REGULATION OF PLATELET PRODUCTION AND FUNCTION BY MEGAKARYOCYTE GROWTH AND DEVELOPMENT FACTOR IN NONHUMAN-PRIMATES

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory r ole of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte gro wth and development factor (rHu-MGDF) on megakaryocytopoiesis. platele t function, and thrombogenesis in nonhuman primates. rHu-MGDF was admi nistered to 10 baboons for 28 days while performing pharmacokinetics a nd repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin ac cumulation on segments of vascular graft and endarterectomized aorta i n vivo. Daily subcutaneous injections of rHU-MGDF (5 mu g/kg/d) attain ed plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after i njection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platel et concentration threefold by day 7 and fivefold by day 28 (P < 10(-4) ) associated with a reciprocal decrease of 25% in mean platelet volume s (P < 10(-3)). Platelet mass turnover, a steady-state measure of plat elet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred i n peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in a ll cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF injections. Marrow megakaryocyte vo lume doubled (P < 10(-3)) three days after initiating rHU-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow meg akaryoctye number increased twofold by day 7, and nearly fourfold by d ay 28 (P < 10(-4)), resulting in a 6.5-fold increase in marrow megakar yocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were d etermined by comparing baseline, day 5, and day 28 rHU-MGDF-treatment measurements of (111)n-platelet deposition and I-125-fibrin accumulati on on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF incre ased In-111-platelet deposition in direct proportion to the circulatin g concentration of platelets for both EA and VG (r = .98 in both cases ), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet a ggregatory responsiveness was enhanced to physiologic agonists (adenos ine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP (1-6)) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu- MGDF enhanced aggregatory responses to low doses of physiologic agonis ts, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl r eceptor. Flow cytometric expression of platelet activation epitopes wa s not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryoc yte growth and development factor regulates platelet production and fu nction by stimulating endoreduplication and megakaryocyte formation fr om marrow progenitor cells, and transiently enhancing platelet functio nal responses ex vivo. rHu-MGDF has the potential for achieving platel et hemostatic protection with minimal thrombo-occlusive risk. (C) 1996 by The American Society of Hematology.