scholarly journals Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 511-521 ◽  
Author(s):  
LA Harker ◽  
UM Marzec ◽  
P Hunt ◽  
AB Kelly ◽  
A Tomer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Dose Response ◽  
Growth And Development ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Platelet Production ◽  
Development Factor ◽  
And Function

Thrombopoietin (TPO) is the physiologic Mpl-ligand regulating platelet production. Pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated polypeptide Mpl-ligand derivitized with poly-(ethylene glycol), induces megakaryocyte endoreduplication and proliferation in vitro and in vivo. In the present study, the dose-response effects of PEG-rHuMGDF on pharmacokinetics, megakaryocytopoiesis, platelet production, and platelet function were characterized for dosing 0.05, 0.10, 0.50, or 2.5 micrograms/kg/d in 22 baboons for 28 days. Daily subcutaneous injections of PEG-rHuMGDF produced linear log-dose responses in (1) steady-state trough plasma levels of PEG-HuMGDF (P < 10(-3)); (2) marrow megakaryocyte volume (P < 10(-3)), ploidy (P <10(-4)), and number (P < .01); and (3) peripheral platelet concentrations (P < 10(- 4)) and platelet mass turnover (P < 10(-3)). Platelet morphology, life span, and recovery were normal, and peripheral leukocyte, neutrophil, and erythrocyte counts were not significantly affected by PEG-rHuMGDF (P > .1 in all cases). PEG-rHuMGDF at 0.5 micrograms/kg/d produced similar blood concentrations of Mpl-ligand and platelets as 10 times the dose of rHu-MGDF (5.0 micrograms/kg/d), reflecting the extended plasma half-life achieved through pegylation. Whereas PEG-rHuMGDF did not induce platelet aggregation in vitro, platelet aggregatory responsiveness induced by thrombin receptor agonist peptide (TRAP1–6) and collagen was transiently enhanced ex vivo during the initial few days of PEG-rHuMGDF administration. However, adenosine diphosphate (ADP)-induced platelet aggregation was not enhanced ex vivo by PEG- rHuMGDF therapy. 111In-platelet deposition on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts increased in direct proportion to the circulating platelet concentration (P < 10(-4) for both EA and VG); 125l-fibrin accumulation was not affected by PEG-rHuMGDF-induced increases in peripheral platelet counts. Changes in platelet production and function produced by PEG-rHuMGDF returned to baseline within 2 weeks after discontinuing treatment. Thus, in nonhuman primates, PEG- rHuMGDF increases platelet production in a linear log-dose-dependent manner by stimulating megakaryocyte endoreduplication and new megakaryocyte formation from marrow hematopoietic progenitors. These findings suggest that appropriate dosing of PEG-rHuMGDF therapy during periods of chemotherapy-induced marrow suppression may maintain hemostatic concentrations of peripheral platelets without increasing the risk of thrombosis.

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.

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 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.

scholarly journals Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Frank Denis Torres-Huaco ◽  
Cláudio C. Werneck ◽  
Cristina Pontes Vicente ◽  
Talita Vassequi-Silva ◽  
Ana Cláudia Coelho Nery-Diez ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Proteinase Inhibitors ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Venom Component ◽  
Purification Method ◽  
Rapid Purification

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

scholarly journals Gastrodia elata Blume and Zanthoxylum schinifolium Siebold & Zucc Mixed Extract Suppress Platelet Aggregation and Thrombosis

Medicina ◽  
2021 ◽  
Vol 57 (10) ◽  
pp. 1128
Author(s):  
Yong-Deok Jeon ◽  
Ji-Hyun Lee ◽  
Mi-Ran Park ◽  
Ji-Ye Lim ◽  
Sa-Haeng Kang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Oral Anticoagulants ◽  
Dependent Manner ◽  
Gastrodia Elata ◽  
Gastrodia Elata Blume ◽  
Wide Range ◽  
Zanthoxylum Schinifolium

Background and objectives: Blood vessel thrombosis causes blood circulation disorders, leading to various diseases. Currently, various antiplatelet and anticoagulant drugs, such as aspirin, warfarin, heparin, and non-vitamin K antagonist oral anticoagulants (NOACs), are used as the major drugs for the treatment of a wide range of thrombosis. However, these drugs have a side effect of possibly causing internal bleeding due to poor hemostasis when taken for a long period of time. Materials and Methods: Gastrodia elata Blume (GE) and Zanthoxylum schinifolium Siebold & Zucc (ZS) are known to exhibit hemostatic and antiplatelet effects as traditional medicines that have been used for a long time. In this study, we investigated the effect of a mixed extract of GE and ZS (MJGE09) on platelet aggregation and plasma coagulation. Results: We found that MJGE09 inhibited collagen-and ADP-induced platelet aggregation in vitro. In addition, collagen- and ADP-induced platelet aggregation were also inhibited in a dose-dependent manner on the platelets of mice that were orally administered MJGE09 ex vivo. However, compared with aspirin, MJGE09 did not prolong the rat tail vein bleeding time in vivo and did not show a significant effect on the increase in the prothrombin time (PT) and activated partial thromboplastin time (aPTT). Conclusions: These results suggest that MJGE09 can be used as a potential anticoagulant with improved antithrombotic efficacy.

scholarly journals Extracellular Tyrosyl-tRNA Synthetase Is a Potent Stimulator of Thrombocytopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1476-1476
Author(s):  
Sachiko Kanaji ◽  
Taisuke Kanaji ◽  
My-Nuong Vo ◽  
Alessandro Zarpellon ◽  
Ryan Shapiro ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Trna Synthetase ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Equity Ownership

Abstract Aminoacyl-tRNA synthetases (aaRSs) are enzymes with a key role in the first step of protein synthesis by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. During evolution, eukaryotic aaRSs have acquired additional domains and motifs conferring non-canonical functions beyond translation, such as expressing multiple cytokine activities. Repurposing aaRSs often requires an activation step and the first reported example was for human tyrosyl-tRNA synthetase (YRS), which is abundant in platelets and released from their α-granules upon thrombin or arachidonic acid stimulation. As shown by previous work, activated YRS (YRSACT) - created by natural proteolysis, alternative splicing, or rational mutagenesis - can express the activity of different cytokines. In the current study, we demonstrate that recombinant YRSACT rendered active by the gain-of-function mutation Tyr341Ala exhibits a previously unrecognized role in megakaryocytopoiesis and thrombocytopoiesis. When administered in vivo in C57BL/6 wild type (WT) mice, recombinant YRSACT caused platelet increase both under baseline conditions as well as in a model of immune-mediated thrombocytopenia in which mice are made thrombocytopenic by injection of rat anti-mouse glycoprotein (GP) Ib monoclonal IgG. When WT mouse bone marrow (BM) cells were cultured ex vivo for 3 days, YRSACT treatment increased the number of megakaryocytes by 3.0-fold, particularly of megakaryocytes with 16N ploidy. This effect was independent of thrombopoietin (TPO) signaling because YRSACT could support the expansion of c-mpl-/- (TPO receptor knock-out) mouse megakaryocytes. YRSACT had no effect on purified mouse CD41+ or Sca1+ hematopoietic progenitor cells, indicating that YRS-dependent stimulation likely required the contribution of other cells present in BM cultures. When mouse BM cells were stimulated with different doses of YRSACT, the number of F4/80+ monocyte/macrophages as well as of megakaryocytes increased in a dose-dependent manner. Mechanistic analysis revealed YRSACT targets the Toll-like receptor (TLR) pathway signaling through MyD88 in monocyte/macrophages, thereby enhancing release of cytokines that influence megakaryocyte development. In vitro binding assay showed that YRSACT is capable of binding to TLR2 and TLR4. The effect of YRSACT was attenuated in the BM cells derived from TLR2-/- mice and was abolished in MyD88-/- mice. Among the cytokines with synthesis induced by YRSACT, IL-6 plays a pivotal role in megakaryocyte development. Thus, we tested the effect of YRSACT on megakaryocytes obtained by culturing BM cell derived from IL-6-/- mice and found that no effect was apparent. The stimulatory effect of YRSACT on megakaryocytopoiesis was confirmed with human CD41+ megakaryocyte progenitors differentiated from CD34+ hematopoietic stem cells derived from peripheral blood. In conclusion, we have documented a previously unrecognized activity of YRSACT that results in enhanced megakaryocytopoiesis and platelet production. These studies document a mechanistically distinct aaRS-directed hematological activity that highlights new potential approaches to stimulating platelet production for treating thrombocytopenia and for improving ex vivo preparation of platelet concentrates for transfusion. Disclosures Belani: aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Do:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Yang:aTyr Pharma: Consultancy, Patents & Royalties, Research Funding. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals The effect of chlorin e6 drug on platelet aggregation activity

2019 ◽  
Vol 8 (3) ◽  
pp. 4-10 ◽  
Author(s):  
N. N. Petrishchev ◽  
M. A. Galkin ◽  
T. G. Grishacheva ◽  
I. N. Dementjeva ◽  
S. G. Chefu
Keyword(s):  
Platelet Aggregation ◽  
Laser Irradiation ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Dependent Manner ◽  
Functional Changes ◽  
Aggregation Activity ◽  
Dose Dependent

The goal of the study is to evaluate the effect of Radachlorin (OOO “RADA-PHARMA”, Russia) (RC) on platelet aggregation in ex vivo and in vivo experiments. The experiments were conducted on male Wistar rats. Platelet aggregation activity was determined in platelet-rich plasma (PRP) using a turbidimetric method and the aggregation inducer was ADP at a final concentration of 1.25 μM. PRP samples containing RC were irradiated with ALOD-Granat laser device (OOO “Alkom Medika”, Russia) at 662 nm wavelength with 0.05 W/cm2 power density. After a 5-minute incubation of PRP with RC in the dark, dose-dependent inhibition of platelet aggregation was observed. Laser irradiation (12.5 J/cm2 and, especially, 25 J/cm2) increased the inhibitory effect of RC. 3 hours after intravenous administration of RC, the rate and intensity of platelets aggregation did not change, while disaggregation slowed down significantly. Irradiation at a dose of 5 J/cm2 did not affect the platelets aggregation kinetics, and disaggregation slowed down even more at 10 J/cm2, and at 20 J/cm2 the rate and intensity of platelets aggregation decreased, and no disaggregation occurred.In vitro, RC inhibited the ADP-induced platelet aggregation in rats in a dose-dependent manner; after laser irradiation, this effect was enhanced significantly. The effect of RC on circulating platelets leads to a change in their functional state, which manifests in slowing down the disaggregation after exposure to ADP. After laser irradiation (10 J/cm2 and, especially, 20 J/cm2), the severity of the functional changes increases. The role of decreasing the disaggregation activity of platelets in the mechanism of vascular thrombosis in the affected area of photodynamic therapy (PDT) is discussed.

Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation

1995 ◽  
Vol 80 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Christopher F. Toombs ◽  
Cindy H. Young ◽  
John A. Glaspy ◽  
Brian C. Varnum
Keyword(s):  
Platelet Aggregation ◽  
Growth And Development ◽  
Development Factor

Inhibition of Thrombosis by a Selective Fibrinogen Receptor Antagonist without Effect on Bleeding Time

1994 ◽  
Vol 72 (01) ◽  
pp. 119-124 ◽  
Author(s):  
Juerg F Tschopp ◽  
Curt Mazur ◽  
Kenneth Gould ◽  
Raymond Connolly ◽  
Michael D Pierschbacher
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Ic50 Values ◽  
Receptor Assays ◽  
Dose Dependent ◽  
Fibrinogen Deposition

SummaryMembrane glycoprotein αIIbβ3 on platelets plays a pivotal role in hemostasis by mediating RGD-(arginine-glycine-aspartic acid)-dependent platelet adhesion and aggregation. Antagonists of αIIbβ3 ligand binding function, such as antibodies, snake venom peptides, or synthetic RGD-containing peptides can completely inhibit platelet aggregation in vitro and cause significant prolongation of bleeding times when injected into experimental animals. The in vitro and in vivo properties of an αIIbβ3 specific RGD-containing peptide 2G (G(Ten)GHRGDLRCA) were compared to two non-specific RGD-containing peptides IN (G(Pen)GRGDTPCA) and 2H (GRGDSPDG). All three peptides have similar IC50 values in human patelet aggregation (14-22 μM) and ELISA-based μIIbβ3 receptor assays (0.2–0.3 αM) but show different inhibitory activity (IC50 values) in the αv㯂5 (2G = 10 μM; IN = 0.06 μM; 2H = 0.05 μM) and receptor assays (2G = 8.3 μM; IN = 0.06 μM; 2H = 0.04). The αIIbβ3 specific peptide 2G had no effect on monolayers of human saphenous vein endothelial cells while IN and 2H caused many cells to detach and contract. Peptides 2G and IN inhibited ADP-stimulated ex vivo platelet aggregation in dogs in a dose dependent manner. When complete inhibition (>90%) of ex vivo platelet aggregation was achieved with either a 10 mg/kg bolus followed by a 16mg/kg/h infusion of 2G or with a 15 mg/kg bolus and 24 mg/kg/h infusion of IN, peptide IN caused a dose-dependent increase of the template bleeding time, while peptide 2G had no effect, even at doses up to 15 mg/kg bolus followed by 24 mg/kg/h infusion. The in vivo properties of peptides 2G and 2H were also examined in a baboon ex vivo shunt model for their ability to block platelet uptake and fibrinogen deposition on small caliber GORE-TEX® grafts and for their effect on the hemostatic system. Systemic administration of peptide 2G at 10 mg/kg bolus followed by 10 mg/kg/h infusion (or at a 2-fold lower dose) abolished platelet uptake and fibrinogen deposition on the graft surface without affecting the hemostasis and template bleeding time of the animal. By contrast, peptide 2H caused a 3-4-fold increase in bleeding time at a dose of 10 mg/kg. The results suggest that efficacy and the effect of specific aIIbp3antagonists on bleeding time can be separated and that selective aIIbP3 receptor blockade may be an efficient and safe approach to improve the patency and the success rate pf small caliber vascular grafts and to treat unwanted platelet-dependent thromboses. While peptide 2G may represent a unique class of antithrombotic agent, the clinical use of this type of molecule would require a significant enhancement in potency.

Chlorobutanol, a Preservative of Desmopressin, Inhibits Human Platelet Aggregation and Release In Vitro

1990 ◽  
Vol 64 (03) ◽  
pp. 473-477 ◽  
Author(s):  
Shih-Luen Chen ◽  
Wu-Chang Yang ◽  
Tung-Po Huang ◽  
Shiang Wann ◽  
Che-ming Teng
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Atp Release ◽  
Free Calcium ◽  
Dependent Manner ◽  
Antiplatelet Effect ◽  
Human Platelet Aggregation ◽  
Acid Pathway ◽  
Arachidonic Acid Pathway

SummaryTherapeutic preparations of desmopressin for parenteral use contain the preservative chlorobutanol (5 mg/ml). We show here that chlorobutanol is a potent inhibitor of platelet aggregation and release. It exhibited a significant inhibitory activity toward several aggregation inducers in a concentration- and time-dependent manner. Thromboxane B2 formation, ATP release, and elevation of cytosolic free calcium caused by collagen, ADP, epinephrine, arachidonic acid and thrombin respectively were markedly inhibited by chlorobutanol. Chlorobutanol had no effect on elastase- treated platelets and its antiplatelet effect could be reversed. It is concluded that the antiplatelet effect of chlorobutanol is mainly due to its inhibition on the arachidonic acid pathway but it is unlikely to have a nonspecitic toxic effect. This antiplatelet effect of chlorobutanol suggests that desmopressin, when administered for improving hemostasis, should not contain chlorobutanol as a preservative.

Sign in / Sign up

Export Citation Format

Share Document