A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis

Abstract

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

Imaging platelet biogenesis in vivo

“…Functional deficiency of Cdc42, or the von Willebrand receptor GPIbα impairs transendothelial proplatelet formation as observed using 2PM. In contrast, lack of RhoA results in increased Cdc42 activity and the transmigration of entire MKs into the vessel lumen …”

“…Two‐photon microscopy has afterwards been used by a few groups interested in MKs to study the effect of drugs (like dasatinib) on thrombopoiesis in vivo or to investigate knock‐out mice for platelet production . MKs were visualized using either reporter strains that express fluorescent proteins specifically in MKs and platelets, or using fluorophore‐conjugated antibody derivatives against the von Willebrand receptor subunit GPIX prior to imaging.…”

“…This antibody is widely used in in vivo platelet studies and is described not to interfere with platelets or megakaryocytes in these settings . The vessel lumen is usually stained with fluorophore‐conjugated high‐molecular weight dextrans, quantum dots (see Figure ) or fluorophore‐conjugated BSA, while we do use anti‐CD105 (anti‐endoglin) antibodies to label the endothelial lining, in addition to the vessel lumen . The optimization of labeling techniques as well as technical improvements of two‐photon microscopes have increased the spatial resolution compared to the initial study .…”

“…Nevertheless, two‐photon microscopy of cranial bone marrow has become the method of choice to study platelet biogenesis in vivo. At the ISTH conference in Berlin several studies were presented that utilized 2PM to assess the effects of gene deficiencies on platelet biogenesis …”

“…With regard to the final step of MK polarization and proplatelet release Pleines presented a recent study that the two small GTPases Cdc42 and RhoA act as central regulators coordinating these processes. Functional deficiency of Cdc42, or the von Willebrand receptor GPIbα impairs transendothelial proplatelet formation as observed using 2PM.…”