Bax activators potentiate coated-platelet formation.

BACKGROUND: Activation of platelets with collagen plus thrombin produces a subset of cells known as coated-platelets. Coated-platelets retain several alpha-granule proteins on their surface, express phosphatidylserine (PS), lose mitochondrial potential and release microparticles. OBJECTIVE: A number of these characteristics are also observed in apoptotic cells, and this similarity leads to the hypothesis that mechanisms controlling initiation of apoptosis might also affect generation of coated-platelets. RESULTS: In this report, we demonstrate that BH3 mimetics, molecules that facilitate apoptosis by releasing pro-apoptotic Bax from its antiapoptotic partner Bcl-2, are able to promote coated-platelet formation as monitored by several different markers of these cells. Specifically, gossypol and methoxy-antimycin (MAM) promote fibrinogen retention, mitochondrial depolarization, and PS exposure upon activation with thrombin plus convulxin, a ligand of the glycoprotein VI collagen receptor. Gossypol also potentiates microparticle release by convulxin plus thrombin activated platelets although MAM does not. In addition, Bax activators together with thrombin generate coated-platelets, effectively bypassing the requirement for convulxin. CONCLUSION: These findings further support a close relationship between apoptotic-like events and the production of coated-platelets.

Quantitation of microparticles released from coated-platelets.

Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also release microparticles. Platelets were prelabeled with 2.5 microm Bodipy-maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3-0.5 microm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy-labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 +/- 5 microparticles per coated-platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated-platelet production, significantly increased the number of microparticles released per coated-platelet.

Collagen response and glycoprotein VI function decline progressively as canine platelets age in vivo.

Clinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define age-dependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxin-binding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.

Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer.

Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigation into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally mediated gene transfer and subsequently detected in cultured MKs with a monoclonal antibody (MoAb) that specifically recognizes the murine protein. CD34+ human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induce MK progenitors into the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depleted medium for 3 to 7 additional days. Flow cytometry analysis using the anti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis was performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because deteriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TAB+ particles were functionally active. Addition of phorbol myristate acetate resulted in the redistribution of P-selectin (CD62) from the alpha granule to the platelet surface as detected by MoAbs S12 and G5 in three-color flow cytometry analyses. These studies showed that up to 76% of the mCD9+ TAB+ particles were functionally active. The data show that retrovirally mediated gene transfer is a viable approach for genetically altering MK progenitors, resulting in platelets that express heterologous proteins.

Erythropoietin potentiates thrombus development in a canine arterio-venous shunt model.

Erythropoietin (EPO) has been previously shown to affect platelet as well as red cell production. In addition, recent studies demonstrated that platelets from EPO-treated dogs are hyperreactive towards thrombin when compared to age-matched, control platelets. This report extends these observations by quantitating the thrombogenic potential of EPO in dogs. Dogs with arterio-venous (A-V) shunts received 100 U EPO/kg/day for 6 days, and thrombogenicity was serially monitored by insertion of a thrombotic surface into the A-V shunt. The resulting experimental thrombi were analyzed for platelet and erythrocyte content after formalin-fixation and chymotrypsin digestion, a technique which allows non-isotopic quantitation of cellular components. By day 5 of EPO-administration all animals demonstrated a significant increase in platelet and red cell content of the experimental thrombi; the average increase in platelet number was 2.94 +/- 0.12 fold (mean +/- 1 SE; n = 3; p = 0.006) above baseline while that for red cells was 2.46 +/- 0.18 fold above baseline (p = 0.023). After cessation of EPO, thrombogenicity returned to normal. During EPO-treatment, the percentage of thiazole orange-positive (TO+) platelets increased significantly to 17.2 +/- 1.6% (mean +/- 1 SE; n = 3) on day 5 compared to a pre-treatment level of 8.5 +/- 0.9% (p = 0.029). Although the percentage of TO+ erythrocytes also increased during the short course of EPO administration, the change was not significant. Despite the increases in TO+ cells, total platelet and erythrocyte counts did not change significantly within the time frame of these experiments. Fibrin/fibrinogen content of the experimental thrombi was unaltered with EPO-treatment. These data demonstrate that human EPO is pro-thrombotic in dogs and, in conjunction with earlier studies, suggest that hyperreactive platelets may be responsible for the potentiated thrombogenicity.

Erythropoietin administration increases production and reactivity of platelets in dogs.

Administration of erythropoietin (EPO) to adult dogs resulted in a dramatic increase in the number of thiazole orange-positive (TO+) platelets, also referred to as reticulated platelets. Pre-treatment level of TO+ platelets was 6.2 +/- 0.5% (mean +/- 1 SE: n = 5); following day 5 of treatment with 500 U EPO/kg/day, the percentage of TO+ platelets peaked at 16.8 +/- 2.3% (n = 5; p <0.02). After cessation of the hormone, the number of TO+ platelets fell rapidly to below starting levels. Unexpectedly, there was a significant decline in total platelet count during EPO administration despite an increased level of TO+ platelets. To assess platelet reactivity, total platelets and TO+ platelets from EPO-treated dogs were analyzed for thrombin-responsiveness as quantitated by P-selectin expression on the cell surface; reactivity was expressed as a thrombin EC50, the thrombin concentration required to activate 50% of platelets. Both total and TO+ platelets were hyperreactive during EPO treatment when compared either to pre-treatment values or to control animals. Thrombin EC50 values for total and TO+ platelets on day 5 fell to 66.5 +/- 5.4% (mean +/- 1 SE; n = 5; p <0.02) and 62.2 +/- 8.7% (n = 5; p <0.025), respectively, of pre-treatment levels. These data indicate that EPO not only promotes the synthesis of increased numbers of TO+ platelets in the dog but that these newly produced platelets are hyperreactive when compared to TO+ platelets from control animals.

Inhibition of the acute-phase response in vivo by anti-gp130 monoclonal antibodies.

The acute-phase response is believed to be an important systemic defence reaction to inflammation during infection, trauma, injury or neoplasia. Although the interleukin-6 (IL-6) family of cytokines appear to be the major regulators of the acute-phase reaction, the exact biological significance of this process remains unknown. In this study, a panel of monoclonal antibodies (Mabs) was raised against the extracellular domain of human gp130 (the common signal transducing chain of the IL-6 cytokine family) in order to inhibit the biological activity of IL-6-like cytokines in vivo. Mabs designated 4B11 and 2H4 were most effective in the inhibition of the in vitro acute-phase response on hepatoma cells and prevented the IL-6-induced growth inhibition of A375 cells. Administration of the antibodies to dogs at a dosage of 8 mg/kg/d showed that 2H4 was a potent inhibitor of the IL-6-induced (40 micrograms/kg/d) acute-phase response, abrogating IL-6-mediated increments in fibrinogen, C-reactive protein and the platelet count. This antibody, the first described to abrogate the acute-phase response in vivo, may not only permit development of a new anti-inflammatory strategy, but provides an excellent tool for defining the function of acute-phase proteins in inflammation and infection.

Relative reactivity of platelets from thrombopoietin- and interleukin-6-treated dogs.

Previous reports have shown that interleukin-6 (IL-6) enhances the responsiveness of platelets to thrombin stimulation and has modest thrombocytopoietic effects in vivo. Thrombopoietin (TPO; mpl ligand) has been shown to have dramatic thrombocytopoietic effect in vivo, but little is known of its capacity to alter platelet function. In this study, a direct comparison of the effects of IL-6 and TPO on platelet function in dogs has been performed, with modest doses of TPO (1 microgram/kg/d) chosen to match or moderately exceed the platelet counts achieved with IL-6 (40 micrograms/kg/d) for 10 days. Platelet responsiveness to thrombin stimulation was assessed in TPO-treated, IL-6-treated, and control dogs by flow cytometric measurement of P-selectin expression. On day 5, the dose of thrombin promoting half maximal stimulation (EC50) of platelets was not significantly changed in TPO-treated dogs, whereas in IL-6-treated dogs the EC50 decreased to 73.1% +/- 6.1% (mean +/- 1 SD; n = 5) of control values (P < 0.01). These experiments were performed on both gel-filtered platelets and washed whole blood, indicating that the observed changes in EC50 were caused by cytokine-mediated alteration of platelets rather than plasma components. Because it has been shown that thiazole orange specifically labels a subpopulation of dog platelets that is less than 24 hours old, the thrombin responsiveness of these young, newly synthesized platelets was determined. The EC50 of thiazole orange-positive platelets from IL-6-treated dogs decreased dramatically by day 5 to 46.5% +/- 13.1% (n = 4) of control values (P < 0.001), whereas TPO-treated dogs did not significantly change. When TPO was directly incubated with platelets ex vivo, no effects on either thrombin-mediated P-selectin expression or adenosine diphosphate-induced fibrinogen binding were observed. These data show that IL-6 alters platelet function, as measured by reactivity to thrombin, whereas TPO does not. This divergence in function is observed even though TPO is equally, or more, effective at promoting platelet production under these experimental conditions.

Quantitation of platelet life span in splenectomized dogs.

Platelet life spans in dogs were measured pre- and post- splenectomy utilizing in vitro whole blood biotinylation. Four splenectomized dogs were found to have significantly lengthened platelet life spans, 193 +/- 7 hours (mean +/- 1 SD;n=4) postsurgery vs. control life spans of 131 +/- 15 hours (n=6; p< 0.01) when analyzed with the multiple-hit model. Additionally, platelets from normal dogs transfused into splenectomized dogs were found to have convex survival curves with extended life spans approximating that of the splenectomized dog. These data indicate that the spleen is a significant determinant of platelet life span in dogs, with survivals increasing approximately 47% upon splenectomy.

Cytokine-induced alteration of platelet and hemostatic function.

A number of nonplatelet-specific cytokines that augment platelet recovery following chemo/radiotherapy have been described. The members of the interleukin 6 (IL-6) family have properties that influence the hematopoietic system beyond their modest thrombocytopoietic effects. Studies performed in a canine model with IL-6 have shown that this factor augments plasma fibrinogen and von Willebrand factor (vWf) concentrations and decreases the level of free protein S. IL-6 appears to decrease the bleeding time in thrombocytopenic dogs, although this effect does not seem to be due to a direct influence of the factor on endothelial vWf or tissue factor production. The factor does not directly alter platelet function in vitro, but when administered to dogs, it increases the sensitivity of the platelets to activation by thrombin. Normal platelets injected into IL-6-treated dogs, and platelets from IL-6-treated dogs injected into normal animals, survive normally. Following injection of either IL-6 or the more specific thrombocytopoietic cytokine thrombopoietin (TPO), IL-6 increases platelet responsiveness to thrombin-induced activation to a greater extent than does TPO. The data show that IL-6 has certain properties that might be construed as prohemostatic, and these properties may prove to be useful clinically.

Demonstration that thiazole-orange-positive platelets in the dog are less than 24 hours old.

Approximately 6% of dog platelets are positive for staining with thiazole orange, a dye frequently used to stain ribonucleic acid. In this report, thiazole-orange positivity is shown to mark platelets that are less than 24 hours old. Dog platelets were derivatized in vivo with N-hydroxysuccinimido biotin such that greater than 95% of all platelets were biotinylated. Newly synthesized, nonbiotinylated platelets were then monitored by flow cytometry for their ability to bind thiazole orange. After biotinylation, the percentage of biotin-negative, thiazole-orange-positive platelets increased gradually from 0.72% at 30 minutes to 5.44% at 24 hours. These data indicate that thiazole-orange staining does label newly synthesized platelets.

Dog platelets accumulate intracellular fibrinogen as they age.

The alpha granules of circulating platelets are dynamic structures that acquire endogenous and exogenous components by synthesis and uptake, respectively. The uptake of exogenous components is a result of either receptor-mediated endocytosis or fluid-phase pinocytosis. Despite many detailed studies on the function and content of alpha-granules, little is known of the impact of platelet age on these organelles. In this report, we describe the use of platelet biotinylation to identify and isolate aged platelets for the analysis of alpha-granule contents. When aged platelets were permeabilized and examined by flow cytometry utilizing fluorescently labeled antibodies, two exogenously acquired proteins, fibrinogen and immunoglobulin G, were found to increase significantly with platelet age. The levels of intracellular fibrinogen were found to be elevated relative to control, 114 +/- 2% and 119 +/- 5% on days 4 and 5 postbiotinylation, respectively; the life span of dog platelets is 6.0 days. Intracellular immunoglobulin G content increased similarly. Levels of two endogenously synthesized proteins, thrombospondin and P-selectin, were not elevated in aged platelets. Confirmation of the flow cytometric data was obtained by isolating aged, biotinylated platelets by fluorescence-activated cell sorting and quantitating the fibrinogen levels with an ELISA assay. For platelets averaging 4.6 days of age, the fibrinogen level was elevated to 128 +/- 23% of the level for the entire platelet population. These data demonstrate that age-dependent changes in exogenously acquired alpha-granule proteins do occur and that the uptake mechanism for these proteins is active throughout the platelet life span.

Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression.

After the intravenous infusion of N-hydroxysuccinimido biotin into dogs, 80.6% +/- 9.7% (n = 5) of platelets were covalently labeled with biotin. The in vivo survival of the biotinylated platelets was monitored by flow cytometry and was normal as compared with previous reports for dog platelets. The ability of the biotinylated platelets to be activated was analyzed by measuring the expression of cell-surface P-selectin after incubation with graded concentrations of thrombin. When P-selectin expression was examined 3 hours after labeling, biotinylated platelets were indistinguishable from the nonlabeled population of platelets, indicating that biotinylation did not adversely affect the cells. On consecutive days after biotinylation, the thrombin dose-response curves for biotinylated and nonbiotinylated platelets were repeated, and as the biotinylated-platelets aged, they became less responsive to thrombin. On days 3, 4, and 5, the thrombin EC50 for the aged, biotinylated platelets as compared with the total population of platelets was 136%, 150%, and 178%, respectively. Increasing age clearly impairs the reactivity of platelets towards thrombin as quantitated by the expression of cell-surface P-selectin.

Alteration of platelet function in dogs mediated by interleukin-6.

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL-6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.

Biotinylated platelets: a new approach to the measurement of platelet life span.

The in vivo life span of dog platelets was determined by derivatizing whole blood with N-hydroxysuccinimido biotin, reinfusing the biotinylated blood and subsequently monitoring the survival of the biotinylated platelets with flow cytometry. We found that the biotinylated platelets had a mean life span of 6.0 +/- 1.1 d as determined by curve-fitting the platelet disappearance data to gamma functions. These data are in good agreement with literature values of platelet life span for canine platelets labelled with either 111Indium-oxine or 51Chromium. Biotinylated platelets were analysed after reinfusion and found to aggregate normally in response to the agonists adenosine diphosphate and phorbol myristate acetate. These experiments demonstrate that biotinylated platelets survive normally in vivo and that this labelling method can be used for determining platelet life spans.

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.

Canine megakaryocytopoiesis: analysis utilizing a monoclonal antibody to a 140-kd dog platelet protein.

The dog, a convenient and relatively large animal whose size permits repetitive blood and marrow sampling, marrow biopsy, and mechanical apheresis, would be a suitable experimental model for the study of in vivo megakaryocytopoiesis. A monoclonal antibody to a 140-kd dog platelet membrane protein has been developed that reacts with canine megakaryocytes. Using the fluoresceinated derivative of this antibody to identify megakaryocytes and propidium iodide staining to measure relative DNA content, the DNA distribution of megakaryocytes in dog bone marrow or in cultured dog marrow cells could be rapidly assessed by flow cytometry. Normal dogs showed a modal ploidy of 16N (54%), with 17% 8N and 16% 32N. In contrast, dogs made thrombocytopenic by plateletpheresis showed a shift in distribution to higher ploidy cells (36% 32N). The data show that use of a specific marker of megakaryocytes in combination with flow cytometric analysis is an accurate and reproducible method of assessing megakaryocytopoiesis in a convenient and easily manipulable animal model.