Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal haematopoietic stem cell disorder characterised by haemolytic anaemia, thrombosis and bone marrow failure. Thrombosis is considered a major cause for high morbidity and mortality in PNH patients (Hillmen P et al N Eng J Med 1995). However, the underlying mechanisms of thrombosis in PNH are still poorly understood. Various factors including defective interactions between the complement system, platelets and coagulation systems have been proposed (Peacock-Young B et al Haematologica 2017). Platelet function and clot formation in this disorder have not been comprehensively evaluated. Here we describe standard and novel techniques to evaluate platelet function in blood from PNH patients, to elucidate the underlying pro-thrombotic mechanisms.


Whole blood was collected from five PNH patients and compared to same-day healthy donor (HD) controls. The samples were collected at trough for those patients who were on eculizumab (Complement C5 inhibitor used in the treatment of PNH). Surface levels of platelet adhesion proteins and activation markers P-selectin and phosphatidylserine (PS) were assessed using flow cytometry. Plasma soluble GPVI (a platelet-specific activation marker) and cytokine levels were measured by ELISA. Clot formation was assessed by viscoelastic testing (ROTEM). Adhesion of platelets to collagen under flow in a whole blood assay was evaluated by Digital Holographic Microscopy (DHM) and thrombus height, surface area and volume quantified using custom-built MatLab-based software.


Clinical characteristics of PNH patients were highly variable: two patients had history of thrombosis, three patients were on eculizumab, four patients were thrombocytopenic (<150x109/L) and three were haemolysing. Platelet surface levels of adhesion/signalling receptor proteins including glycoproteins (GP) Iba, GPVI, aIIbb3, and ADAM10 (membrane expressed enzyme responsible for shedding GPVI from the surface) were all at lower ends of HD ranges. P-selectin and PS levels under resting and activated conditions and plasma soluble GPVI levels were comparable to same day HD. There were no differences between HD and PNH groups for levels of interleukin (IL) -6, IL-1β, tumour necrosis factor α, IL-17A, interferon γ or monocyte chemoattractant protein-1 (MCP-1). ROTEM analysis revealed slower formation of smaller clots in PNH patients, which correlated with their platelet count. Peak thrombus height and volume analysed by DHM were not different from data obtained with HD blood at both venous and arterial shear rates. However, both parameters were increased in PNH samples at arterial shearrate, when adjusted for platelet count (p<0.01).


Analysis of these PNH patients with highly variable clinical characteristics did not identify a unifying platelet lesion. DHM could detect and quantify parameters of small thrombi in real time, in PNH patient samples and these data were consistent with enhanced thrombogenic potential in PNH patients. Mechanisms beyond platelet activation that contribute to increased thrombosis in these patients and the impact of eculizumab therapy on thrombotic propensity need to be explored further.


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Author notes


Asterisk with author names denotes non-ASH members.

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