Platelets Exposed to Elevated Levels of Endogenous Thrombopoietin In Vivo Have a Reduced Response to Megakaryocyte Growth and Development Factor In Vitro

Abstract

SummaryThrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

Thrombopoietin Contributes to Enhanced Platelet Activation in Patients With Unstable Angina

“…In addition, rhTPO is able to phosphorylate c-Mpl only in platelets from SA patients and healthy control subjects but not in those from UA patients, further supporting this hypothesis. Platelets from patients with UA also showed reduced reactivity to rhTPO ex vivo, possibly related to their exposure in vivo to increased levels of endogenous TPO, analogously to what was previously shown for thrombocytopenic mice (33) and humans with the syndrome of congenital thrombocytopenia with absent radii (33,34).…”

Decreased prothrombotic effects of pegylated recombinant human megakaryocyte growth and development factor in thrombocytopenic state in a rat thrombosis model

“…Such decreased prothrombotic effects of PEG‐rHuMGDF in thrombocytopenic rats might be related not only to a low platelet number but also to decreased platelet reactivity to PEG‐rHuMGDF in the thrombocytopenic state. The latter possibility was based on our previous findings that platelets from thrombocytopenic mice had decreased surface levels of c‐Mpl and decreased reactivity to TPO in a platelet aggregation response in vitro , possibly because of increased endogenous TPO concentration [12]. Consistent with these observations, this study confirmed that serum TPO concentrations were elevated in thrombocytopenic rats (Fig.…”

“…We used the mouse TPO ELISA [12] to measure TPO concentrations in rat plasma with some modifications. Nunc‐Immuno plates (Nunc, Roskilde, Denmark) were incubated with 5 μg mL −1 rabbit immunoglobulin G antibody against recombinant mouse TPO at 4 °C overnight and washed.…”

“…The supernatants of platelet lysates were prepared, and immunoprecipitated and analyzed by Western blotting as described previously [12]. Briefly, an aliquot of the supernatant was incubated with rat c‐Mpl antiserum (1 : 1000 dilution) or anti‐rat CD61 antibody (10 μg mL −1 ) at 4 °C overnight.…”

Transgene Insertion in Proximity to the c-myb Gene Disrupts Erythroid-Megakaryocytic Lineage Bifurcation

“…Serum thrombopoietin (TPO) concentrations were measured using sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (22). A double-staining technique and flow cytometry were used to measure megakaryocyte ploidy (35).…”