OC-03 - Modelisation of the procoagulant properties of cancer cells and their capacity to alter the antithrombotic efficiency of LMWHs and specific inhibitors of factor Xa.

INTRODUCTION: The risk of venous thromboembolism varies according to the histological type of cancer. The failure of antithrombotic treatment is more frequent in cancer patients as compared to non-cancer ones. AIM: We aimed to elucidate the mechanism of activation of blood coagulation induced by cancer, the impact of chemo-resistance phenotype on the capacity of cancer cells to trigger thrombin generation and the alterations of the efficiency of LMWHs and the specific inhibitors of factor Xa (fondaparinux and apixaban) in the presence of cancer cells. MATERIALS AND METHODS: Thrombin generation of human plasma was assessed in the presence of various cancer cell lines. The model of cancer-induced hypercoagulability was coupled to the research for the expression of procoagulant molecules by cancer cells. RESULTS: The pancreatic adenocarcinoma cells BXPC3 and the breast adenocarcinoma cells MCF7 were initially tested. The BXPC3 cells induce significantly higher thrombin generation as compared to the MCF7 cells. In the same line Marchetti et al. showed that malignant hematologic cells (NB4, HEL, and K562) and H69 small cell lung cells express different procoagulant potential on triggering thrombin generation of human plasma. The comparison of the procoagulant activity has been extended to cancer cell lines from various cancers (i.e. colon, ovarian and prostatic cancer) as well as to different cell lines of the same type of cancer. The differences of the cancer cell lines to trigger thrombin generation are mainly due to the expression of TF. The acquisition of chemoresistant phenotype by cancer cells is correlated with increased TF expression and enhancement of theit procoagulant activity. The ability of cancer cells to activate FXII is an alternative pathway of significant importance for some cancer cell lines (i.e. MCF7). Clinically relevant concentrations of LMWH and specific direct and indirect inhibitors of FXa (apixaban and fondaparinux) inhibit thrombin generation induced by cancer cells. The synergy between the anti-Xa and anti-IIa activities of LMWHs rather than the AT-dependent selective inhibition of FXa results in profound inhibition of thrombin generation induced by BXPC3 cells. This experimental model allowed the functional distinction between the two specific FXa inhibitors (apixaban and fondaparinux). CONCLUSIONS: The cancer cell-based model of hypercoagulability is suitable for the identification of the prothrombotic fingerprint of various cancer types. This experimental model allows to perform pharmacological studies for the evaluation of the efficiency of the antithrombotic drugs in cancer-induced hypercoagulability. It is suitable for the study of the impact of anticancer drugs on the procoagulant properties of the cancer cells.

PO-53 - Prospective evaluation of risk assessment models and biological markers of hypercoagulability for the identification of high VTE risk patients with lung adenocarcinoma. The ROADMAP study.

INTRODUCTION: In patients with lung adenocarcinoma (LA), metastasis (MTS), advanced stage and chemotherapy (CTx) are risk factors for thromboembolism (VTE). Routine thromboprophylaxis is not recommended but individualized risk assessment is encouraged. AIM: The selection of the most relevant hypercoagulability biomarkers (HB) for incorporation into the risk assessment models (RAM) for VTE. MATERIALS AND METHODS: Patients with documented LA eligible for CTx at distance of at least 3months from surgery or hospitalization were included. They were either CTx naive (NG) or had received CTx (OTG). Control group (CG) consisted of 30 healthy age & sex-matched individuals. We assessed them for thrombin generation (TG), P-Selectin, heparanase (HPA), procoagulant phospholipids (PPL), factor VIIa, D-Dimers (DDi) and Tissue Factor activity (TFa). RESULTS: Patients showed significantly shortened PPL and higher levels of TFa, DDi and HPA as compared to the CG. FVIIa levels were lower in patients compared to CG. The NG showed significantly shorter lag-time and lower ETP as compared to the OTG. It also showed significantly higher levels of HPA as compared to the OTG.The increase of TG and of HPA, P-Selectin, FVIIa was associated with the stage. Patients with MTS had higher levels of P-Selectin, TFa, DDi, FVIIa, TGT and HPA than those with localized or advanced disease.Patients with VTE had higher baseline levels of DDi, TGT, shorter PPL and lower levels of HPA as compared to those without. Patients who died within 3-months had higher baseline levels of DDi and lower HPA levels as compared to those who were alive. CONCLUSIONS: Increased PPL, TF pathway up regulation, DDi and HPA increase is a universal phenomenon in LA. CTx has an impact on TGT and HPA levels. Baseline values of TGT, PPL, HPA, DDi were related with mortality and thrombosis. The incorporation of HB in VTE-RAMs might improve their predictive value. This concept is being studied on an ongoing trial.

The factor V gene A4070G mutation and the risk of venous thrombosis.

The A4070G polymorphism in exon 13 of the factor V (FV) gene, which replaces His by Arg at position 1299 of the B domain, was recently shown to influence circulating FV levels and to contribute to the activated protein C (APC) resistance phenotype. We examined the impact of this polymorphism in a population of unselected patients with venous thromboembolic disease (VTE). The prevalence of the G4070 (R2) allele was determined in 205 patients and 394 healthy subjects of similar age and sex distribution. Thirty-seven patients (18%) were heterozygous for the R2 allele and 1 (0.5%) was homozygous. Forty-four controls (11.2%) were heterozygous for the R2 allele and 1 (0.2%) was homozygous. Thus, the allelic frequency was significantly higher in the patients with VTE than in the healthy controls, with respective values of 9.5% and 5.8%. The odds ratio was 1.8 (95% CI: 1.1-2.8, p = 0.02), pointing to an increased risk of VTE in carriers of the R2 allele. After excluding subjects with putative or confirmed gene defects (mainly the FV R506Q mutation), the R2 allele was still a risk factor for VTE in the remaining patients, with an odds ratio of 2.0 (95% CI: 1.2-3.5, p = 0.01), demonstrating that this polymorphism is itself a risk factor. This study also confirms that the R2 allele influences APC resistance (APCR) in the absence of the FV R506Q mutation.

Assessment of activated protein C resistance using a new and rapid venom-based test: STA Staclot APC-R.

Activated protein C (APC) resistance is related to a single point mutation in the factor V gene (FV:Q506) and appears to be the most common inherited risk factor for venous thromboembolism. A reliable screening test is therefore useful. We aimed to evaluate a new APC resistance test, on the basis of the procoagulant activity present in one snake venom of a crotalidae family: STA Staclot APC-R. We studied 36 consecutive patients with an acute deep venous thrombosis (DVT) confirmed by compression ultrasonography and carrying the FV:Q506 allele, assessed by DNA analysis, 103 of their family members and 35 consecutive patients with a proven DVT but who did not carry the FV:Q506 allele. Blood samples were collected within 24 h of admission for the DVT cases and on the day of medical registration for the family members. Tests were performed blind. The STA Staclot APC-R test, using a cut-off value of 0.80, had an overall sensitivity of 100% (95% CI, 95-100) and a specificity of 98.8% (95% CI, 92.0-99.6). An acute thrombosis process did not influence the performance of the test. We conclude that this test is easy and rapid to perform in every day practice and fulfills the criteria for a screening test.

Problems of oral anticoagulation in an adult with homozygous protein C deficiency and late onset of thrombosis.

We describe a 57-year-old woman with homozygous protein C deficiency and mild thrombotic manifestations consisting of three spontaneous distal deep vein thromboses occurring after the age of 45. Previous surgery and pregnancies had been uneventful. Low but detectable protein C antigen and activity levels (both 20%) were discovered on the occasion of skin necrosis induced by oral anticoagulation. This therapy was interrupted because of skin necrosis and several episodes of disseminated intravascular coagulation (DIC) at the initiation of treatment despite a cautious protocol. No recurrent thromboembolic event has occurred in our patient using prophylactic doses of low molecular weight heparin for 24 months. New therapeutic approaches might be the administration of low molecular weight heparin or oral anticoagulation associated with protein C replacement in the induction period. This case reflects the variability of expression of protein C deficiency as well as the potential hazards of antivitamin K anticoagulation in this disorder.

Protein composition of follicular fluid and oocyte cleavage occurrence in in vitro fertilization (IVF).

The present study was carried out from in vitro fertilization (IVF) attempts to analyze further the total and specific protein contents of 47 follicular fluids yielding one oocyte. The aim was to find correlations between the follicular concentrations of such proteins and the occurrence of coupled oocyte cleavage. These findings would be used as markers of IVF outcome. Two groups of follicular samples were distinguished: one group with cleavage occurrence (25 cases) and another group without cleavage or even fertilization (22 cases). In the group with cleavage a significantly higher level was observed for six proteins: C3 complement fraction and ceruleoplasmin (P less than 0.02), alpha-antitrypsin and transferrin (P less than 0.01), and alpha 2-macroglobulin and beta 2-microglobulin (P less than 0.001). The data are discussed with respect to changes in follicle permeability with advancing maturity.

Human seminal fibrinolytic activity: specific determinations of tissue plasminogen activator and urokinase.

The determination of total fibrinolytic activity of ejaculates was realized by fibrin plate method. For the same seminal samples, tissue plasminogen activator (t-PA), urinary plasminogen activator (uPA) antigens and uPA activity were specifically quantified. The seminal values were fifty times higher than in the blood for t-PA and fifteen times for uPA. There was no correlation between the both levels but from split ejaculates measurements a higher concentration was observed in all first fractions. By zymography assays, it was shown that seminal plasminogen activators are under active forms. The lack of proUrokinase in semen was also demonstrated.

Fructose and proteins in human semen.

The aim of this study was to correlate human seminal fructose determinations by enzymatic UV method with protein content, using three different methods (Biuret, Lowry, Meulemans). On one hand, the incidence on fructose levels of partial deproteinization after treatment by perchloric acid was tested: the fructose levels values increased after deproteinization 14.1 +/- 3.7 versus 4.2 +/- 1.7 mMol/l. On the other hand, physiologically, semen proteolysis occurs for many hours after collection and concerns mainly low molecular weight proteins. No modifications were found in protid levels and fructose concentrations during the first four hours, at 20 degrees C. All these data seem to indicate a better linkage of fructose to the highest molecular weight proteins and might explain the absence of fructose rise during the first four hours.

Age-related changes in factor VII activation in healthy women.

1. To investigate the age-related changes in factor VII activation in healthy women, a regular factor VII clotting assay (factor VIIc) was carried out simultaneously with a new enzyme immunoassay for the quantification of antigen factor VII (factor VIIag). 2. Both factor VIIc and factor VIIag levels were positively correlated with age (r = 0.79 and r = 0.62, respectively, n = 25, P less than 0.001). The rise with age in factor VIIc was steeper than in factor VIIag and the ratio of factor VIIc to factor VIIag (an indicator of the activity state of factor VII) increased with age (r = 0.42, P less than 0.05). 3. The results show that an increased conversion of native single-chain factor VII to the fully active double-chain form is associated with advancing age in women. 4. This finding is consistent with a possible role of activated factor VII in the pathogenesis of thrombo-occlusive vascular disease in women.

Determination of tissue plasminogen activator (t-PA) antigen in seminal plasma by modified enzyme-linked immunosorbant assay (ELISA).

The determination of tissue plasminogen activator (t-PA) antigen in semen by a new immunoenzymatic method was done in 152 men without genito urinary pathology. The seminal value is one hundred higher than the blood value with a log normal distribution. These levels are not influenced by freezing temperature, by the sequences freezing-thawing, by centrifugation. From split ejaculates a correlation has been established between t-PA antigen and zinc and not with fructose. These results indicate the major prostatic origin of the t-PA antigen.