Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

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

Blood ◽

10.1182/blood.v86.1.54.bloodjournal86154 ◽

1995 ◽

Vol 86 (1) ◽

pp. 54-59 ◽

Cited By ~ 145

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.

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Platelets Exposed to Elevated Levels of Endogenous Thrombopoietin In Vivo Have a Reduced Response to Megakaryocyte Growth and Development Factor In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612918 ◽

2001 ◽

Vol 85 (01) ◽

pp. 152-159 ◽

Cited By ~ 5

Author(s):

Uichi Nishiyama ◽

Haruhiko Morita ◽

Yoshifumi Torii ◽

Tomoaki Kuwaki ◽

Eiko Shimizu ◽

...

Keyword(s):

Growth And Development ◽

Platelet Reactivity ◽

Biochemical Properties ◽

Human Platelets ◽

Janus Kinase ◽

Intravenous Injections ◽

Development Factor ◽

Lower Reactivity

SummaryThrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

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Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice

Blood ◽

10.1182/blood.v86.12.4486.bloodjournal86124486 ◽

1995 ◽

Vol 86 (12) ◽

pp. 4486-4492 ◽

Cited By ~ 132

Author(s):

MM Hokom ◽

D Lacey ◽

OB Kinstler ◽

E Choi ◽

S Kaufman ◽

...

Keyword(s):

Growth And Development ◽

Granulocyte Colony Stimulating Factor ◽

Potent Inducer ◽

Development Factor ◽

Internal Bleeding ◽

Life Threatening ◽

Stimulating Factor ◽

Sublethal Irradiation

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

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The Effects Of Prostacyclin (PGI2) On Intravascular Platelet Aggregation

10.1055/s-0038-1653317 ◽

1981 ◽

Author(s):

G G Duncan ◽

G Mallarkey ◽

G M Smith

Keyword(s):

Platelet Count ◽

Guinea Pigs ◽

Adenosine Diphosphate ◽

Before And After ◽

Dependent Inhibition ◽

Induced Aggregation ◽

Dose Dependent ◽

Anaesthetised Rats

Intravascular aggregation can be measured by counting the number of circulating platelets before and after the injection of aggregation agents. The Technicon Autocounter was modified to count platelets continuously and connected via a double cannula in a carotid artery to an anaesthetised animal.Adenosine diphosphate (ADP) and collagen gave dose- dependent falls in the circulating platelet count when injected into rats, guinea pigs and rabbits. This enabled aggregation to be accurately quantitated in vivo.The infusion of PGI2 (0.25-1 ug/kg/min) in anaesthetised rats and rabbits produced a dose-dependent inhibition of the fall in platelet count produced by ADP and collagen. The formation of PGI2 can be inhibited in vitro by 15- hydroperoxyarachidonic acid (15HPAA). When 20 ug/kg/min of 15HPAA was infused into rats, aggregation produced by collagen was significantly increased suggesting that PGI2 is continuously formed by the rat vascular endothelium. This observation was confirmed by infusing 6-keto PGF1α antiserum. This antibody also prevented the inhibitory activity of PGI2 on collagen-induced aggregation. The study of continuous platelet counting in guinea pigs has been hampered by the occurrence of thrombocytopenia in certain animals. When 2 ug/kg/min of PGI2 was infused for 10 mins, a rise in the circulating platelet count to a steady plateau 4-5 × 105 platelets occurredThese experiments have shown that PGI2 will prevent aggregation by ADP and collagen and will reverse spontaneous thrombocytopenia and that PGI2 is continuously released from the vessels of anaesthetised rats.

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Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

Blood ◽

10.1182/blood.v77.4.770.770 ◽

1991 ◽

Vol 77 (4) ◽

pp. 770-779 ◽

Cited By ~ 124

Author(s):

AD Michelson ◽

PA Ellis ◽

MR Barnard ◽

GB Matic ◽

AF Viles ◽

...

Keyword(s):

Whole Blood ◽

Actin Polymerization ◽

Flow Cytometric Analysis ◽

Adenosine Diphosphate ◽

Surface Expression ◽

Surface Glycoprotein ◽

Platelet Surface ◽

Granule Secretion

Abstract In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

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A New Model for Antiplatelet Drug Discovery

10.1055/s-0039-1682679 ◽

1977 ◽

Author(s):

R.N. Saunders ◽

T.S. Burns ◽

E.R. Waskawic ◽

M.R. Stelzer

Keyword(s):

Platelet Count ◽

Platelet Rich Plasma ◽

Antiplatelet Agents ◽

Adenosine Diphosphate ◽

Male Rats ◽

Antiplatelet Drug ◽

Preliminary Evaluation ◽

Transient Ischemic Attacks

Circulating platelet aggregates (CPA) have been observed by Wu and Hoak (Lancet 2: 294, 1974) in the blood of patients with transient ischemic attacks, acute myocardial infarction and acute peripheral arterial insufficiency. We have discovered that retired breeder (RB) male rats have spontaneous CPA which respond to therapy with clinically proven antiplatelet drugs. CPA are described as the ratio of the platelet count from blood drawn into a citrate/formalin solution compared to the platelet count from blood drawn into a citrate solution. A platelet aggregate ratio (PAR) of 1.0 is indicative of the absence of CPA. The PAR of 24 virgin male rats (0.94 ± .02, S.E.M.) was significantly greater (P<0.01) than that of 34 male RB rats (0.77 ± .02)-indicating the presence of increased CPA in the RB rats. Acetylsalicylic acid (ASA) and sulfinpyrazone (S) have been successfully used clinically in patients with CPA and transient ischemic attacks (Stroke 6: 521, 1975).RB rats treated with ASA or S (100 mg/kg/ day, i.g. for 8 days) demonstrated significantly (P < 0.01) increased PAR of 0.88 ± 0.02 and 0.99 ± 0.02 respectively. In vitro responses of RB platelets in citrate-treated platelet-rich plasma to adenosine diphosphate (0.4 yM to 3 uM/109 platelets) and collagen (420 μg to 1470 μg/109 platelets) were similar in sensitivity to platelets from virgin rats. This suggests that the hyperactivity of RB rat platelets expressed as CPA is related to an increased aggregate stimulation in vivo. The results indicate that the male RB rat may be a useful model for the preliminary evaluation of therapeutically useful antiplatelet agents.

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Role of P-Selectin and GPIbα in the Fast and Delayed Clearance of Transfused Platelets.

Blood ◽

10.1182/blood.v104.11.3625.3625 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3625-3625

Author(s):

Valery Leytin ◽

David J. Allen ◽

Adam Gwozdz ◽

Marie B. Garvey ◽

John J. Freedman

Keyword(s):

Platelet Activation ◽

Survival Curve ◽

Rabbit Model ◽

Reticuloendothelial System ◽

Surface Glycoprotein ◽

In Vitro Assays ◽

Platelet Surface ◽

Adhesive Interactions

Abstract Platelets become activated during preparation and storage of platelet concentrates (PCs) for transfusion. Flow cytometric assays of platelet activation can be employed for quantifying in vitro quality of PCs. It remains, however, unclear whether the level of in vitro platelet activation in stored PCs correlates with in vivo survival of the platelets after transfusion. Platelet surface glycoprotein (GP) Ibα and P-selectin (CD62) can be involved in regulation of posttransfusion PC clearance, mediating adhesive interactions of platelets with counter-receptors on leukocytes and endothelial cells. Recently, we described a rabbit model for analyzing posttransfusion kinetics of human PCs (Leytin et al, Transfusion42:711, 2002, Transfusion43:983, 2003). In the present work, we used this validated model for studying the implication of CD62 and GPIbα expression in posttransfusion PC clearance. Platelet activation in vitro was determined by flow cytometry using anti-CD62 and anti-GPIbα antibodies. PC clearance in vivo was evaluated in rabbits with inhibited reticuloendothelial system, as measured by 0.5 hr (R0.5), 24 hr (R24) and total (R∑) platelet recoveries, and survival time (ST). Correlations were analysed between in vitro assays of platelet activation and in vivo clearance of conventional (Day 2–5) and outdated (Day 7–8) PCs stored at 22°C, and refrigerated PCs. We found that the binding of anti-CD62 antibody was significantly increased in outdated and refrigerated PCs compared to conventional PCs, reflecting an increased exposure of CD62 on the platelet surface. In contrast, binding of anti-GPIbα antibody was significantly decreased during prolonged and refrigerated PC storage. The clearance of conventional (Day 2–5) PCs from the circulation can be described by a biphasic survival curve. The first (early) phase of platelet clearance is characterized by fast (≥ 14 x 109 platelets per hour) platelet removal, whereas the second (delayed) phase has a much slower rate of platelet clearance (approximately 0.4 x 109 platelets per hour). The biphasic survival curves were also obtained for outdated and refrigerated PCs, and were employed for determining fast (R0.5), delayed (ST and R24) and overall (fast + delayed; R∑) platelet clearance in vivo. We found that when stored PCs are cold-damaged, their in vivo viability decreased significantly, in comparison to conventional PCs, as reflected by the fast, delayed and overall platelet clearances. Viability of Day 7–8 PCs is also decreased, compared to Day 2–5 PCs, but only the fast and overall platelet clearance increased significantly. Negative correlation was observed between in vitro anti-CD62-binding to platelets and their fast, but not delayed, clearance. In contrast, anti-GPIbα-binding showed positive correlations with delayed, but not fast, platelet clearance. Overall clearance correlated better with anti-GPIbα- than with anti-CD62-binding. We also demonstrated that CD62 is shed from the platelet surface after transfusion, whereas GPIbα remains unchanged on the surface of circulating platelets. The data suggest that CD62 exposure during PC storage triggers fast CD62-mediated PC clearance. However, after CD62 shedding during platelet circulation, in vitro GPIbα alterations, such as cleavage, clustering or conformation changes, may determine long-term GPIbα-mediated PC clearance.

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Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound [see comments]

Blood ◽

10.1182/blood.v77.4.770.bloodjournal774770 ◽

1991 ◽

Vol 77 (4) ◽

pp. 770-779 ◽

Cited By ~ 1

Author(s):

AD Michelson ◽

PA Ellis ◽

MR Barnard ◽

GB Matic ◽

AF Viles ◽

...

Keyword(s):

Whole Blood ◽

Actin Polymerization ◽

Flow Cytometric Analysis ◽

Adenosine Diphosphate ◽

Surface Expression ◽

Surface Glycoprotein ◽

Platelet Surface ◽

Granule Secretion

In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.

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Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647788 ◽

1973 ◽

Vol 29 (02) ◽

pp. 490-498 ◽

Cited By ~ 6

Author(s):

Hiroh Yamazaki ◽

Itsuro Kobayashi ◽

Tadahiro Sano ◽

Takio Shimamoto

Keyword(s):

Platelet Count ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

The Other ◽

Endothelial Surface ◽

Important Interaction ◽

Blood Coagulability ◽

The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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