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Coagulation is an interaction between tissue factor (TF) and clotting factors, assembled on a negatively-charged phospholipid surface. Previously it was reported that malignant T-lymphoblastoid cells might have the ability to support procoagulant activity (PCA). In the present study human T-cell lines (CEM-CCRF, Jurkat, Molt-4, A3.01) representing different stages of differentiation were cultured and their basal PCA compared with that of a monocytoid cell line (THP-1). Various phospholipid or TF-dependent coagulation tests were used to investigate this PCA. The effect of TF on the phospholipid-dependent assays was also investigated.;The phospholipid dependent PCA was reported as phospholipid units/mL by comparison with a bovine brain phospholipid standard. There was variation in the procoagulant activity between the cell lines. Using calcium ionophore to stimulate this activity, and annexin A5 and an inhibitory phosphatidylserine antibody (3G4) to inhibit it, along with flow cytometry data, it was concluded that this activity was due to the exposure of anionic phospholipid.;Two pathophysiological processes, apoptosis and lipid peroxidation significantly enhanced the PCA of T-Lymphoblastoid cells. This enhanced PCA was higher than that observed following calcium ionophore treatment, suggesting the increase in activity was not solely due to anionic phospholipid exposure. Whilst annexin A5FITC and 3G4 bound to cells undergoing apoptosis only annexin A5FITC bound to cells exposed to oxidative stress. This implies that apoptosis increases PCA by causing the translocation of oxidised/native phosphatidylserine to the outer membrane, whilst lipid peroxidation appears to increase the PCA possibly due to malondialdehyde-adducts altering the net charge on the cell surface, which allows phospholipids other than phosphatidylserine to participate in thrombin generation. The possible pathophysiological significance of these observations with regard to thromboembolic complications of leukaemia, chemotherapy and atherosclerosis is discussed.

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An active particle tracking microrheology technique has been developed to study the viscoelastic properties of human fibrin and plasma clots. In order to perform microrheology measurements, a magnetic microrheometer device has been adapted and a technique developed, following the procedure of Evans et al., to measure the frequency dependent viscoelastic moduli (G() and G()). This technique has been supported by complementary investigation methodologies, such as protein analysis, turbidity, and multiple microscopy techniques. As a result of this study new insights into the viscoelastic dynamics of fibrin have been revealed. Three stress relaxation mechanisms, as predicted by Morse et al. for networks of semi-flexible fibres, were observed and occur on distinctly different timescales. The scaling of the tension dominated contribution was measured to scale as G ~ c2.7 0.2 in agreement with the prediction of Mackintosh. The presence of FXIII resulted in stiffer less deformable clots but was found to have no effect on the viscoelastic dynamics of clots. Frequency measurements of the loss tangent revealed that on timescales intermediate between stress relaxation modes clots were much more susceptible to permanent deformation. The effect of fibrinogen, thrombin and calcium on the viscoelastic behaviour of clots was also investigated. Increased fibrinogen levels produced clots which displayed predominantly elastic behaviour on shorter time-scales. The molecular mechanism underpinning the role of fibrinogen γ in fibrin clot polymerisation, structure and viscoelasticity was also investigated. We report new data which show that fibrinogen γ is associated with the formation of mechanically weaker, non-uniform clots composed of thinner fibres. This is caused by direct disruption of protofibril formation by and not through thrombin inhibition or binding to FXIII. In addition, the effects of the plasma proteins FVIIa, FIXa, FXIIa and FXIII on clot properties are also reported.

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