Effects of Recombinant Human Megakaryocyte Growth and Development Factor (rHuMGDF) on Platelet Production, Platelet Aggregation, and Thrombosis.
Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a c-mpl ligand that promotes the differentiation of CD34+ precursor cells into megakaryocyte, and then platelets. In experimental animals, injection of this and other c-mpl ligands leads to profound increases in the circulating platelet count in a matter of days. However, c-mpl ligands have also been shown to sensitize platelets to aggregating agents in vitro, raising the possibility that c-mpI ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defining the in vivo pharmacology of this novel and important hernatopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcutaneous injections of rHuMGDF at 0.1, 1.0, or 10 µg/kg were administered ever 7 days. Daily injection of 10 µ/kg produced an approximate fourfold increase in platelet count and 1.0 µ/kg doubled platelet count over the injection period, both of which were statistically significant. The serum concentrations of rHuMGDF were determined 10 minutes following a single intravenous injection with 0.1, 1.0, and 10 µ/kg, and were 0.05 +/- 0.02, 0.98 +/- 0.07, and 21.32 +/- 21.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immediately before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 µ/kg). Intravenous injection of 10 µ/kg produced measurable changes in platelet aggregometry ex vivo, as evidenced by an increased sensitivity of platelets to adenosine diphosphate (ADP). To assess. the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR slope and frequency. Intravenous administration of rHuMGDF had no effect on CFR slope or frequency when administered in doses ranging from 0.1 to 10 µ/kg. Control experiments demonstrated that CFR slope and frequency can be enhanced by intravenous infusion of epinephrine and can be abolished by the combined administration of aspirin and ketanserin, indicating that potentially prothrombotic and antithrombotic agents can be identified in this model. We conclude that biologically active doses of rHuMGDF used in this study (1.0 and 10 µ/kg) produce measurable serum levels, induce a thrombopoietic effect, and sensitize platelets in vivo, as determined by ex vivo aggregometry, at 10 µ/kg. Despite the sensitization of platelets to aggregation induced by ADP, it is clear that rHuMGDF does not alter the pattern of CFRs observed in the rabbit carotid artery, whereas agents known to sensitize platelets (epinepherine) and to inhibit platelets (aspirin and ketanserin) readily affected the CFR pattern. These findings indicate that intravenous rHuMIGDF administration, while capable of sensitizing pIatelets, does not enhance platelet-dependent thrombosis in vivo.
Lott
FA
Department of Pharmacology, Amgen Inc., Thousand Oaks, California, USA.
Nelson
AG
Toombs
CF