Abstract

Backgound: Sickle cell disease (SCD) is characterized by a hypercoagulable state involving multiple factors, including chronic hemolysis and circulating cell-derived microparticles (MPs). There is still no consensus on the cellular origin of such MPs and the exact mechanism by which they may support coagulation activation in SCD.

Aim: In the present study, we aimed to analyze the origin of circulating MPs and their pro-coagulant phenotype in 25 consecutive SCD patients during painful crises and steady state. conditions

Methods: MPs were identified by flowcytometry on basis of their size and density and on their ability to bind annexin V. Measurements were corrected for auto-fluorescence. Labeling with cell-specific monoclonal antibodies was corrected with isotype-matched control antibodies. For thrombin generation experiments patient derived MPs were reconstituted in defibrinated (reptilase-treated) normal pool (MP-free) plasma. For the inhibition experiments, the defibrinated plasma and the MPs were separately incubated with antibodies against coagulation factors VII or XI, or tissue factor pathway inhibitor (TFPI).

Results: The majority of MPs originated from platelets (GPIIIa, CD61+: 86.1%) and erythrocytes (Glycophorin A, CD235+: 9.7%), and their numbers did not differ significantly between crisis and steady state. A distinct subset of transferrin receptor (CD71+)-exposing MPs was present, but neither MPs originating from monocytes (CD14+) nor endothelial cells (CD144+, CD146+, CD62E+) were detectable, and also no MPs exposing TF could be identified. The number of erythrocyte-derived MPs strongly correlated with plasma levels of hemolytic markers, i.e. hemoglobin (r=−0.58, P<0.001) and lactate dehydrogenase (r=0.59;P<0.001), von Willebrand factor as a marker of endothelial activation (r=0.44;P<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52;P<0.001 and r=0.59;P<0.001, respectively) as markers of fibrinolysis and coagulation activation. In the thrombin generation test, the total amount of thrombin generated (represented by the area under the curve (AUC) of the thrombin generation curve) correlated with the total number of circulating MPs (R=0.63, P<0.001). Thrombin generation was unaffected by pre-incubation with anti-human factor VII but increased slightly in the presence of anti-TFPI (16%; P=0.01) while the presence of anti-factor XI suppressed thrombin generation by about 50% (P<0.001). The extent of this inhibition (as a percentage of thrombin generation without antibody) was significantly correlated with the circulating number of erythrocyte-derived MPs (r=0.50, P=0.023), but not with platelet-derived MPs or reticulocyte-derived MPs. Also the absolute difference in thrombin generation between the experiments with and without factor XI antibody correlated with the absolute number of glycophorin A+ MPs (r=0.55, P=0.002).

Conclusion: We conclude that the procoagulant state in SCD is, at least in part, due to the procoagulant effects of circulating erythrocyte-derived MPs. Their relation to the ability of anti-factor XI to block thrombin generation in SCD patients suggests an important role of factor XI-dependent thrombin generation in these patients.

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author