Ramón Montes

Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity

Summary.  Background: Endothelial cell protein C receptor (EPCR) binds protein C through its γ‐carboxyglutamic acid (Gla) domain and enhances its thrombin–thrombomodulin complex‐dependent activation. So far, only protein C/activated protein C has been shown to interact with EPCR. Factor VII (FVII), the coagulation trigger upon tissue factor (TF) interaction, is a serine protease whose Gla domain is highly homologous to the Gla domain of protein C. Objectives:  To characterize the binding of FVII/FVIIa to EPCR and its functional consequences. Methods and results:  We demonstrated by surface plasmon resonance (SPR) that FVII/FVIIa binds to EPCR through its Gla domain. At therapeutic concentrations, FVIIa reduced the activation of protein C by 40%. Soluble EPCR (sEPCR) was also able to prolong dose‐dependently the clotting time induced by the FVIIa–TF complex. SPR and amidolytic experiments showed that FVIIa is able to interact simultaneously with TF and EPCR, thus ruling out the possibility that the effect of EPCR on clotting time was due to the inhibition of the binding between FVIIa and TF. sEPCR inhibited dose‐dependently the activation of FX by the FVIIa–TF complex. Notably, blocking the binding site of EPCR on the endothelial surface increased the generation of FXa 2‐fold. Conclusions: EPCR binds to FVII/FVIIa and inhibits the procoagulant activity of the FVIIa–TF complex.

The c.−1639G > A polymorphism of the VKORC1 gene is a major determinant of the response to acenocoumarol in anticoagulated patients

Much of the variability in the sensitivity to warfarin in anticoagulated patients is associated with the c.−1639G > A polymorphism of the vitaminK‐epoxide reductase (VKORC1) gene. However, its association with the acenocoumarol dose in patients under anticoagulant therapy has not been studied. The c.−1639G > A genotype of VKORC1 was determined in 113 patients on stable anticoagulation requiring low (n = 42), medium (n = 42) or high (n = 21) acenocoumarol doses. To evaluate the association between acenocoumarol requirements and the c.−1639G > A variant, multivariate logistic regression models were fitted, adjusting for age, gender, and the c.430C > T and c.1075A > C variants of cytochrome P450 2C9 (CYP2C9). A total of 90·5% of the patients in the low acenocoumarol dose group carried the A allele of VKORC1:c.−1639G > A. The A allele independently increased the odds of requiring a low acenocoumarol dose [odds ratio (OR) 9·4; 95% confidence interval (CI) 1·9–46·4; P = 0·006], especially when the homozygous form was present (OR 44·2; 95% CI 5·5–354·6; P < 0·001). The A allele was less frequent in the high dose group showing an inverse association with the requirement for high doses (OR 0·04; 95% CI 0·01–0·22; P < 0·001). The A allele of the c.−1639G > A polymorphism of VKORC1 is therefore associated with a low‐dose requirement for acenocoumarol in patients receiving anticoagulant therapy.

Development and clinical application of a new ELISA assay to determine plasmin–α2‐antiplasmin complexes in plasma

Plasmin–α2‐antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine‐sepharose and elution with ε‐aminocaproic acid. This material, characterized by SDS‐PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme‐linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified α2‐antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3.5% (intra‐assay) and 10.6% (interassay). A mean value of 573.5 ± 131.4 ng/ml was obtained from PAP concentration in a healthy population (n = 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels.

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

BackgroundDifferent isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.ResultsRecombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found.ConclusionsOur results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.

Usefulness of High-Sensitivity C-Reactive Protein to Predict Mortality in Patients With Atrial Fibrillation (from the Atherosclerosis Risk In Communities [ARIC] Study)

High-sensitivity C-reactive protein (hs-CRP) is a marker for the risk of cardiovascular and overall mortality. However, information about the association between hs-CRP and mortality in patients with atrial fibrillation is scarce. A total of 293 participants of the Atherosclerosis Risk In Communities study with a history of AF and hs-CRP levels available were studied. During a median follow-up of 9.4 years, 134 participants died (46%). The hazard ratio of all-cause mortality associated with the highest versus the lowest tertile of hs-CRP was 2.52 (95% confidence interval 1.49 to 4.25) after adjusting for age, gender, history of cardiovascular diseases, and cardiovascular risk factors. A similar trend was observed for cardiovascular mortality (57 events; hazard ratio 1.90, 95% confidence interval 0.81 to 4.45). The Congestive heart failure, Hypertension, Age >75 years, Diabetes, and previous Stroke or transient ischemic attack (CHADS2) score was also associated with all-cause and cardiovascular mortality, with an adjusted hazard ratio of 3.39 (95% confidence interval 1.91 to 6.01) and 8.71 (95% confidence interval 2.98 to 25.47), respectively, comparing those with a CHADS2 score >2 versus a CHADS2 score of 0. Adding hs-CRP to a predictive model including the CHADS2 score was associated with an improvement of the C-statistic for total mortality (from 0.627 to 0.677) and for cardiovascular mortality (from 0.700 to 0.718). In conclusion, high levels of hs-CRP constitute an independent marker for the risk of mortality in patients with atrial fibrillation.

The increase of plasminogen activator inhibitor activity is associated with graft occlusion in patients undergoing aorto-coronary bypass surgery

Early graft occlusion is a common complication in patients undergoing aorto‐coronary bypass surgery. Both mechanical and haemostatic factors play a role in the pathogenesis of thrombotic occlusion. Several studies have demonstrated a relationship between fibrinolytic activity and venous or arterial thrombosis. We undertook this study to evaluate the possible contribution of the fibrinolytic system to postoperative occlusion in patients undergoing aorto‐coronary bypass graft (CABG).

Prothrombin A19911G and G20210A polymorphisms' role in thrombosis

Summary. The prothrombin G20210A polymorphism, which correlates with the plasmatic prothombin levels, is the second genetic risk factor for deep venous thrombosis (DVT), although its prothrombotic role is mild. Recently, the prothrombin A19911G polymorphism, also associated with slight variations of the prothrombin level, has been suggested to modulate the thrombotic risk of the G20210A polymorphism in a preliminary study including few patients and controls. Our study evaluated the effect of the A19911G polymorphism in the arterial and venous thrombotic risk of the prothrombin 20210G/A genotype, analysing 204 consecutive DVT patients and 204 matched controls. Moreover, we analysed 213 carriers of the 20210G/A genotype (152 with DVT, 26 with arterial thrombosis and 35 healthy subjects) and 10 homozygous 20210 A/A carriers. We developed a simple method to simultaneously determine the genotype of both polymorphisms. In accordance with our case/control study, the A19911G polymorphism did not play a significant role in the development of DVT. Analysis of 120 20210 A alleles demonstrated a complete linkage disequilibrium with the 19911 A allele. These polymorphisms (alone or combined) did not modify the risk of arterial thrombosis. However, the 19911A/G genotype slightly increased the risk of developing DVT in carriers of the 20210G/A genotype (OR 3·34 vs 5·86), supporting that the prothrombin 19911 polymorphism could modulate the risk of the G20210A polymorphism in developing DVT.

Endotoxin-induced disseminated intravascular coagulation in rabbits: Effect of recombinant hirudin on hemostatic parameters, fibrin deposits, and mortality

We evaluated the effect of r-hirudin on an experimental model of disseminated intravascular coagulation (DIC) in rabbits, through the continuous infusion of 100 microg/kg/hr of Escherichia coli endotoxin for a period of 6 hours. r-Hirudin (0.05, 0.3, and 0.6 mg/kg/hr) as treatment, or saline solution as placebo, were administered simultaneously with endotoxin. Severe DIC in the endotoxin control group was shown by impairment in hemostatic parameters, kidney fibrin deposition, and a high mortality rate. Medium and high doses of r-hirudin led to an improvement in such DIC-related parameters as platelet numbers and fibrinogen and protein C concentrations. High-dose r-hirudin also reduced consumption of antithrombin III (ATIII). All doses of r-hirudin prevented decreases in tissue plasminogen activator (t-PA) and reduced the increase in plasminogen activator inhibitor-1 (PAI-1) activity observed at 2 hours after endotoxin administration. A significant reduction in kidney fibrin deposition was seen in medium- and high-dose r-hirudin groups. Additionally, the mortality rate in rabbits receiving medium- and high-dose r-hirudin was 10%, and that in rabbits receiving low-dose r-hirudin was 20%, as compared with a mortality rate of 70% in the control group. Protein C activity was significantly lower (p < 0.001) in nonsurviving rabbits. Moreover, there was a strong positive correlation (r = 0.68, p < 0.001) between protein C consumption and kidney fibrin deposition. We conclude that r-hirudin can be a useful drug in the clinical treatment of DIC.

Hemostatic Disturbances in Patients With Systemic Inflammatory Response Syndrome (SIRS) and Associated Acute Renal Failure (ARF)

Endothelial damage plays a central role in the development of an SIRS-related Multiple Organ Dysfunction Syndrome (MODS) as a consequence of the establishment of a hemostatic imbalance between coagulation and fibrinolysis systems. Until now, sepsis is the SIRS model that has been most studied. The aim of this study was to assess the endothelial damage and the hemostatic imbalance in early stages of an SIRS of different origins, and to study if there are any differences in these disturbances between infectious and noninfectious SIRS. The endothelial damage and hemostatic changes were studied in 40 patients with SIRS (with less than 12 h of evolution) and an acute renal failure. Infectious SIRS was diagnosed in 19 cases and noninfectious SIRS in the remaining 21 patients. Patients with SIRS presented significantly higher values (p<0.001) for factors related to endothelial damage [von Willebrand factor (vWF), thrombomodulin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) antigen], hypercoagulability [prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes (TAT)], and fibrinolysis (D-dimer and PAI activity) with respect to the control group. However, although the group with infectious SIRS presented higher values for all the factors except for the t-PA and D-dimer with respect to SIRS of other origins, none of these differences reached statistical significance (p>0.05). Our data show that patients with SIRS and associated acute renal failure, irrespective of the origin (infectious or noninfectious), show signs of intense endothelial damage and hypercoagulability throughout the process.

Effect of the administration of recombinant hirudin and/or tissue-plasminogen activator (t-PA) on endotoxin-induced disseminated intravascular coagulation model in rabbits

We evaluated the effect of r‐hirudin and/or tissue‐plasminogen activator (t‐PA) in a model of DIC in rabbits induced by i.v. infusion of 100 μg/kg/h/6 h endotoxin. Rabbits were treated with saline (endotoxin control group), r‐hirudin at 0.3 mg/kg/h/6 h, t‐PA at 0.3 mg/kg for 90 min and r‐hirudin plus t‐PA at the doses described above. The best results were achieved when r‐hirudin and t‐PA were infused together. This treatment reduced the consumption of platelets and protein C and attenuated the increase of PAI‐1 more efficiently than r‐hirudin or t‐PA alone. r‐Hirudin plus t‐PA also resulted in the lowest formation of fibrin deposits in the kidneys. Finally, mortality at 24 h dropped from 70% in the endotoxin control group to 40%, 10% and 0% in the t‐PA, r‐hirudin and r‐hirudin plus t‐PA groups respectively. None of the t‐PA‐infused rabbits which had died by 24 h showed macroscopic signs of haemorrhage. r‐Hirudin alone was better than t‐PA alone, as was shown by fibrin deposits and mortality. We conclude that r‐hirudin and t‐PA given simultaneously were more efficient than either given alone in this model of DIC. Effective thrombin inhibition, which could influence other pathophysiological mechanisms apart from coagulation, together with the improvement in fibrinolysis, would explain these results.