Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

This study reports on the frozen stability of all commonly measured coagulation proteins in normal citrated plasma: activated partial thromboplastin time, prothrombin time (%), thrombin time and fibrinogen (Clauss); clotting assays for factors II, V, VII, VIII, IX, X, XI and XII; functional assays for protein C (clotting), protein S (clotting), antithrombin (chromogenic) and plasminogen (chromogenic); and immunological assays for von Willebrand factor and D-dimer. All these factors listed are stable for up to 3 months if frozen at -24 degrees C or lower. At -74 degrees C, all these factors (allowing for 10% variation) were stable for at least 18 months, most were stable for 24 months. The number of proteins showing > 5% variation over baseline after 6 months storage indicates that some decay does occur even at -74 degrees C. There was no clear advantage in snap freezing at -74 degrees C and then storing at -24 degrees C over both freezing and storing at -24 degrees C; therefore, the freezing process itself is not responsible for the loss of stability. The best stability, especially at -24 degrees C, was obtained when small samples (1 ml) were stored in screw-cap tubes with a minimum dead space. The decrease in stability of the coagulation proteins directly correlates with the effect of temperature and time.

SummaryThe monoclonal antibody RFF-VIII:R/1 recognises an epitope on von Willebrand factor involved in its interaction with GPIbα. A two-site, solid phase ELISA has been established using RFF-VIII:R/1 as the solid-phase, capture antibody and an enzyme-conjugated, polyclonal antibody to human VWF, which provides an assay for VWF functional activity with a detection limit of 0.5 U/dl VWF and an interassay %CV<10. Plasma from 192 VWD patients (48 studied retrospectively; 144 prospectively) showed VWF levels of <50 U/dl in type 1 patients (n = 156), <25 U/dl in type 2A (n = 26) and <35 U/dl in type 2B (n = 8) which, in type 1 and 2A patients, correlated with RiCoF activity (r >0.82). In plasma from patients with type 1 VWD values of VWF in the Mab-based ELISA were similar to levels of VWF:Ag measured in a polyclonal antibody-based ELISA (r >0.87) but were significantly lower than VWF:Ag in type 2A and 2B plasmas (p <0.0005), allowing discrimination of variant VWD. The Mab-based ELISA has advantages of sensitivity and reproducibility over the RiCoF assay to measure VWF activity and can be used to analyse stored samples. In conjunction with an ELISA for VWF:Ag and VWF multimer analysis, it provides a reliable method, for the laboratory diagnosis of VWD.

The endothelial protein C receptor (EPCR) facilitates protein C activation and plays a protective role in the response to Escherichia coli-mediated sepsis in primates. Previously, a soluble form of EPCR (sEPCR) in human plasma was characterized, and several studies indicated that generation of sEPCR is regulated by inflammatory mediators, including thrombin-mediated up-regulation of surface metalloproteolytic activity in vitro. This study addressed the question of whether plasma sEPCR levels reflect changes in thrombin generation in patients undergoing anticoagulant treatment. The sEPCR levels in patients treated with coumarintype oral anticoagulants were significantly lower than those in healthy asymptomatic adult volunteers (105.3 ؎ 70.8 ng/mL [n ‫؍‬ 55] versus 165.8 ؎ 115.8 ng/mL [n ‫؍‬ 200]; P < .0001). A similar decline in plasma sEPCR levels was found in patients treated with unfractionated heparin. In healthy volunteers, sEPCR levels declined to about 100 ng/mL within 3 days after initiation of an 8-day period of warfarin administration and increased within 2 days after its cessation. Plasma sEPCR levels returned to pretreatment values within 1 week, and the changes in plasma sEPCR levels mirrored changes in values for international normalized ratios. A similar decline in sEPCR levels with time was observed in 7 patients beginning treatment with warfarin for a thrombotic disorder. Prothrombin fragment 1 ؉ 2 levels also decreased in volunteers and patients given warfarin. These results show that plasma sEPCR levels decline in response to treatment with anticoagulants whose mechanism of action is known to decrease in vivo thrombin production. (Blood. 2002;99:526-530)

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.

Many subjects carrying inherited thrombophilic defects will never experience venous thromboembolism (VTE) while other individuals developed recurrent VTE with no known additional risk factors. High levels of circulating microparticles (MP) have been associated with increased risk of VTE in patients with factor V Leiden and prothrombin G20210A mutation, suggesting a possible contribution of MP in the hypercoagulability of mild genetic thrombophilia. The role of MP as additional risk factor of VTE in carriers of natural clotting inhibitors defects (severe thrombophilia) has never been assessed. Plasma levels of annexin V-MP, endothelial-derived MP (EMP), platelet-derived MP (PMP), tissue factor-bearing MP (TF+) and the MP procoagulant activity (PPL) were measured in 132 carriers of natural anticoagulant deficiencies (25 antithrombin, 63 protein C and 64 protein S defect) and in 132 age and gender-matched healthy controls. Carriers of natural anticoagulant deficiencies, overall and separately considered, presented with higher median levels of annexin V-MP, EMP, PMP, TF+MP and PPL activity than healthy controls (p< 0.001, < 0.001, < 0.01, 0.025 and 0.03, respectively). Symptomatic carriers with a previous episode of VTE had significantly higher median levels of annexin-V MP than those without VTE (p=0.027). Carriers with high levels of annexin V-MP, EMP and PMP had an adjusted OR for VTE of 3.36 (95% CI, 1.59 to 7.11), 9.26 (95% CI, 3.55 to 24.1) and 2.72 (95%CI, 1.16 to 6.38), respectively. Elevated levels of circulating MP can play a role in carriers of mild and severe inherited thrombophilia. The clinical implications of this association remain to be defined.

SummarySickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

This study evaluates the functional procoagulant features of plasma MP in order to explore the MP contribution to the hypercoagulable state of patients with Essential Thrombocythemia (ET). Platelet-free plasma samples were obtained from 73 ET patients (37 positive for the JAK2V617F mutation) and 72 control subjects. The calibrated automated thrombogram (CAT) was performed in plasma samples to determine thrombin generation of MP-associated tissue factor (TF) and procoagulant phospholipid (PPL) activity, and the STA Procoag PPL assay to measure MP-PPL activity only. Both thrombin generation and PPL procoagulant activities were found significantly elevated in ET patients compared to controls, and were associated to significantly higher levels of TF antigen and FVIIa/AT complex. Thrombin generation was significantly greater in JAK2-V617F positive compared to JAK2-V617F negative patients and normal subjects. Significant correlations were found between the PPL-assay and the different parameters of the CAT assay. No difference was seen between the thrombosis and no thrombosis group. Prospective studies are needed to test whether MP-associated thrombin generation and procoagulant activity may predict for thrombosis in these patients.