The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI

by Selleckchem | Nov 28 2023

Introduction: Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis.

Methods: We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin.

Results: We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions.

Conclusion: In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Comments:

**Introduction:**
Hemolysis, the process of red blood cell destruction, leads to the release of free hemoglobin and hemin from erythrocytes. Previous studies have indicated that hemin has the ability to activate platelets and trigger thrombosis, a condition where blood clots form within blood vessels. This research aims to investigate the effects of hemin on platelet function and the expression of glycoprotein VI (GPVI), a collagen receptor on the surface of platelets.

**Methods:**
Isolated platelets were exposed to varying concentrations of hemin to assess its impact on platelet activation and the expression of GPVI. The study included experiments to measure platelet aggregation, the formation of aggregates on collagen surfaces under flow conditions, and the levels of GPVI on the platelet surface. Various inhibitors were employed to identify the underlying mechanisms of hemin-induced changes in platelet behavior.

**Results:**
Hemin was found to significantly enhance platelet activation, aggregation, and the formation of aggregates on immobilized collagen surfaces. Surprisingly, the study revealed that high concentrations of hemin led to a reduction in the surface expression of GPVI. This reduction did not hinder platelet aggregation, suggesting that the loss of GPVI caused by hemin did not interfere with platelet function. Further analysis revealed that hemin-induced shedding of the GPVI ectodomain was responsible for the decrease in GPVI surface expression. This shedding was independent of certain signaling pathways and metalloproteinases previously associated with GPVI degradation. Unexpectedly, the research identified furin, a subtilisin-like proprotein convertase, as a key regulator of hemin-induced GPVI shedding and platelet functions.

**Conclusion:**
In summary, this study has uncovered a novel mechanism involving furin-mediated shedding of GPVI, triggered by hemin exposure. This shedding does not impede platelet aggregation and thrombus formation, indicating a complex interplay between hemin, GPVI shedding, and platelet function. These findings provide valuable insights into the understanding of hemin-induced platelet activation and may pave the way for future research and therapeutic interventions targeting hemin-associated thrombotic disorders.