Development of platelet function analysis for use in haematological and clinical investigations

Platelets are highly specialised cells that play a pivotal role in the regulation of haemostasis and thrombosis. Accurate measurement of platelet function is important in identifying patients with platelet abnormalities: for example, platelet hyperfunction, which may result in hyperthrombotic risk, or platelet hypofunction, which may lead to enhanced bleeding. Also, accurate measurement is becoming crucial for assessing the adequacy of treatment with antiplatelet therapy. Platelet function testing to assess the efficacy of antiplatelet drugs is becoming widely used and a range of assays has been developed. However, assays such as Light Transmittance Aggregometry (LTA), VerifyNow and Multiplate Electrode Aggregometry (MEA) are cumbersome for regular clinical use and have many limitations. Only a limited number of assays offer the advantage of assessing multiple platelet activation pathways simultaneously. In this thesis, I describe the development of a 96-well plate-based assay carried out in whole blood, where flow cytometry is used concomitantly to assess platelet aggregation (measured as the decrease in number of single platelets in the blood) together with platelet leucocyte conjugate (PLCs) formation, using lysing and non-lysing conditions (fluorescence triggering) – both measurements performed on the same fixed sample of whole blood. First, this thesis evaluates the effect of blood volume and different fixation approaches, double fixation flow cytometry (DFF) and single fixation Ultra-Flo 100 (SFU), on measuring platelet aggregation in whole blood. The smallest blood volume that was appropriate to study platelet aggregation was 125µl. Both fixation methods were shown to be highly comparable. The preliminary results revealed the suitability of using the 96-well plate format to evaluate the platelet response to a range of different platelet agonists. The second part of this thesis explores the suitability of the 96-well plate format to study platelet aggregation, and to assess inhibition, using aspirin and the P2Y12 antagonist, cangrelor. The 96-well plate format has successfully demonstrated dose-dependent inhibition of adenosine diphosphate (ADP)-induced platelet aggregation with cangrelor and of arachidonic acid (AA)-induced platelet aggregation with aspirin, except when using high concentrations of AA. The apparent failure of aspirin to inhibit AA-induced platelet aggregation at a high concentration could have been due to the fact that endogenous ADP, which may have leaked from red blood cells (RBCs), may have overcome the inhibition. Dual antiplatelet therapy, using aspirin in conjunction with cangrelor, has confirmed this explanation and also demonstrated that more inhibition is obtained when antiplatelet agents are used in pairs. When the glycoprotein (GP) IIb/IIIa antagonist MK-0852 was used to block the final aggregation pathway, it failed to achieve a complete inhibition of platelet aggregation, when measured using the single platelet counting technique. This could be due to the binding of platelets to leucocytes, as demonstrated previously by our group. To investigate whether it was PLCs formation that was responsible for the decrease in the number of single platelets in the presence of MK-852, platelet leucocyte interaction was studied using lysing and non-lysing approaches, based on a 96-well plate format. The findings demonstrated that PLCs formation in the presence of MK-0852 tends to increase, especially platelet-monocyte conjugate formation. As expected, there was inhibition of PLCs formation with KPL-1, an agent that blocks P-selectin glycoprotein ligand-1 (PSGL-1) on leucocytes, to which P-selectin binds. In this regard, thrombocytopenia, which sometimes occurs after administration of a GPIIb/IIIa antagonist, and the accompanied increase in mortality, could be thereby explained and also implies a potential limitation to the use of GPIIb/IIIa antagonists. The final part of this thesis focused on the possibility of studying more than one platelet function and the effect of antiplatelet therapy. PLC formation can be measured using fluorescence triggering to capture the leucocyte population, instead of lysing RBCs, to avoid manipulation of the cells. The results have indicated that the use of more than one antiplatelet agent can achieve more inhibition by blocking more activation pathways than a single antiplatelet agent. In conclusion, 96-well plate methods have shown the advantage of assessing multiple platelet activation pathways using a small volume of whole blood. This method has the advantage of using only one sample of fixed whole blood to assess both aggregation and PLCs formation. Moreover, development of this assay can also be useful to testing the effects of new compounds on platelet function