Marcello Baroni

Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles

Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (micropar‐ticles, MPs). Here, we show that monocyte‐derived human dendritic cells (DCs) stimulated with a P2X7 receptor (P2X7R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane‐bound form of tissue factor (TF), a glycop‐rotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane‐bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti‐TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full‐length TF form. Activity of MP‐bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti‐human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are “deliverable” after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.—MarcelloBaroni, CinziaPizzirani, MirkoPinotti, DavideFerrari, ElenaAdinolfi, SaraCalzavarini, PierpaoloCaruso, FrancescoBernardi, FrancescoDi Virgilio. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor‐bearing microparticles. FASEB J. 21, 1926–1933 (2007)

Tissue Factor and Coagulation Factor VII Levels During Acute Myocardial Infarction: Association With Genotype and Adverse Events

Objective—We investigated in patients with ongoing myocardial infarction (MI) whether coagulation factor VII (FVII) and tissue factor (TF) levels are affected at admission by genetic components and whether they may predict subsequent cardiovascular events. Methods and Results—256 patients admitted for MI were evaluated for FVII and TF antigen levels before any treatment at entry, and were genotyped for FVII and TF polymorphisms. FVII gene insertions at −323, 11293 and the −402G/A change predicted FVII levels and explained 14% of variance. The −603 TF gene polymorphism failed to affect significantly TF levels (P=0.07). These variables were correlated with the incidence of death (36 patients) and reinfarction (9 patients) after a median follow-up of 397 days. Events were independently predicted by FVII (HR 2.1, 95% CI 1.2 to 5.7) and TF (HR 4.1, 95% CI 2 to 11) levels. Composite end point was significantly worse when both parameters were above the receiver-operating characteristics (ROC) values (HR 8.3, 95% CI 5 to 18, compared with FVII and TF below), and above the ROC value of TF (>630 pg/mL) it differed among FVII genotype groups. Conclusions—Admission FVII and TF antigen levels, partially predicted by polymorphisms, are independent predictors of mortality and reinfarction in patients with acute MI.

Impaired prothrombinase activity of factor X Gly381Asp results in severe familial CRM+ FX deficiency.

We investigated three members of a large Omani family affected by severe factor X (FX) deficiency (coagulant activity <1%) and showing marked differences in the onset of severe hemorrhagic symptoms. All patients were homozygous for a novel FX mutation (Gly381Asp) in the structurally conserved region of the serine protease active site. Expression levels of recombinant 381D-FX were similar to those of wt-FX, indicating the presence of a severe CRM+ FX deficiency, a poorly investigated condition. The 381D-FX was normally activated and did not show a detectable amidolytic activity. Instead, we observed a residual activity in a prothrombin-time based assay (1%) and in prothrombinase assays both in plasma (1%) and in purified systems (3%). Comparison with FX variants characterized by reduced activation suggests that mutations affecting FX activity might result in a more pronounced impairment of coagulation and thus in severe hemorrhagic phenotype. In addition, this study indicates that the hemorrhagic heterogeneity observed in FX deficiencies is only partially explained by molecular analysis of FX gene.

Factor II Activity is Similarly Increased in Patients With Elevated Apolipoprotein CIII and in Carriers of the Factor II 20210A Allele

Background Few studies have so far investigated the relationship between apolipoprotein CIII (Apo CIII) and coagulation pathway in subjects with or without coronary artery disease (CAD). Methods and Results Serum Apo CIII concentrations and plasma coagulant activities of factor II (FII:c), factor V (FV:c), and factor VIII (FVIII:c), and activated factor VII (FVIIa) were analyzed in a total of 933 subjects, with (n=687) or without (n=246) angiographically demonstrated CAD and not taking anticoagulant drugs. Activated factor X (FXa) generation assay was performed on plasma from subgroups of subjects with low and high levels of Apo CIII. A statistical incremental concentration of FII:c, FV:c, and FVIIa levels was observed through the quartiles of Apo CIII distribution in the population considered as a whole. Significant results were confirmed for FII:c in CAD and CAD‐free subgroup when separately considered. Subjects within the highest Apo CIII quartile (>12.6 mg/dL) had high FII:c levels not statistically different from those of carriers of 20210A allele (n=40; 4.28%). In a multiple linear model, Apo CIII was the best predictor of FII:c variability, after adjustment for age, gender, plasma lipids, CRP, creatinine, diagnosis, and carriership of 20210A allele. FXa generation was increased and its lag time shortened in plasmas with high Apo CIII levels. However, after thrombin inhibition by hirudin, differences between low and high Apo C‐III samples disappeared. Conclusions Elevated concentrations of Apo CIII are associated with an increase of thrombin activity to an extent comparable with the carriership of G20210A gene variant and mainly modulating the thrombin generation.

Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function

Summary.  Objective: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium‐binding module with a poorly defined functional role. Patients: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. Results: Reverse transcription‐polymerase chain reaction analysis showed the presence of the IVSg‐2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from probands plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C‐independent activities (24–38%) when compared with wild‐type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 ± 30.8 nm, rPSwt; Kd 15.2 ± 0.9 nm) as well as to Ca2+‐dependent conformation‐specific monoclonal antibodies for GLA domain was significantly reduced. Conclusions: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.

Activated factor VII–antithrombin complex predicts mortality in patients with stable coronary artery disease: a cohort study

Essentials Activated factor VII–antithrombin complex (FVIIa‐AT) in plasma may reflect tissue factor exposure. FVIIa‐AT levels were assessed in an angiographically controlled coronary artery disease (CAD) cohort. High FVIIa‐AT levels correlated with an increased thrombin generation. High FVIIa‐AT levels were associated with a greater risk of mortality in patients with stable CAD.

Membrane binding and anticoagulant properties of protein S natural variants

INTRODUCTION Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency. MATERIALS AND METHODS Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems. RESULTS AND CONCLUSIONS Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800 nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17). In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7+/-1.6 nM, rPS217S 146.0+/-16.1 nM and rPSDelI203D204 234.1+/-28.1 nM) was substantially increased by membrane oxidation (10.9+/-0.6, 38.2+/-3.5 and 81.4+/-6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration. These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation.

The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X

The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl‐terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion.

Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation

Alterations in coagulation factor X (FX) activation, mediated by the extrinsic VIIa/tissue factor (FVIIa/TF) or the intrinsic factor IXa/factor VIIIa (FIXa/FVIIIa) complexes, can result in hemorrhagic/prothrombotic tendencies. However, the molecular determinants involved in substrate recognition by these enzymes are poorly defined. Here, we investigated the role of arginine 386 (chymotrypsin numbering c202), a surface-exposed residue on the FX catalytic domain. The naturally occurring FX386Cys mutant and FX386Ala variant were characterized. Despite the unpaired cysteine, recombinant (r)FX386Cys was efficiently secreted (88.6±21.3% of rFXwt) and possessed normal clearance in mice. rFX386Cys was also normally activated by FVIIa/TF and displayed intact amidolytic activity. In contrast, rFX386Cys activation by the FIXa/FVIIIa complex was 4.5-fold reduced, which was driven by a decrease in the kcat (1.6∗10(-4) s(-1) vs 5.8∗10(-4) s(-1), rFXwt). The virtually unaltered Km (70.6 nM vs 55.6nM, rFXwt) suggested no major alterations in the FX substrate exosite. Functional assays in plasma supplemented with rFX386Cys indicated a remarkable reduction in the thrombin generation rate and thus in coagulation efficiency. Consistently, the rFX386Ala variant displayed similar biochemical features suggesting that global changes at position 386 impact the intrinsic pathway activation. These data indicate that the FXArg386 is involved in FIXa/FVIIIa-mediated FX activation and help in elucidating the bleeding tendency associated with the FX386Cys in a rare FX deficiency case. Taking advantage of the unpaired cysteine, the rFX386Cys mutant may be efficiently targeted by thiol-specific ligands and represent a valuable tool to study FX structure-function relationships both in vitro and in vivo.

Coagulation Factor XII Levels and Intrinsic Thrombin Generation in Multiple Sclerosis

Background Factor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS. Objective To investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG). Methods In plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers. Results Higher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients. Conclusion In MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression.