Elena Campello

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism.

BACKGROUND Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited. OBJECTIVES To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF(+)MPs) in plasma may contribute to cancer-associated thrombosis. PATIENTS/METHODS EMPs, PMPs and TF(+)MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500). RESULTS Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/μL, respectively) than controls (299 ± 102 and 495 ± 241 MP/μL; p<0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF(+)MPs (927 ± 415 MPs/μL) than controls (204 ± 112 MPs/μL; p<0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF(+)MPs plasma levels (1019 ± 656 MPs/μL) than cancer patients without VTE (755 ± 391 MPs/μL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF(+)MPs and VTE in cancer patients. CONCLUSIONS Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.

Extracellular vesicles, tissue factor, cancer and thrombosis – discussion themes of the ISEV 2014 Educational Day

Although the association between cancer and venous thromboembolism (VTE) has long been known, the mechanisms are poorly understood. Circulating tissue factor–bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV) and International Society on Thrombosis and Haemostasis (ISTH) held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Hypercoagulability in overweight and obese subjects who are asymptomatic for thrombotic events

The role of circulating microparticles (MP) of different origin and tissue factor (TF)-bearing in overweight and obese patients with and without metabolic syndrome is still a matter of debate. In a case-control study, the presence of hypercoagulability was evaluated in overweight and obese patients by measuring MP, thrombin generation (TG) and FVIIa-AT complexes. Twenty overweight patients (body mass index [BMI] range 25-29.9 kg/m²), 20 with I degree (30-34.9 kg/m²), 20 with II degree (35-39.9 kg/m²) and 20 with III degree obesity (≥ 40 kg/m²) were enrolled and compared to 40 age and gender-matched normal weight individuals. A significant increase in median levels of all MP subtypes was observed in the three degrees of obese patients compared to controls. Overweight patients had higher levels of annexin V-MP (AMP), endothelial-derived, leukocyte-derived and TF-bearing MP than controls. Obese patients had a significantly shorter median lag time (p< 0.05), higher median peak thrombin (p< 0.01) and increased median endogenous thrombin potential [ETP] (p< 0.001) compared to controls. Overweight subjects had significantly increased ETP compared to controls (p< 0.05). Both AMP levels and ETP were found to positively correlate with BMI, waist circumference, and inflammatory parameters. No significant increase in FVIIa-AT complex was seen in cases compared to controls. We conclude that obesity is associated with overproduction of procoagulant MP and increase TG. Interestingly, hypercoagulability is found in overweight patients free of metabolic syndrome and increases with the severity of obesity. Assessment of MP and TG may be helpful in the early characterisation of the prothrombotic state in obese patients.

Factor VIIa-antithrombin complexes in patients with arterial and venous thrombosis

Antithrombin (AT), in the presence of heparin, is able to inhibit the catalytic activity of factor VIIa bound to tissue factor (TF) on cell surfaces. The clinical meaning of FVIIa-AT complexes plasma levels is unknown. It was the objective of this study to evaluate FVIIa-AT complexes in subjects with thrombosis. Factor VIIa-AT complexes plasma levels in 154 patients consecutively referred to our Department with arterial or venous thrombosis and in a group of 154 healthy subjects, were measured. Moreover, FVIIa-AT complexes were determined in: i) n = 53 subjects belonging to 10 families with inherited factor VII deficiency; ii) n = 58 subjects belonging to seven families with AT deficiency; iii) n = 49 patients undergoing oral anticoagulant therapy (OAT). Factor VIIa-AT levels were determined by a specific ELISA kit (R&D, Diagnostica Stago, Gennevilliers, France). Factor VIIa-AT complexes mean plasma levels were lower in patients with either acute arterial (136 +/- 40 pM) or venous (142 +/- 53 pM) thrombosis than subjects with previous thrombosis (arterial 164 +/- 33 pM and venous 172 +/- 61 pM, respectively) and than healthy controls (156 +/- 63 pM). Differences between acute and previous thrombosis, were statistically significant (p < 0.05). Subjects with inherited and acquired (under OAT) factor VII deficiency had statistically significant lower FVIIa-AT complexes plasma levels (80 +/- 23 pM and 55 +/- 22 pM, respectively) than controls (150 +/- 51 pM, p < 0.0001 and 156 +/- 63 pM, p < 0.00001, respectively). Factor VIIa-AT complexes are positively correlated with plasma factor VII/VIIa levels. Further investigations are needed to verify the possible role of higher FVIIa-AT complex plasma levels in predicting hypercoagulable states and thrombosis.

New Prothrombin Mutation (Arg596Trp, Prothrombin Padua 2) Associated With Venous Thromboembolism

Objective—Two different prothrombin variants, p.Arg596Leu and p.Arg596Gln, conferring antithrombin resistance to patients with venous thromboembolism have been recently reported. Here, we describe a novel substitution affecting Arg596 of prothrombin molecule (Arginine596 to Tryptophan or p.Arg596Trp or Arg221aTrp in the chymotrypsinogen numbering system or prothrombin Padua 2) in 2 Italian families with venous thromboembolism. Approach and Results—Prothrombin Padua 2 has been characterized either in plasma of carriers or using Arg596Trp recombinant prothrombin. Routine coagulation tests, thrombin generation, and antithrombin resistance tests were performed, as well as measurement of the levels of thrombin–antithrombin complexes. All carriers were heterozygotes and presented with a mild reduction of the prothrombin activity. Thrombin generation in carriers showed only a markedly prolonged decay. This finding was confirmed in plasma reconstituted with Arg596Trp recombinant prothrombin mimicking a homozygous condition, which showed longer decay and higher endogenous thrombin potential in thrombin generation than wild-type recombinant prothrombin reconstituted plasma. Patient’s plasma as well as Arg596Trp recombinant prothrombin showed a clear thrombin resistance to antithrombin inactivation. These findings were supported by the assessment of thrombin–antithrombin complexes formation, which was strongly reduced for Arg596Trp recombinant prothrombin as compared with wild-type recombinant prothrombin. In a series of 400 unrelated consecutive patients with venous thromboembolism, 2 carriers of prothrombin Padua 2 were found (estimated prevalence of 0.5%). Conclusions—Our study showed that prothrombin Padua 2 induces antithrombin resistance and is associated with an increased risk of venous thromboembolism. Codon 596 (CGG) of prothrombin is a hot spot for mutations, which constitute a new and relatively frequent cause of inherited thrombophilia.

The clinical performance of a chemiluminescent immunoassay in detecting anti-cardiolipin and anti-β2 glycoprotein I antibodies. A comparison with a homemade ELISA method

Abstract Background: Fully automated chemiluminescence immunoassays (CLIAs) are emerging technologies for the detection of anti-cardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies for anti-phospholipid syndrome (APS) classification, which is commonly based on an enzyme-linked immunosorbent assay (ELISA) test result. CLIA and a homemade ELISA were used in this study to detect these antibodies, and their performances were compared. Methods: Sera were collected from 104 patients with primary APS, 88 seronegative subjects who met the clinical but not the laboratory criteria for APS, and 150 control subjects. IgG/IgM aCL and IgG/IgM anti-β2GPI antibodies were determined in the sera using a CLIA (HemosIL AcuStar®) and a homemade ELISA. Results: CLIA had a significantly lower comparative sensitivity for IgM aCL and IgG/IgM IgG anti-β2GPI antibodies; its comparative specificity was higher with respect to ELISA for IgM aCL and IgM anti-β2GPI antibodies. The two techniques showed a high, significant agreement (p<0.001) and a significant titer correlation (p<0.001). CLIA also detected IgG/IgM aCL and IgG anti-β2GPI antibodies in the seronegative patients. There was a significantly higher prevalence of IgG aCL and IgG anti-β2GPI antibodies (p<0.001 and p=0.01, respectively) in those patients with respect to that in the control population. Conclusions: Despite a lower comparative sensitivity, CLIA showed a higher comparative specificity for some aPL and a good level of agreement and correlation with a homemade ELISA. CLIA also detected some aCL and anti-β2GPI antibodies in the seronegative patients not usually identified by homemade ELISA.

Hypercoagulability detected by whole blood thromboelastometry (ROTEM®) and impedance aggregometry (MULTIPLATE®) in obese patients

INTRODUCTION Obesity has been associated with hypercoagulability and to increased risk of both arterial and venous thromboembolic events. Many different and complex changes in plasma coagulation factors have been described in patients with obesity. The aim of this case-control study is to evaluate hypercoagulability in a group of overweight and obese subjects by whole blood rotation thromboelastometry (ROTEM®) and impedance aggregometry (Multiplate®). METHODS ROTEM® and Multiplate® analyses were performed in 80 subjects with a BMI ≥ 25 Kg/m(2), of whom 20 overweight [BMI = 25-29.9 Kg/m(2)], 20 with I degree obesity [BMI = 30-34.9 Kg/m(2)], 20 with II degree obesity [BMI = 35-39.9 Kg/m(2)] and 20 with III degree [BMI > 40 Kg/m(2)] and compared with 80 age and gender-matched normal weight healthy individuals. RESULTS Thromboelastometry. In INTEM and EXTEM tests MCF and AUC were significantly increased in III degree obese compared with controls. MCF in FIBTEM was significantly higher in I, II and III degree obesity than controls (p = 0.027, 0.002 and < 0.001, respectively). Impedance aggregometry. A significant difference in platelet aggregation was found between III degree obese subjects and healthy controls in each of the tests considered. A significant correlation between FIBTEM-MCF and aggregometry parameters with BMI, waist circumference, leptin levels and high sensitive-C reactive proteins was also found. CONCLUSIONS A relationship between hypercoagulability detected by whole blood thromboelastometry and aggregometry and increased fat mass is shown. Hypercoagulability also correlated with inflammatory markers. Point-of-care tests can be used to assess the degree of hypercoagulability and hyperaggregability in obese patients. Wider studies are needed to confirm our observations.

Optimal duration of anticoagulation Provoked versus unprovoked VTE and role of adjunctive thrombophilia and imaging tests

Once anticoagulation is stopped, the risk of recurrent venous thromboembolism (VTE) over years after a first episode is consistently around 30%. This risk is higher in patients with unprovoked than in those with (transient) provoked VTE, and among the latter in patients with medical than in those with surgical risk factors. Baseline parameters that have been found to be related to the risk of recurrent VTE are the proximal location of deep-vein thrombosis, obesity, old age, male sex and non-0 blood group, whereas the role of inherited thrombophilia is controversial. The persistence of residual vein thrombosis at ultrasound assessment has consistently been shown to increase the risk, as do persistently high values of D-dimer and the early development of the post-thrombotic syndrome. Although the latest international guidelines suggest indefinite anticoagulation for most patients with the first episode of unprovoked VTE, strategies that incorporate the assessment of residual vein thrombosis and D-dimer have the potential to identify subjects in whom anticoagulation can be safely discontinued. Moreover, new opportunities are offered by a few emerging anti-Xa and anti-IIa oral compounds, which are likely to induce fewer haemorrhagic complications than vitamin K antagonists while preserving the same effectiveness; and by low-dose aspirin, which has the potential to prevent the occurrence of both venous and arterial thrombotic events.

ABO blood groups and the risk of venous thrombosis in patients with inherited thrombophilia.

BACKGROUND Although having a non-O blood type is now regarded as a risk factor for venous thromboembolism, the strength of this association is poorly defined, as is its interaction with inherited thrombophilia. MATERIALS AND METHODS The prevalence of non-O blood group and inherited thrombophilia (deficiencies of natural anticoagulants, factor V Leiden and prothrombin G20210A mutation) was assessed in a series of 712 consecutive patients with proximal deep vein thrombosis of the lower limbs who were referred to our Institution between 2004 and 2010, and in 712 age- and gender-matched healthy volunteers. Odds ratios (OR) of deep vein thrombosis and their 95% confidence intervals (CI) were computed for non-O group and thrombophilia, both separately and in combination. RESULTS A non-O blood group was present in 492 cases and 358 controls (OR 2.21; 95% CI, 1.78 to 2.75). A thrombophilic abnormality was present in 237 cases and 105 controls (OR 2.82; 2.18 to 3.66). The combination of non-O group and thrombophilia was present in 152 cases and 51 controls (OR 7.06; 4.85 to 10.28). DISCUSSION Having a non-O blood group is associated with an increased risk of proximal deep vein thrombosis of the lower limbs with or without pulmonary embolism. The addition of inherited thrombophilia increases the thrombotic risk conferred by non-O group alone by almost 3-fold.

Circulating microparticles in carriers of factor V Leiden with and without a history of venous thrombosis

Although factor V Leiden (FVL) is a major determinant of thrombotic risk, the reason why less than 10% of carriers eventually develop venous thromboembolic (VTE) events is unknown. Recent observations suggest that circulating levels of microparticles (MP) may contribute to the thrombogenic profile of FVL carriers. We measured the plasma level of annexin V-MP (AMP) platelet-MP (PMP), endothelial-MP (EMP), leukocyte-MP (LMP) and tissue factor-bearing MP (TF(+)MP), and the MP procoagulant activity (PPL) in 142 carriers of FVL (of these 30 homozygous and 49 with prior VTE), and in 142 age and gender-matched healthy individuals. The mean (± SD) level of AMP was 2,802 ± 853 MP/μl in carriers and 1,682 ± 897 in controls (p<0.0001). A statistically significant difference between homozygous and heterozygous carriers of FVL was seen in the level of PMP, EMP and LMP, but not in that of the remaining parameters. When the analysis was confined to carriers with and without a VTE history, the mean level of AMP was 3,110 ± 791 MP/μl in the former, and 2,615 ± 839 MP/μl in the latter (p<0.005). The mean level of all subtypes of circulating MP showed a similar pattern. The PPL clotting time was 39 ± 9 seconds (sec) in carriers, and 52 ± 15 sec in controls (p=0.003); and was 35 ± 8 sec in carriers with prior thrombosis, and 41 ± 10 sec in thrombosis-free carriers (p<0.005). Our study results suggest that circulating MP may contribute to the development of thrombosis in carriers of FVL mutation.