Abstract
Platelet disorders encompass abnormalities in platelet function as well as alterations in platelet counts, including thrombocytopenia and thrombocythemia. Patients with these disorders are at an increased risk of hemorrhage or thrombosis. Megakaryocytes (MKs), the precursors of platelets, reside in the bone marrow and undergo a tightly regulated maturation program known as megakaryopoiesis, accompanied by cytoskeletal changes that drive thrombopoiesis (proplatelet formation). However, the mechanisms regulating megakaryopoiesis and thrombopoiesis remain poorly understood. G protein-coupled receptor kinases (GRKs) are serine/threonine kinases that attenuate signaling from agonist-occupied G protein-coupled receptors (GPCRs) in a phosphorylation-dependent manner. In addition, GRKs also phosphorylate non-GPCR substrates, thereby influencing various cellular processes. We previously demonstrated that GRKs are critical negative regulators of platelet activation and thrombus formation through thrombin and/or ADP receptors. Importantly, GRK5 and GRK6 expression is upregulated during hematopoietic stem cell differentiation into MKs, leading us to hypothesize that GRKs play essential roles in MK development and platelet biogenesis.
To test this hypothesis, we generated MK- and platelet-specific double knockouts (DKO) for GRK5 and GRK6 in both human and mouse cells. Compared to Grk5 and Grk6 individual KOs,DKO mice displayed severe thrombocytopenia with platelet counts reduced to ~5% of normal. DKO platelets showed a significantly reduced survival (t1/2=36 hours), clearing faster than either single knockouts or controls (t1/2=48 hours). Furthermore, DKO platelets displayed increased integrin activation and granule secretion in both the resting and activated states, which may contribute to their enhanced clearance. However, splenectomy failed to elevate platelet counts in these DKO mice. Bone marrow analysis of DKO mice revealed abnormally shaped MKs clustered near blood vessels, along with myelofibrosis, osteosclerosis, and increased angiogenesis. Notably, plasma levels of TPO and TGF-β1 were 2-3-fold higher in DKO mice than in WT. Additionally, transmission electron microscopy demonstrated pronounced alterations in the demarcation membrane system in DKO bone marrow MKs. Ex vivo cultured DKO MKs displayed normal size, ploidy, and CD41a+CD42d+ surface expression. Importantly, machine learning-assisted image segmentation revealed that DKO MKs had approximately 64% fewer proplatelet-forming MKs, 60% fewer proplatelet tips per cell, and proplatelet tips that were 40% larger compared to WT MKs. To delineate the molecular mechanisms, we performed proteomics and phosphoproteomics using WT and DKO induced pluripotent stem cell-derived MKs (iMKs) and mouse bone marrow-derived MKs. After normalization, five proteins were consistently dephosphorylated in DKO samples compared to WT samples in both iMKs and mouse MKs. Among these, GPIbβ was dephosphorylated at serine166 compared with WT, confirmed by immunoblot using a site-specific antibody. GPIbβ protein expression was also decreased in DKO iMKs, coinciding with the loss of serine166 phosphorylation, though causality remained to be determined. Finally, endogenous interaction between the GPIb complex and GRK6 was detected in WT iMKs.
In conclusion, thrombocytopenia in DKO mice results from both impaired platelet production and enhanced platelet clearance. These findings identify GRK5 and GRK6 as dual regulators of platelet homeostasis, by (1) ensuring efficient thrombopoiesis, and (2) preventing premature platelet clearance by maintaining platelet quiescence and limiting excessive activation. Building on prior reports highlighting the critical role of GPIbβ in thrombopoiesis, this study reveals a potential regulatory pathway linking GRK5/6 to GPIbβ signaling, with implications for understanding the pathogenesis of thrombocytopenia, particularly in Bernard-Soulier syndrome and related platelet disorders.
