To Bleed or Not to Bleed: MCS-Related Shear Stress Promotes Redistribution of GPIb and αIIbβ3 from Platelets to Microparticles Thus Impairing Platelet Hemostatic Function

Abstract

Purpose Bleeding is a leading cause of readmission and 1-year mortality for mechanical circulatory support (MCS) patients. Mechanical damage of platelets and coagulation proteins induced by MCS-generated high shear stress is the main driver of bleeding. We previously demonstrated that hypershear promotes platelet pro-apoptotic behavior resulting in membrane reorganization and generation of platelet-derived microparticles (PDMP). Here, we test the hypothesis that hypershear alters surface expression and shedding of adhesion receptors GPIb and αIIbβ3 from platelets and PDMPs thus affecting platelet aggregability - all potential bleeding contributors. Methods Human platelets were exposed to shear stress in a hemodynamic shearing device (30-70 dyn/cm2, 10 min). Integrin GPIb and αIIbβ3 surface expression and PDMP generation were quantified by flow cytometry. Single platelets and PDMPs were distinguished by FS/SS characteristics. Shedding of soluble GPIb and αIIbβ3 fragments was quantified by ELISA. Platelet aggregation was measured by optical aggregometry. Results Platelet exposure to hypershear led to a reduction of platelet GPIb surface expression, with a significant drop of GPIb+ platelet number and fluorescence (73.5 ± 2.9% and 953.1 ± 101.0 AU vs. 94.3 ± 0.7% and 1620 ± 72.4 AU for intact platelets; M ± SEM, ANOVA, p ≤ 0.05). Yet, no shear-mediated shedding of soluble GPIb fragment or β3 subunit was found. Downregulation of platelet GPIb expression coincided with a progressive increase of GPIb- & αIIbβ3-positive PDMPs after shear (up to 3.3 ± 0.6% & 12.5 ± 2.8%, correspondingly). Surface density of GPIb and αIIbβ3 on PDMPs was 2-4x higher than on platelets, post-shear. Platelet aggregation to increasing concentrations of ADP and TRAP-6 was significantly impaired by 70 dyn/cm2 shear stress. Conclusion MCS-mediated hypershear and platelet stress accumulation 1) induces downregulation of platelet adhesion receptors via ejection of receptor-enriched PDMPs; 2) does not induce shedding of soluble fragments of GPIb and β3, and; 3) mechanistically limits platelet aggregatory and hemostatic responses. Shear-mediated redistribution of primary adhesion receptors from platelets to PDMPs coupled with impaired aggregability may mechanistically contribute to MCS-related bleeding.