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+ precurs or cells into megakaryocytes and then platelets. In experimental anima ls, injection of this and other c-mpl ligands leads to profound increa ses 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-mpl ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defin ing the in vivo pharmacology of this novel and important hematopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcu taneous injections of rHuMGDF at 0.1, 1.0, or 10 mu g/kg were administ ered over 7 days. Daily injection of 10 mu g/kg produced an approximat e fourfold increase in platelet count and 1.0 mu g/kg doubled platelet count over the injection period, both of which were statistically sig nificant. The serum concentrations of rHuMGDF were determined 10 minut es following a single intravenous injection with 0.1, 1.0, and 10 mu g /kg, and were 0.05 +/- 0.02, 0.98 +/- 0.07, and 21.32 +/- 2.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immedia tely before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 mu g/kg). Intravenous injection of 10 m u g/kg produced measurable changes in platelet aggregometry ex vivo, a s evidenced by an increased sensitivity of platelets to adenosine diph osphate (ADP). To assess the in vivo prothrombotic potential of rHuMGD F, a rabbit carotid artery model of cyclic flow reduction (CFR) was us ed to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR slope and frequency. Int ravenous administration of rHuMGDF had no effect on CFR slope or frequ ency when administered in doses ranging from 0.1 to 10 mu g/kg. Contro l experiments demonstrated that CFR slope and frequency can be enhance d by intravenous infusion of epinephrine and can be abolished by the c ombined administration of aspirin and ketanserin, indicating that pote ntially prothrombotic and antithrombotic agents can be identified in t his model. We conclude that biologically active doses of rHuMGDF used in this study (1.0 and 10 mu g/kg) produce mea surable serum levels, i nduce a thrombopoietic effect, and sen sitize platelets in vivo, as de termined by ex vivo aggregometry, at 10 mu g/kg. Despite the sensitiza tion of platelets to aggregation induced by ADP, it is clear that rHuM GDF does not alter the pattern of CFRs observed in the rabbit carotid artery, whereas agents known to sensitize platelets (epinephrine) and to inhibit platelets (aspirin and ketanserin) readily affected the CFR pattern. These findings indicate that intravenous rHuMGDF administrat ion, while capable of sensitizing platelets, does not enhance platelet -dependent thrombosis in vivo.