Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists

Abstract

Background Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. Methods Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbβ3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. Results Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbβ3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. Conclusion Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers—with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.

Impella 5.5 Versus Centrimag: A Head-to-Head Comparison of Device Hemocompatibility

“…Externalization of anionic phospholipids on platelet surface, as a specific marker of shear-mediated platelet activation, 20 was examined using annexin V binding assay and quantified as MFI of annexin V–positive population. We showed that platelet annexin V binding gradually increased over the shear exposure time for both devices (Figure S2, Supplemental Digital Content, http://links.lww.com/ASAIO/A548 ).…”

“… 37 , 38 Yet, we have previously shown that externalization of anionic phospholipids (as indicated by annexin V binding) is a characteristic signature of shear-mediated platelet activation as opposed to platelet activation by biochemical agonists, for example, ADP and thrombin. 20 Indeed, Mondal et al 39 , 40 reported that markers of platelet apoptosis and oxidative stress were elevated in bleeders versus non-bleeders among patient cohort supported by CF-VADs. However, we noted that annexin V binding was also elevated in the nonsheared control (see Figure S2, Supplemental Digital Content, http://links.lww.com/ASAIO/A548 , “240 Still” sample) suggesting that the shear-mediated increase of platelet annexin V binding observed in our study could be at least partially attributed to in vitro platelet “aging” occurring in blood samples during long-term storage and handling.…”

cAMP prevents antibody-mediated thrombus formation in COVID-19

“…Our study showed that IgGs from patients with severe COVID- (21). In contrast to PS, conventional markers of PLT activation were declared in this study to fail the detection of early activation events leading to thrombosis.…”

Thrombosis and Haemostasis 2020 Editors' Choice Papers

“…57 devices. 58 Albeit the anticoagulation properties of diverse sea cucumber species had already been identified, attempts had failed to isolate its oligosaccharides. Zhou et al 59 achieved this goal, allowing them to comprehensively characterize their individual structures in relation to biological activities.…”