Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2514-2522 ◽  
Author(s):  
Laurence A. Harker ◽  
Lorin K. Roskos ◽  
Ulla M. Marzec ◽  
Richard A. Carter ◽  
Judith K. Cherry ◽  
...  
Keyword(s):  
Life Span ◽  
Binding Sites ◽  
Growth And Development ◽  
Annexin V ◽  
Normal Function ◽  
Healthy Human ◽  
Platelet Production ◽  
Platelet Counts ◽  
Development Factor ◽  
Human Volunteers

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P< .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P > .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P > .6 in both cases); or density of platelet TPO-receptors (P > .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P < .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P < .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.

Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2514-2522 ◽  
Author(s):  
Laurence A. Harker ◽  
Lorin K. Roskos ◽  
Ulla M. Marzec ◽  
Richard A. Carter ◽  
Judith K. Cherry ◽  
...  
Keyword(s):  
Life Span ◽  
Binding Sites ◽  
Growth And Development ◽  
Annexin V ◽  
Normal Function ◽  
Healthy Human ◽  
Platelet Production ◽  
Platelet Counts ◽  
Development Factor ◽  
Human Volunteers

Abstract The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P&lt; .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P &gt; .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P &gt; .6 in both cases); or density of platelet TPO-receptors (P &gt; .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P &lt; .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P &lt; .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.

Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4427-4433 ◽  
Author(s):  
Laurence A. Harker ◽  
Ulla M. Marzec ◽  
Francis Novembre ◽  
I. Birgitta Sundell ◽  
Edmund K. Waller ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth And Development ◽  
Serum Levels ◽  
Platelet Production ◽  
Platelet Counts ◽  
Viral Loads ◽  
Development Factor ◽  
Immunodeficiency Virus ◽  
Baseline Values ◽  
Normal Controls

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.

scholarly journals Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1833-1844 ◽  
Author(s):  
LA Harker ◽  
P Hunt ◽  
UM Marzec ◽  
AB Kelly ◽  
A Tomer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Growth And Development ◽  
Vascular Graft ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Platelet Production ◽  
Development Factor ◽  
Platelet Deposition ◽  
And Function

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and 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 accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating 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 aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1–6) (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 agonists, 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 receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.

Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4427-4433 ◽  
Author(s):  
Laurence A. Harker ◽  
Ulla M. Marzec ◽  
Francis Novembre ◽  
I. Birgitta Sundell ◽  
Edmund K. Waller ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth And Development ◽  
Serum Levels ◽  
Platelet Production ◽  
Platelet Counts ◽  
Viral Loads ◽  
Development Factor ◽  
Immunodeficiency Virus ◽  
Baseline Values ◽  
Normal Controls

Abstract Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P &gt; .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P &lt; .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P &gt; .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P &lt; .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P &gt; .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.

scholarly journals Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1833-1844 ◽  
Author(s):  
LA Harker ◽  
P Hunt ◽  
UM Marzec ◽  
AB Kelly ◽  
A Tomer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Growth And Development ◽  
Vascular Graft ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Platelet Production ◽  
Development Factor ◽  
Platelet Deposition ◽  
And Function

Abstract The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and 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 accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating 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 aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1–6) (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 agonists, 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 receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.

scholarly journals Prevention of Thrombocytopenia and Neutropenia in a Nonhuman Primate Model of Marrow Suppressive Chemotherapy by Combining Pegylated Recombinant Human Megakaryocyte Growth and Development Factor and Recombinant Human Granulocyte Colony-Stimulating Factor

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Laurence A. Harker ◽  
Ulla M. Marzec ◽  
Andrew B. Kelly ◽  
Ellen Cheung ◽  
Aaron Tomer ◽  
...  
Keyword(s):  
Platelet Count ◽  
Neutrophil Count ◽  
Growth And Development ◽  
Granulocyte Colony Stimulating Factor ◽  
Serum Concentrations ◽  
Colony Stimulating Factor ◽  
Platelet Counts ◽  
Development Factor ◽  
Trough Serum ◽  
Stimulating Factor

Abstract This report examines the effects on hematopoietic regeneration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF ) (2.5 μg/kg/d) alone and in combination with recombinant human granulocyte colony stimulating factor (rHu-GCSF ) (10 μg/kg/d) for 21 days in rhesus macaques receiving intense marrow suppression produced by single bolus injections of hepsulfam (1.5 g/m2). In six hepsulfam-only control animals thrombocytopenia (platelet count <100 × 109/L) was observed between days 12 and 25 (nadir 39 ± 20 × 109/L on day 17), and neutropenia (absolute neutrophil count <1 × 109/L) occurred between days 8 and 30 (nadir 0.167 ± 0.120 × 109/L on day 15). PEG-rHuMGDF (2.5 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.9 ± 0.2 ng/mL and increased the platelet count twofold over basal prechemotherapy levels (856 ± 594 × 109/L v baseline of 416 ± 88 × 109/L; P = .01). PEG-rHuMGDF alone also shortened the period of posthepsulfam neutropenia from 22 days to 12 days (P = .01), although the neutropenic nadir was not significantly altered (neutrophil count 0.224 ± 0.112 × 109/L v 0.167 ± 0.120 × 109/L; P < .3). rHu-GCSF (10 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.4 ± 1.1 ng/mL, and reduced the time for the postchemotherapy neutrophil count to attain 1 × 109/L from 22 days to 4 days (P = .005). The postchemotherapy neutropenic nadir was 0.554 ± 0.490 × 109neutrophils/L (P = .3 v hepsulfam-only control of 0.167 ± 0.120 × 109/L). However, thrombocytopenia of <100 × 109 platelets/L was not shortened (persisted from day 12 to day 25), or less severe (nadir of 56 ± 32 × 109 platelets/L on day 14; P = .7 compared with untreated hepsulfam animals). The concurrent administration of rHu-GCSF (10 μg/kg/d) and PEG-rHuMGDF (2.5 μg/kg/d) in four animals resulted in postchemotherapy peripheral platelet counts of 127 ± 85 × 109/L (P = .03 compared with 39 ± 20 × 109/L for untreated hepsulfam alone, and P = .02 compared with 856 ± 594 × 109/L for PEG-rHuMGDF alone), and shortened the period of neutropenia <1 × 109/L from 22 days to 4 days (P = .8 compared with rHu-GCSF alone). Increasing PEG-rHuMGDF to 10 μg/kg/d and maintaining the 21-day schedule of coadministration with rHu-GCSF (10 μg/kg/d) in another four animals produced postchemotherapy platelet counts of 509 ± 459 × 109/L (P < 10−4compared with untreated hepsulfam alone, and P = .04 compared with 2.5 μg/kg/d PEG-rHuMGDF alone), and 4 days of neutropenia. Coadministration of rHu-GCSF and PEG-rHuMGDF did not significantly alter the pharmacokinetics of either agent. The administration of PEG-rHuMGDF (2.5 μg/kg/d) from day 1 through day 22 and rHu-GCSF (10 μg/kg/d) from day 8 through day 22 in six animals produced peak postchemotherapy platelet counts of 747 ± 317 × 109/L (P < 10−4 compared with untreated hepsulfam alone, and P = .7 compared with PEG-rHuMGDF alone), and maintained the neutrophil count < 3.5 × 109/L (P = .008 v rHu-GCSF therapy alone). Thus, both thrombocytopenia and neutropenia are eliminated by initiating daily PEG-rHuMGDF therapy on day 1 and subsequently adding daily rHu-GCSF after 1 week in the rhesus model of hepsulfam marrow suppression. This improvement in platelet and neutrophil responses by delaying the addition of rHu-GCSF to PEG-rHuMGDF therapy demonstrates the importance of optimizing the dose and schedule of cytokine combinations after severe myelosuppressive chemotherapy.

scholarly journals Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 54-59 ◽  
Author(s):  
AM Farese ◽  
P Hunt ◽  
T Boone ◽  
TJ MacVittie
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Growth And Development ◽  
Nonhuman Primates ◽  
Normal Male ◽  
Platelet Counts ◽  
Development Factor ◽  
Development In Vitro

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

The Effect of Eltrombopag on Human Platelet Resistance to Apoptosis: The Role of the Bcl-Xl Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2520-2520
Author(s):  
W. Beau Mitchell ◽  
Michele N Edison ◽  
Mariana P Pinheiro ◽  
Nayla Boulad ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Platelet Count ◽  
Life Span ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Apoptotic Pathway ◽  
Platelet Production ◽  
Akt Activation ◽  
Platelet Counts ◽  
Ic50 Values ◽  
Apoptotic Factors

Abstract Abstract 2520 Introduction: Immune thrombocytopenia (ITP) is typically characterized by increased platelet destruction and reduced platelet production. Eltrombopag is a thrombopoietin receptor (TPO-R) agonist that is known to increase platelet counts in patients with ITP by stimulating thrombopoiesis, but it is unknown whether it has any effect on platelet life span. Platelet survival is mediated by two molecular intermediates in an apoptotic pathway, Bcl-xL and Bak. Bcl-xL protein expression in megakaryocytes is thought to be regulated in part by TPO-mediated activation of Akt pathways through Jak2 and Stat5. Although controversial, in a very small number of ITP patients, Eltrombopag may increase platelet counts in 2–5 days. We hypothesized that any increase in platelet count in the first week of treatment might be due to effects of Eltrombopag on platelet survival. Therefore, this study explored whether Eltrombopag treatment has anti-apoptotic effects in patients with ITP. Methods: Following a treatment wash out period, 75 mg of Eltrombopag once daily was initiated for 2 weeks. Blood counts were measured on days 1, 3, 5, 8, 10, 12, and 15. Platelet function and survival was assessed on days 1, 8, and 15 by: immature platelet fraction (IPF); glycocalicin index; Bcl-xL inhibitor (ABT-737) IC50, a novel assay adapted for human platelets; measurement of Bcl-xL by western blot; measurement of several members of the Bcl-xL Akt mediated, apoptotic pathway by flow cytometry (FACS); bleeding score; measurement of thrombin-anti-thrombin complexes (TATs); and quantification of microparticles. Results: Seven of the 9 patients responded to treatment with Eltrombopag with a platelet count ≥ 50,000/μL, and 6 of the 7 responders at least doubled their counts during the 2 weeks of treatment. There was a significant increase in median platelet count (p<0.001), median large platelet count (p<0.01), and median absolute IPF (A-IPF, p<0.01), while there was a significant decrease in median % IPF (p<0.05). The dose of ABT-737 required to kill half of the platelets in the sample (IC50) did not differ significantly between patients or between patients and controls during the first two weeks of treatment, and remained stable over the 2 weeks of study. However, the relative IC50 values (% of day 1 IC50 value) increased after the first week of treatment but returned to baseline after the second week. While these changes were not significant, their kinetics were similar to those seen in the AKT/ Bcl-xL signal transduction pathway (Figure). There was no significant correlation between the platelet counts and the IC50 values. FACS analysis of members of the AKT signal transduction pathway revealed significant increase in each of the markers between days 1 and 8 (p<0.01), followed by a significant decrease between days 8 and 15 (p<0.05), with no difference between days 1 and 15 (Figure). The other lab tests are pending. Discussion: Because the A-IPF increased by less than the platelet increase and because the life span of the A-IPF is not known, it is unclear if the overall platelet count increase is entirely a result of increased platelet production. Platelet life span may be enhanced by Eltrombopag treatment as there was a parallel albeit transient increase in AKT activation markers and platelet apoptosis resistance. Our data suggest that platelets are more resistant to apoptosis when the levels of anti-apoptotic factors (eg. PTEN, Phospho-GSK3β) involved in the AKT/Bcl-xL pathway are greatest despite a concomitant increase in pro-apoptotic factors (eg. Bak, Bax). Since both increased AKT activation and apoptotic resistance returned to baseline at day 15, megakaryocytes and platelets already present at the start of treatment may respond differently than those generated de novo in the presence of Eltrombopag. Disclosures: No relevant conflicts of interest to declare.

Thrombopoietin therapy increases platelet yields in healthy platelet donors

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1339-1345 ◽  
Author(s):  
David J. Kuter ◽  
Lawrence T. Goodnough ◽  
John Romo ◽  
John DiPersio ◽  
Randolph Peterson ◽  
...  
Keyword(s):  
Platelet Count ◽  
Growth And Development ◽  
Serious Adverse Events ◽  
Platelet Counts ◽  
Development Factor ◽  
Healthy Donors ◽  
To Receive ◽  
Apheresis Platelets ◽  
Dose Limiting Toxicity ◽  
Median Maximum

The recombinant thrombopoietins have been shown to be effective stimulators of platelet production in cancer patients. It was therefore of interest to determine if one of these, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), could be used to increase platelet counts and consequently platelet yields from apheresis in healthy platelet donors. In a blinded, 2-cycle, crossover study, 59 platelet donors were randomized to receive a single subcutaneous injection of PEG-rHuMGDF (1 μg/kg or 3 μg/kg) or placebo and 15 days later undergo platelet apheresis. Donors treated with placebo had a median peak platelet count after PEG-rHuMGDF injection of 248 × 109/L compared with 366 × 109/L in donors treated with 1 μg/kg PEG-rHuMGDF and 602 × 109/L in donors treated with 3 μg/kg PEG-rHuMGDF. The median maximum percentage that platelet counts increased from baseline was 10% in donors who received placebo compared with 70% in donors who received 1 μg/kg and 167% in donors who received 3 μg/kg PEG-rHuMGDF. There was a direct relationship between the platelet yield and the preapheresis platelet count: Placebo-treated donors provided 3.8 × 1011 (range 1.3 × 1011-7.9 × 1011) platelets compared with 5.6 × 1011 (range 2.6 × 1011-12.5 × 1011) or 11.0 × 1011 (range 7.1 × 1011-18.3 × 1011) in donors treated with 1 μg/kg or 3 μg/kg PEG-rHuMGDF, respectively. Substandard collections (&lt;3 × 1011 platelets) were obtained from 26%, 4%, and 0% of the placebo, 1 μg/kg, and 3 μg/kg donors, respectively. No serious adverse events were reported; nor were there events that met the criteria for dose-limiting toxicity. Thrombopoietin therapy can increase platelet counts in healthy donors to provide a median 3-fold more apheresis platelets compared with untreated donors.

scholarly journals Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry

2021 ◽  
Author(s):  
Samuel Kemble ◽  
Amanda L Dalby ◽  
Gillian C Lowe ◽  
Phillip LR Nicolson ◽  
Steve P Watson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ex Vivo ◽  
Super Resolution ◽  
Thiazole Orange ◽  
Healthy Human ◽  
Platelet Counts ◽  
Human Volunteers ◽  
Immature Platelets ◽  
Super Resolution Microscopy

Circulating large ″preplatelets″ undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry (ISFC) and super-resolution microscopy. Immature platelets, preplatelets and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia (ITP) or undergoing chemotherapy. Preplatelets and barbells were 1.9%±0.18/1.7%±0.48 (n=6) and 3.3%-+1.6/0.5%±0.27 (n=12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of HLA-I with high thiazole orange and mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence upon the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with ITP had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent post-chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.

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