The platelet function defect of paroxysmal nocturnal haemoglobinuria

Abstract

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired stem cell disorder, characterised by an abnormal susceptibility of red blood cells to complement induced lysis, resulting in repeated episodes of intravascular haemolysis and haemoglobinuria, thromboembolic events at atypical locations and, to a much lesser extent, bleeding complications. Platelet function is assumed to be abnormal, however, a defect has not yet been characterised and underlying mechanisms remain elusive. To explore these issues, we investigated platelet function in PNH patients using assays for clot formation under low and high shear force (thrombelastography and PFA100 device), adhesion to glass beads in native whole blood (Hellem method), aggregometry using various agonists (Born method), and flow cytometric assays for baseline and agonist-induced surface expression density of alpha-granule (CD62P) and lysosomal granule proteins (CD63), ligand binding to surface receptors (thrombospondin), and expression density of activation-induced neoepitopes of the fibrinogen receptor complex (PAC-1). Platelet PNH clone size determined by CD55 and CD59 labelling was compared to the clone sizes of granulocytes, monocytes, erythrocytes, and reticulocytes. A profound reduction of platelet reactivity was observed in PNH patients for all "global function" assays (clot formation, adhesion, aggregation). Platelet hyporeactivity was confirmed using flow cytometric assays. Whereas baseline levels of flow cytometrically determined platelet activation markers did not differ significantly between controls and PNH patients, agonist-induced values of all markers were distinctly reduced in the PNH group. Moreover, significantly reduced white blood cell counts (3.1/nl vs. 5.9/nl), haemoglobin values (9.5 vs. 14.3/g per dl), and platelet counts (136 vs. 219/nl) delineate profound tricytopenia in PNH patients. The fraction of particular cell types lacking the surface expression of GPI-anchored glycoproteins is referred to as the respective PNH clone; median PNH clone sizes of cells with short life spans (reticulocytes, platelets, granulocytes) was 50-80% of total cell populations compared to 20% of red blood cells. The results of our laboratory investigations show, that in PNH, reduced platelet counts coincide with reduced platelet reactivity. The foremost clinical complication in PNH, however, is venous thromboembolism, very probably induced by an activated and dysregulated plasmatic coagulation system. From these seemingly contradictory findings we infer, that part of the platelet hyporeactivity is probably due to reactive downregulation of platelet function in response to chronic hyperstimulation. The overall result is thought to be an unsteady balance, associated with thromboembolism in a larger proportion of patients, and with bleeding in a smaller proportion.

PIGO deficiency: palmoplantar keratoderma and novel mutations

“…PNH is an acquired stem cell disorder due to somatic variants in PIGA and causes an abnormal susceptibility of erythrocytes to complement induced lysis, resulting in episodes of intravascular haemolysis, haemoglobinuria and both thromboembolic events and bleeding complications. Platelets from PNH cases showed platelet hyporeactivity using in vitro assays possibly due to chronic hyperstimulation in the circulation [27]. Though functional platelet defects have not been reported in the other patients with PIGO variants, we detected a multi- agonist platelet defect in our patient as a subclinical phenotype.…”

Paroxysmal nocturnal hemoglobinuria: an acquired X-linked genetic disease with somatic-cell mosaicism

“…The GPI-linked molecule CD157 has been shown to be an important mediator of neutrophil adhesion and migration, and PNH-patient neutrophils, the large majority of which are GPIÀ even when PNH red cells are a minority, are severely defective in these functions [37 ]. An attractive hypothesis to explain the marked tendency of PNH patients to develop venous thrombosis has been that it is a consequence of the presence of large numbers of GPI-platelets [38]: however, definitive evidence has been difficult to obtain even in a recent detailed study [39].…”

The prothrombotic state in paroxysmal nocturnal hemoglobinuria: a multifaceted source

“…50 It is possible that the failure of activated platelets to bind fibrinogen, VWF and to aggregate is due to receptor GpIIb-IIIa complexes in proximity to MAC pores becoming uncoupled from the intracellular transduction mechanisms normally involved in their activation. 37 This observation was consistent with the findings of Gralnick et al, who reported variable amounts of platelet activation and further observed reduced VWF binding.…”

“…30 Grünewald et al hypothesized a mechanism of dual causality responsible for platelet hyporeactivity. 50 One mechanism is hyperstimulation of platelets due to sustained complement attack. 16,37,50 Chronic hyperstimulation of the coagulation system was hypothesized to further downregulate the activity of activated platelets.…”

“…16,37,50 Chronic hyperstimulation of the coagulation system was hypothesized to further downregulate the activity of activated platelets. 4,50 This highlights the complex variable nature of thrombosis in PNH, suggesting that platelets post activation possibly have a diminished role in PNH-induced thrombosis in comparison to other thrombotic diseases.…”