrombin III increased up to 117-fold in the presence of glycosaminoglycans. These data predicted that considering the plasma concentration of the inhibitors, C1 inhibitor would be the main inhibitor of FXIa in plasma in the presence of glycosaminoglycans. Results of experiments in which the formation of complexes between serine protease inhibitors and FXIa was studied in plasma agreed with this prediction. Glycosaminoglycans did not enhance the inhibition of alpha-FXIIa, beta-FXIIa, or kallikrein by C1 inhibitor. Thus, physiological glycosaminoglycans selectively enhance inhibition of FXIa without affecting the activity of FXIIa and kallikrein, suggesting that glycosaminoglycans may modulate the biological effects of contact activation, by inhibiting intrinsic coagulation without affecting the fibrinolytic potential of FXIIa/kallikrein.

Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces.

The binding site of human factor XII (FXII) for negatively charged surfaces has been proposed to be localized in the N-terminal region of factor XII. We have generated two recombinant factor XII proteins that lack this region: one protein consisting of the second growth-factor-like domain, the kringle domain, the proline-rich region and the catalytic domain of FXII (rFXII-U-like), and another consisting of only 16 amino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-1pc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII-U-like and, to a lesser extent, rFXII-lpc bound to glass in a concentration-dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of non-labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glass-bound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but less efficiently than rFXII, rFXII-U-like and rFXII-lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N-terminal part of the heavy-chain region of factor XII contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal-growth-factor-like domain and/or the kringle domain, contribute to the binding of factor XII to these surfaces.

Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Estrogen induction and contact phase activation of human factor XII.

This paper reviews data reported in the literature and results of our experiments on the transcriptional control of Factor XII by estrogens and on the activation of Factor XII in the plasma. Coagulation Factor XII (Hageman factor, FXII) is a serine protease secreted by the liver and activated by negative charged surfaces to play roles in fibrinolysis, coagulation, and inflammation. Multiple effects on hemostasis involving these processes via Hageman factor have been reported in relation to estrogen therapy. The nucleotide sequence of 3,174 base pair (bp) DNA at the 5' end of the Factor XII gene indicates that the Factor XII promoter is typical of TATA-less, liver-specific, and serine protease-type eukaryotic genes involved in clotting. In addition the Factor XII promoter contains at position -44/-31 a palindrome similar, but not identical, to an estrogen-responsive element (ERE) together with four hemisite EREs between positions -1314 and -608. These promoter regions may underlie the mechanism by which estrogens enhance Factor XII concentrations in plasma. In vivo, a 6-fold stimulation of FXII gene transcription by 17 beta-estradiol was observed in ovariectomized rats. In vitro a 230-bp promoter fragment of Factor XII (-181/+49) confers a strong 17 beta-estradiol responsiveness onto a chlorampenicol acetyltransferase reporter when transiently co-transfected with the human estrogen receptor. The domain structure of Factor XII allows identification of those parts of the protein with particular functions. cDNA constructs, in which sequences coding for selected domains were deleted, were used to produce recombinant deleted Factor XII proteins in a vacinia virus expression system. To identify the domain(s) responsible for contact phase activation, these recombinant proteins were tested for their capacity to bind to negatively charged substrates, to become activated by kallikrein, and to sustain blood clotting and amidolytic activity. In addition to the N-terminal domain, the growth factor and kringle domains and, to a lesser extent, the polyproline region also interact with negatively charged surfaces and presumably thus contribute to activation.

Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein.

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.

Molecular basis of estrogen regulation of Hageman factor XII gene expression.

Estrogen therapy has been reported to cause multiple alterations in hemostasis and to increase blood levels of several procoagulants, including Hageman factor [factor XII (FXII)]. Liver FXII gene expression has been investigated in ovariectomized rats, treated or not with 17 beta-estradiol. A 6-fold stimulation of FXII gene transcription was observed in treated compared to untreated animals, indicating that 17 beta-estradiol is able to induce FXII gene expression in vivo. We have recently shown that human FXII promoter contains an imperfect palindrome, 5'-GGGCAnnnTGACC-3', at position -43/-31 resembling the consensus estrogen-responsive element (ERE). Portions of different length of the FXII promoter were fused to the chloramphenicol acetyltransferase (CAT) coding sequence and transiently cotransfected with human estrogen receptor (ER) into NIH3T3 and HepG2 cells in the presence or absence of 17 beta-estradiol. A 230-base pair fragment of FXII promoter, spanning nucleotides - 181/49, conferred a strong estrogen responsiveness to the CAT reporter gene, suggesting that a functional ERE resides in this region. Cognate receptors, such as those for thyroid hormone or retinoic acid, did not stimulate CAT activity. Gel mobility assays demonstrated a specific interaction between ER and the 230-bp FXII promoter fragment containing the putative ERE palindrome. Similar results were obtained when an oligonucleotide spanning the consensus ERE was used; the complex between ER and FXII promoter sequences was supershifted after the addition of an anti-ER monoclonal antibody. Insertion of FXII-ERE into the heterologous thymidine kinase promoter conferred a strong estrogen responsiveness that was abolished by mutations of the 5'-half of the palindrome. These results represent the first demonstration at the molecular level of the regulation of a blood coagulation factor gene by 17 beta-estradiol as well as the first identification of a functional ERE within this class of genes.