PB0295 - A Novel Assay to Investigate Thrombogenesis in TTP Patients

Background: Thrombotic thrombocytopenic purpura (TTP) patients with sustained low ADAMTS13 activity could benefit from a new assay to better monitor risk of relapse.

Aims: To develop a novel dynamic assay that better resembles the conditions in TTP, for improved monitoring of TTP patients at risk of relapse.

Methods: Microfluidic channels were coated with collagen or an antibody against VWF-A3 domain (82D6A3). Blood was collected in citrate or lithium heparin from healthy volunteers, congenital cTTP, or immune iTTP patients with normal platelet counts. Informed consent and ethical approval were obtained. Fluorescently-labelled blood was perfused at a shear of 1800s-1. Platelet binding was recorded in real-time and analysed using software developed in-house.

Results: Twenty-one patient samples (13 cTTP and 8 iTTP) and ten controls were analysed. Time-dependent platelet capture was observed in all conditions, forming extensive thrombi on collagen, and small aggregates on anti-VWF A3 antibody. After 3 minutes of flow, platelet surface coverage on anti-VWF A3 antibody was significantly increased in TTP samples with low ADAMTS13 activity (Fig.1). On collagen, thrombi adopted different geometries, with elongated thrombi in patient samples with low ADAMTS13 activity as indicated by FRETS analysis, and compact thrombi in controls (Fig.2). These differences were most evident in heparin samples and indicate the presence of high ultra-large VWF levels. Patients receiving Caplacizumab exhibited no platelet binding on either collagen or anti-VWF A3 antibody.

Conclusion(s): We present a new method to investigate VWF-dependent platelet recruitment under flow using an antibody against VWF-A3 domain to capture plasma VWF. This removes platelet activation by collagen, being more representative for TTP. The assay discriminates between TTP samples and controls, exhibiting significantly higher platelet coverage in patient samples anticoagulated with either citrate or heparin. This assay could represent a new rapid and sensitive diagnostic tool to monitor TTP patients, and a new research tool to study thrombogenesis.