The Role of Platelets in Increased Thrombogenicity Following Splenectomy: Key Platelet-Specific Receptors and Activation Markers

Aim

The mechanisms underlying increased thrombogenicity in splenectomy are poorly understood. We assessed a potential role for platelets by analysing surface expression of key platelet-specific receptors glycoprotein (GP)Iba, GPVI and aIIbb3, and a platelet activation marker, P-selectin. GPIba, that binds von Willebrand factor and other ligands, is constitutively shed from aging platelets, and its surface density is crucial for both thrombus formation and platelet clearance through interactions with phagocytic cells. GPIba also forms a non-covalent complex with GPVI, which binds collagen and fibrin. GPIba/GPVI initiate platelet adhesion, leading to platelet activation and aggregation, secretion of procoagulant factors and thrombus formation.

Methods

Whole blood from same-day healthy controls (n=15) or splenectomy (n=30) was centrifuged to obtain platelet-rich-plasma, and stained with phycoerythrin (PE)-labelled antibodies against GPIba (PE-AK2), GPVI (PE-1G5), aIIb (PE-CD41a), CD9 (PE-CD9), and P-selectin (PE-CD62P) using standardized protocols, and analysed using a FACSCalibur.

Results

Preliminary analysis suggested that compared to healthy controls, platelets from splenectomy cases showed significantly decreased surface expression of GPIba (p <0.0001) and GPVI (p=0.0035). In contrast, there was no significant difference in relative surface expression of aIIbb3 (p=0.759) and CD9 (p=0.112). Further, consistent with increased platelet activation/secretion, expression of surface P-selectin was more variable and significantly elevated in splenectomy cf . control platelets (p=0.0005). ELISA analysis of stored plasma will be used to confirm whether decreased platelet GPVI is likely due to increased shedding, resulting in increased plasma sGPVI.

Conclusion

The results of this pilot study support a role for hyperactivated platelets post-splenectomy, and identify platelet-specific markers related to these changes. Methods and results developed here will enable future larger temporal studies of platelet-related factors, and determine if such measurable changes correlate with clinical haemostatic/thrombotic dysfunction. This can also help explain basic mechanism(s) of thrombotic propensity in this population, and/or indicate potential therapeutic targets for prevention of thrombosis.