Phosphatidylserine-Exposing Extracellular Vesicles after Splenectomy Are Associated with Increased D-Dimers and Fibrin Generation in Hereditary Hemolytic Anemia
Authors: Van Straaten Stephanie , Hau Chi , Hajji Najat , Verhoeven Jill , Schutgens Roger , van Solinge Wouter , van Wijk Richard , Nieuwland Rienk , van Beers Eduard J. 
Published: October 2019

Abstract
Background
Thrombosis is a common complication of hereditary hemolytic anemia (HHA). Etiology of a hypercoagulable state in patients with HHA involves inflammation and splenectomy, although the etiology of the latter is insufficiently established. Because the concentration of circulating extracellular vesicles (EVs) has been reported to increase after splenectomy, and because in patients with sickle cell disease (SCD) circulating EVs are associated with coagulation, we analyzed the concentration of circulating EVs and their procoagulant activity in plasma from splenectomized and non-splenectomized patients with HHA.
Methods
This is a cross sectional, observational study in adult patients with HHA (SCD, other hemoglobin disorders, red cell enzyme disorders, red cell membrane disorders). Blood samples were collected with a 21-gauge butterfly needle and collected in 9 mL citrate phosphate dextrose adenine (CPDA) vacutainers, without use of a tourniquet. The tubes were mixed gently and the time between blood collection and centrifugation was maximum one hour. EVs in platelet-depleted plasma were labeled for Heat Shock Protein 70, CD14 (monocyte-derived EVs), CD61 (platelet EVs), CD62e (endothelial EVs), CD62p (P-selectin-exposing platelet EVs), CD71 (reticulocyte EVs), CD144 (endothelial EVs), CD235a (erythrocyte EVs) and lactadherin (phosphatidylserine (PS)-exposing EVs), and measured with a dedicated flow cytometer for EVs (A60-micro, Apogee Flow systems; lower limit of detection 170-180 nm single EVs). The coagulant activity of EVs was studied by a fibrin generation test, which measures the EV-dependent clotting time of plasma. The time to fibrin formation (1/2max) was measured using optical densitometry (λ = 405 nm) and an arbitrary cut off of V1/2max <1,500s was used to consider FGT as positive. Samples with >25% difference between duplicates or from patients that used anticoagulant medication were excluded from analysis
Results
Ninety seven patients were included in the study. Baseline characteristics are shown in Table 1. FGT of 63 patients were included. Thirteen patients (21%) had a positive FGT. Patients with positive FGT had increased concentrations of circulating EVs (CD61, CD71, lactadherin: p=<0.001; HSP70, CD62p, CD61/CD62p, CD62e, CD235a: p=<0.05) compared to patients with a negative FGT. Of the patients with a positive FGT, 11 patients (85%) were splenectomized, versus 2 patients (28%) in the FGT-negative group (p=0.002, patients with SCD regarded as splenectomized). Splenectomized patients had increased concentrations of lactadherin-binding EVs (p<0.001), as well as increased concentrations of CD61- and CD61/CD62p-exposing EVs (p<0.001) and of CD235a- and CD71-positive EVs (p<0.01, Table 2). FGT V1/2max and D-dimer correlated with lactadherin-binding EVs (ρ=-0.631, p=<0.001, and ρ=0.331, p=0.001).
Conclusion
In this study we show that in HHA patients the plasma concentration of lactadherin-binding and thus PS-exposing EVs correlates with fibrin generation in vitro and plasma D-dimer concentration, indicating that EVs may be associated with the hypercoagulable state that is observed in patients with HHA. Splenectomized patients had higher concentrations of lactadherin-binding EVs, and their plasma samples were prone to clot, as shown by fibrin generation in vitro. As the spleen is the main organ removing PS-exposing cells, higher levels of PS-exposing EVs in such patients may be due to reduced clearance, which in turn may contribute to the increased risk of thrombosis in patients after splenectomy.

Disclosures
Schutgens: Novo Nordisk: Research Funding; Uniqure BV: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding; Bayer: Research Funding; Baxalta/Shire: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.