Barry Woodhams

Changes in coagulation and fibrinolysis during pregnancy: Evidence of activation of coagulation preceding spontaneous abortion

In order to monitor physiological changes in coagulation and fibrinolysis that occur during normal pregnancy, blood samples were collected in each trimester of pregnancy from 17 volunteers. Control samples were collected from 12 non-pregnant female volunteers. As pregnancy advanced there was a rise in the basal levels of fibrinopeptide A, cross linked D-dimer fragment and the B beta 15-42 fragment and an increase in the in vitro rate of fibrinopeptide A generation. These results were consistent with an increased activation of coagulation during normal pregnancy, compensated for by a concomitant rise in fibrinolytic activity. In two patients who spontaneously aborted, evidence of uncompensated activation of coagulation could be detected before the manifestation of any clinical signs. In a second pregnancy in one of these patients similar changes were observed, but were reversed by heparin treatment and the pregnancy progressed to full-term delivery of a normal infant.

The simultaneous measurement of total and free protein S by ELISA

An ELISA for the simultaneous measurement of total and free protein S is described. Polyethylene glycol precipitation was used to remove the C4b -binding protein/protein S complex from plasma. This allows free protein S to be measured. The total protein S was measured directly on plasma samples that had been diluted to dissociate the C4b binding protein/protein S complex. The concentration of total and free protein S found in normal subjects (n = 24) was 92.9 (SD = 10) u/dl and 40.3 (SD = 8.8) u/dl respectively. The within assay coefficient of variation was 5.1%. The between assay coefficient of variation was 5.7%. Warfarin therapy caused a reduction in the level of both total and free protein S but this reduction correlated poorly with the INR value.

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.

Clinical evaluation of a new functional test for detection of plasma procoagulant phospholipids.

The objective of this study was to validate a simple factor Xa-based clotting test developed to monitor procoagulant phospholipids (PPLs) and platelet-derived microparticles (PMPs). This assay is easily automated, giving it a major advantage over the more laborious and expensive flow cytometry, electron microscopy and ELISA techniques in general usage at present. The intra-assay and inter-assay variation coefficients were less than 5% at both low and high levels of PPLs. The test is not affected by other clotting factors is assured by the use of a phospholipid-free animal plasma, which provides excess factor V, fibrinogen and prothrombin. This test was evaluated in apparently healthy volunteers and in selected patient groups associated with increased levels of PMPs in the circulation (diabetes mellitus, sickle cell disease, thyroid cancer and patients with multiple trauma). The study showed that XaCT has a high discriminating power for PPLs and that the patient groups have significantly highly increased PPLs activities when compared with healthy volunteers. Although of a preliminary nature, the test has shown that it has the sensitivity for discriminating severity of disease, as it could detect patients in sickle cell crisis and differentiate between type 1 and 2 diabetes. In conclusion, the combination of reliability, reproducibility and easy performance makes the XaCT assay a simple test to screen for PPLs in plasma samples.

Rapid radioimmunoassay for fibrinopeptide a in human plasma

Abstract A rapid method for the assay of fibrinopeptide A (FPA) in human plasma has been developed which allows results to be obtained within 2 1 2 hours of blood collection. Bentonite is used to remove fibrinogen from plasma. The assay is simple and sensitive. The normal range for plasma FPA (0.3 – 1.5 pmols/ml) is similar to that reported using the original method. There was a good correlation between results obtained by the rapid and original methods on samples obtained from patients with raised levels of circulating FPA and/or fibrinogen/fibrin degradation products, although the rapid assay tended to give slightly lower values. FPA levels in sequential samples drawn through 19-gauge ‘Butterfly’ needles become progressively lower in successive samples. A larger than usual discard is therefore needed to accurately assess basal levels. Comparisons between two antisera showed differences in affinity for FPA and cross-reaction with desaminotyrosyl FPA, but such differences are unlikely to cause problems when assaying clinical samples.

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.

The application of polyethylene glycol to radioimmunoassays used in haemostasis

Use of polyethylene glycol 6000 in the second stages of double antibody radioimmunoassays for fibrinopeptide A, B-thromboglobulin and platelet factor 4 facilitates the more rapid separation of free from bound antigen, and allows precipitating antiserum to be used in greater dilution. At a final PEG 6000 concentration of 5%, separation of free from bound antigen was complete within 1 hour, and antisera could be used in dilutions 3-9 times greater than those recommended by the manufacturers. PEG 6000 had a negligible effect on the affinity of first stage antibodies for their respective antigens. Radioimmunoassays using PEG 6000 were sensitive to protein concentration, failure to adjust standards to similar protein concentrations as those of test samples causing artefactually low results.

Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity.

Microparticles are now considered as critical effectors involved in numerous biological processes (coagulation, inflammation, and vascular biology). Microparticle can be measured using a quantitative and descriptive approach by flow cytometry (FCT) or by a functional approach assessing microparticle biological activities by a factor Xa-based clotting assay. FCT can be used to determine the cellular origin of the different microparticles, although there are concerns about the detection limit of this approach. Functional assays measure only the procoagulant activity of isolated microparticles and give no information on the cellular source or the physical properties of the microparticles. The advantage of the functional assays is that the assays use well defined reagents, and they are readily automated with high sensitivity and simplicity. In this study, we analyzed samples from 60 patients with active cancer of different type, 60 patients with a BMI more than 25 kg/m2, and 49 carriers of Factor V Leiden with and without a prior venous thromboembolic episode. The study showed a significant correlation (P < 0.05) between microparticles determined by annexin V-microparticle-based FCT and a procoagulant activity-based clotting assay. This indicates that the procoagulant assay could be used as a routine screening test to screen out the normal samples so that only the abnormal samples need further testing by FCT.

Circulating microparticles in umbilical cord blood in normal pregnancy and pregnancy with preeclampsia

INTRODUCTION Placenta microthrombi being one of the prevalent recurrent histological findings in women with preeclampsia (PE), it is reasonable to think that the study of coagulation alterations in cord blood could be more informative than that observed in maternal blood. The aim of the present study was to measure different subtypes of microparticles (MP) plasma levels in the maternal peripheral blood at labour and in the venous cord blood of pregnant women with PE compared to those in a group of women without PE. MATERIALS AND METHODS Thirty-two pregnant women in labour, 16 with and 16 without PE, were enrolled. Blood samples were collected immediately after delivery from cord blood and from maternal peripheral blood. Total, cellular-derived and tissue factor- bearing MP were analyzed using flow-cytometry. Procoagulant activity of MP was assessed using the STA® Procoag PPL assay. RESULTS Total MP, platelet activated-derived (P-Selectin+), leukocyte-derived and TF+MP were higher in pregnancies complicated by PE as compared with normotensive women (p<0.05). Platelet-derived MP (CD61+) levels were lower in PE than in healthy women and no difference was found in endothelial-derived MP levels between the two groups. The PPL clotting time was significantly shorter in PE compared with controls. When only venous cord blood was analysed, all MP detected were significantly higher in PE than in healthy normotensive women (p<0.05). CONCLUSIONS MP are very likely involved in the hypercoagulable and pro-inflammatory intravascular reactions during PE. Prospective studies in a larger population are needed to define the clinical meaning of MP measurement in the PE setting.