PB1440 - Lysis of In Vitro Whole Blood Clots Is Altered in Sickle Cell Disease

Background: Sickle cell disease (SCD) patients have a high frequency of developing recurrent venous thromboembolism (VTE) with an increased risk of mortality. Moreover, these patients often present with a hypercoagulable state. SCD is a genetic blood disorder that results in the development of a defective hemoglobin in red blood cells (RBCs), causing the RBCs to sickle. The prominent presence of RBCs in a venous clot and the shape changes in SCD RBCs might affect clot stability.

Aims: To assess lysis of in vitro prepared blood clots from SCD patients.

Methods: Clots were formed on the bottom edge of a 96-wells plate by incubating human recalcified citrated blood with tissue factor. Tissue plasminogen activator (tPA)-induced lysis of whole blood clots was assessed with a spectrophotometer plate reader-based assay (HALO assay). The absorbance from the blood covering the center of the well was measured over time at 510 nm. In addition, contracting clots were visualized by confocal microscopy and real-time tPA-induced lysis was assessed using time-lapse imaging.

Results: Whole blood clots from SCD patients showed a decreased lysis compared to healthy controls. Furthermore, titration or substitution of healthy RBCs rescued the lysis rate in SCD patient blood. Furthermore, the reduced lysis of SCD RBCs could not be rescued by healthy control plasma. Real-time lysis showed a normal degradation of the fibrin network, but the patient RBCs remained in the clot. This demonstrates that the patient RBCs were the cause of the decreased lysis rate. A defective lysis might explain the higher risk of developing VTE in SCD patients. Finally, blocking ICAM-4, a cell adhesion molecule expressed by RBCs, by the addition of an anti-ICAM-4 antibody had a positive effect on the lysis of clots of SCD patient blood.

Conclusion(s): RBCs of sickle cell patients are important for the stability of a clot and contribute to decreased lysis.