THROMBOCYOPENIC EFFECT ON MICE

• Effects of reduced dietary magnesium on platelet production and function in hams

http://www.ionchannels.org/showabstract.php?pmid=2232716

The increased vulnerability of animals fed a magnesium (Mg)-deficient (MD) diet to ischemia-induced myocardial
necrosis has been attributed to changes in myocardial electrolyte metabolism. However, a variety of hematologic
changes have also been reported in MD and some of these, such as an increase in platelet aggregability, may
contribute to the increased myocardial vulnerability. In the present study, we quantified the effect of MD on platelet
and megakaryocyte abundance as well as on platelet aggregability with and without an administered calcium
channel blocker (nifedipine). Hamsters were fed either a "minimal Mg" diet, in which the level of Mg was kept just
high enough to prevent seizures, or a "preset Mg" diet containing precisely known amounts of Mg. Animals fed the
minimum Mg diet showed an initial increase in the platelet count, which returned to control range when the
dietary Mg was increased to 9 mmoles/kg. Animals on the preset Mg diet showed an increased platelet count if
the Mg level was 10 mmoles/kg or less. In addition to the increase in number, platelets from MD animals were less
responsive to the aggregation-inhibiting effect of nifedipine than were platelets from control animals, although MD
itself did not result in an increased aggregability under the conditions used here. Animals with an increase in
circulating platelets showed decreased megakaryocyte abundance in the femoral marrow, but megakaryocytes that
were present were larger than those in control animals. These results indicate a profound effect of dietary Mg
deficiency on platelet biology. The observed changes could contribute to the increase in myocardial vulnerability to
injury found in MD animals.

• C3H mice have larger spleens, lower platelet counts, and shorter platelet lifespans than C57BL
mice: an animal model for the study of hypersplenism.
http://www.ncbi.nlm.nih.gov/pubmed/9293898

• Study of thrombopoietin for gene therapy of thrombocytopenia

Song Chong, Daru Lu, Changben Li, Xinfang Qiu and Jinglun Xue
http://www.springerlink.com/content/h101634t61615rp0/

Abstract
Thrombopoietin (TPO) is likely to he a potent, specific and reliable medication in the treatment of
thrombocytopenia. A TPO-highly-expressed plasmid pcDNA3-TPO was constructed and a primary study was made
on the expression of TPO cDNAin vitro and gene transfer study for thrombocytopeniain vivo. rhTPO showed
complete and stable bioactivity by a series of indicators. High expression of TPO was detected in plasma from
healthy mice or thrombocytopenia mice model receiving direct intramuscular injection of pcDNA3-TPO. And the
platelet level of healthy mice peaked to 1.9-fold of baseline. Mice with CTX-induced thrombocytopenia achieved
profound nadirs, acceleration of recovery, even 1.8–2.0-fold supranormal levels of peripheral platelet
counts. The results offered experimental support for clinical application of gene therapy for thrombocytopenia via
direct intramuscular injection of TPO cDNA. (cyclophosphamide)

• Picogram doses of lipopolysaccharide exacerbate antibody-mediated thrombocytopenia and reduce the

therapeutic efficacy of intravenous immunoglobulin in mice
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2141.2007.06777.x/abstract

Summary
Exacerbation of antibody-mediated thrombocytopenia following infection with viruses has recently been
demonstrated in a mouse model of the disease. The phenomenon was caused by an increased activation of
phagocytes through gamma-interferon secretion in response to infection. Endotoxins from Gram-negative bacteria
are also known to be potent activators of phagocytic cells. The objective of the present work was to determine
whether lipopolysaccharide (LPS) could exacerbate antibody-mediated thrombocytopenia in vivo and so alter the
therapeutic efficacy of intravenous immunoglobulin (IVIg), using a mouse model of thrombocytopenia. Very low
doses of LPS (picogram range) and of anti-platelet antibodies (nanogram range), which did not induce
thrombocytopenia individually, could synergize in vivo, resulting in significant decreases in platelet counts. The
therapeutic efficacy of IVIg in antibody-mediated thrombocytopenia was significantly reduced in presence of LPS.
These in vivo observations further support a role for bacterial infections in the aetiology of immune
thrombocytopenic purpura (ITP) and may contribute to better understand the recognized lack of efficacy of IVIg in a
significant proportion of patients with ITP.

• Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an
 inhibitor of Syk
http://bloodjournal.hematologylibrary.org/cgi/content/full/113/14/3154

TP. With the use of a well-described murine model of ITP,12 we tested the ability of R788 (the prodrug of R406) to
ameliorate thrombocytopenia. Mice injected with an antibody directed to integrin {alpha}IIb were profoundly
thrombocytopenic when platelets were enumerated 24 hours after injection (Figure 1A column 1). However,
mice pretreated with 25 or 40 mg/kg R788 were protected from thrombocytopenia (Figure 1A columns 3 and 4,
respectively; P < .05 for both compared with Nil). Mice treated with IVIg responded as expected (Figure 1A column
5), whereas those pretreated with the vehicle alone (Figure 1A column 2) displayed no protection, that is, profound
thrombocytopenia.

• Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage
http://www.pnas.org/content/103/4/855.ful

Platelet Depletion Led to the Formation of Fewer Vessels and, Most Notably, Highly Hemorrhagic Vessels in a
Cornea Angiogenesis Model. To determine whether platelet depletion affects experimental angiogenesis, we used
the cornea micropocket assay. Pellets containing the slow-release polymer hydron and bFGF were surgically
implanted into the micropockets of mouse corneas, which are avascular. Thrombocytopenia was induced 1 h after
implantation of the pellets. A single i.p. injection of antiplatelet antibody in the mice resulted in a profound
thrombocytopenia within 1 h, with a >95% reduction in the number of circulating platelets. The
thrombocytopenia induced by this single injection was transient, and platelet levels started to return to
normal by day 3. Sustained thrombocytopenia was induced by a repeated injection of the antibody on the third
day. Sprouting of the limbal vessel into the cornea was observed. Newly formed vessels in the corneas of the
control mice were clearly visible and nonhemorrhagic at 72 and 96 h (Fig. 1A Left) and also at 5 days (data not
shown). The angiogenic vessels in the corneas of the platelet-depleted animals were less well defined and were
surrounded by extravascular RBCs (Fig. 1 A Right). The length of new vessels was therefore difficult to define in
corneas of the platelet-depleted animals. However, there was a significant decrease in clock hours (length of the
limbal vessel showing sprouts) 5 days after pellet implantation in the platelet-depleted group when compared with
the control group (2.21 ± 0.16 for IgG and 1.80 ± 0.11 for antiplatelet group, P < 0.05). Animals treated with the
antiplatelet antibody also had lower numbers of corneal vessels when compared with the control animals (Fig.
1B ). The number of vessels was significantly decreased in the antiplatelet antibody group at 72 and 96 h after
pellet implantation (P < 0.05), and the difference was close to significant at 120 h after pellet implantation (P =
0.057). To compare the RBC leakiness of the newly formed vessels, a blinded observer assigned a hemorrhagic
score to the eyes. The score was significantly different in the two sets of animals at all time points (P < 0.001)

• Antimetastatic effects associated with platelet reduction

http://www.pnas.org/search?
fulltext=thrombocytopenic+mice&sortspec=date&submit=Submit&andorexactfulltext=phrase

...units) and bled at different intervals from the right heart. Because serum was difficult to obtain from the
neuraminidase-thrombocytopenic mice, heparinized plasma (0.05-0.5 ml) was incubated at 37?C for 5 min in
the presence of 0.2-0.4 ml of sialyllactose as substrate...

• Anagrelide metabolite induces thrombocytopenia in mice by inhibiting megakaryocyte maturation

without inducing platelet aggregation
http://www.exphem.org/article/S0301-472X%2801%2900742-1/abstract

Abstract
Objective
The mechanism for anagrelide's potent platelet lowering activity in human subjects is not well defined. Studies
related to anagrelide function have been hampered by its lack of activity in nonhuman primates and water
insolubility. In an effort to define the mechanism whereby anagrelide exerts its therapeutic effect, we identified a
water-soluble metabolite (anagrelide.met). The availability of anagrelide.met allowed, for the first time, parallel in
vitro and in vivo animal studies centered on the mechanisms by which anagrelide lowers platelet levels.

Materials and Methods

The effects of anagrelide.met on proliferation and maturation of mega-karyocytes (MKs) as well as platelet
production were studied both in vitro and in vivo.

Results
Anagrelide.met is capable of blocking in vitro MK migration by 20% to 40%. At 100 ng/mL, anagrelide.met
selectively blocked in vitro MK maturation, resulting in a 50% decrease in the total number of CD41a+ MKs,
corresponding with a 30% decrease in MK ploidy by day 10 and a 60% decrease by day 20. Daily intraperitoneal
injections of anagrelide.met 100 μg into BALB/c mice was sufficient to significantly decrease platelet counts within
24 to 48 hours, stabilizing to 40 to 50% of normal levels by day 5. This was associated with a 45% decrease in the
number of developing MKs and an increase in thrombopoietin levels. Anagrelide.met did not alter WBC counts,
hematocrit, or bleeding time, or lead to any apparent signs of toxicity. Furthermore, unlike the parent anagrelide
compound, anagrelide.met did not inhibit ADP-induced platelet aggregation even at high concentrations (10
μg/mL).

Conclusion
We describe a cross-species reactive anagrelide metabolite that selectively inhibits MK maturation and migration,
lowering platelet levels without influencing platelet aggre-gation.

• Effects of a novel pyridylsulphonyl thiazole derivative, FR115092, on autoimmune and mitomycin C-

induced thrombocytopenia in mice. (obat kemoterapi)
http://www.ncbi.nlm.nih.gov/pubmed/10467963

Abstract
Dapsone (4,4'-diaminodiphenyl sulphone), an antileprotic and antimalarial drug, has been reported to be of
therapeutic benefit in idiopathic thrombocytopenic purpura in the clinic. However, adverse reactions such as
haemolytic anaemia have often been observed. In this study, we found that dapsone increased the number of
platelets and decreased the number of red blood cells in male (NZWxBXSB)F1 (W/BF1) mice, an animal model of
idiopathic thrombocytopenic purpura. In studies to prepare derivatives of dapsone with weaker side effects than the
parent compound, FR115092 (2-[5-(2-pyridylsulphonyl)thiazolyl]amine) was discovered. The effect of FR115092 on
the number of blood cells was studied and compared with dapsone in mice. FR 115092 increased the number of
platelets without reducing the number of red blood cells in W/BF1 mice. This drug significantly suppressed the
increase in circulating autoantibodies against platelets and increased the number of megakaryocytes. Furthermore,
FR115092 inhibited the reduction of the number of platelets in mitomycin C-induced thrombocytopenic mice, as a
consequence of its enhancement of growth and maturation of megakaryocytes. These findings suggest that
FR115092 may be effective against various thrombocytopenias, without inducing haemolytic anaemia.

• Platelets of the Wistar Furth Rat have Reduced Levels of Alpha-Granule Proteins
An Animal Model Resembling Gray Platelet Syndrome
(journal_PDF)

Rats of the Wistar Furth (WF) strain have hereditary macro-thrombocytopenia (large mean platelet volume
[MPVJ with in-creased platelet size heterogeneity and reduced platelet count).

• Picogram doses of lipopolysaccharide exacerbate

antibody-mediated thrombocytopenia and reduce the
therapeutic efficacy of intravenous immunoglobulin in mice
(journal_PDF)

Preliminary experiments determined the time course of platelet clearance following intravenous injection of
MWReg30, and indicated that at 6 h postinjection, platelet counts were already decreased and were comparable to
those measured at 24 h postinjection (data not shown). In addition, previous studies in mice showed that,
following injection of very high doses of LPS (>0Æ1 mg/kg), platelet counts were significantly decreased
within 4 h of injection (Shibazaki et al, 1996; Andonegui et al, 2005), indicating that the effects of LPS were very
rapid. We thus selected the 6-h postinjection time- point to perform our analyses in the subsequent experiments.
We next determined the dose of MWReg30 that did not significantly reduce platelet counts per se. Mice were
injected with different doses of MWReg30 and platelet counts were determined by flow cytometry. The results (Fig
1A) showed that injection of 0Æ25 lg of MWReg30 did not reduce platelet
counts, although the presence of IgG bound to platelets was readily detected following staining with Alexa Fluor
488 goat anti-rat IgG (Molecular Probes, Invitrogen, Burlington, Canada) (Fig 1B).

• Neuraminidase-induced Thrombocytopenia in Rats

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2141.1972.tb08790.x/abstract

Summary. The purpose of this study was to elucidate the effect of neuraminidase on circulating platelets in rats
and the pathogenesis of this effect. Injection of bacterial, purified bacterial and viral neuraminidase caused
thrombocytopenia of various degrees. Thrombocytopenia was less pronounced in rats given viral neuraminidase
and in splenectomized animals. A dose-response effect was seen only with purified bacterial neuraminidase
injected intravenously. Ultrastructural studies of blood platelets were performed after injection of neuraminidase.
Fifteen minutes after injecting neuraminidase, before a definite fall in the count, platelets showed evidence of
structural damage such as irregular shapes, centripetal migration and fusion of granules and bleb formation. The
structural alterations were progressive, reaching a maximum at about 2–4 hr. At no time were large platelet
aggregates observed in the circulating blood. Four hours after injection of neuraminidase, when the platelet count
dropped significantly, many platelets had normal ultrastructurc. By 24 hr, the majority of circulating platelets
appeared normal; however, the splenic sinusoids and macrophages contained platelets with the same structural
abnormalities seen in the circulation 15 min to 2 hr after injection of neuraminidase. We conclude that
neuraminidase alters the platelet membrane resulting in its rapid and permanent removal by the reticuloendothelial
system.

• Additional information about Xagrid

http://www.flexyx.com/X/Xagrid.html

Xagrid Indication: For the treatment of patients with thrombocythemia, secondary to myeloproliferative disorders, to
reduce the elevated platelet count and the risk of thrombosis and to ameliorate associated symptoms including
thrombo-hemorrhagic events.
Mechanism Of Action: The mechanism by which anagrelide reduces blood platelet count is still under
investigation. Studies in patients support a hypothesis of dose-related reduction in platelet production resulting from
a decrease in megakaryocyte hypermaturation. In blood withdrawn from normal volunteers treated with anagrelide,
a disruption was found in the postmitotic phase of megakaryocyte development and a reduction in megakaryocyte
size and ploidy. At therapeutic doses, anagrelide does not produce significant changes in white cell counts or
coagulation parameters, and may have a small, but clinically insignificant effect on red cell parameters. Xagrid
inhibits cyclic AMP phosphodiesterase III (PDEIII). PDEIII inhibitors can also inhibit platelet aggregation. However,
significant inhibition of platelet aggregation is observed only at doses of anagrelide higher than those required to
reduce platelet count.
Drug Interactions: Not Available
Food Interactions: Take without regard to meals.
Food appears to reduce the area under the curve by 13.8%, without clinical consequence.
Generic Name: Anagrelide
Synonyms: Anagrelide HCL; Anagrelide Hydrochloride; BL-4162A
Drug Category: Fibrinolytic Agents; Platelet Aggregation Inhibitors; Antithrombotic Agents
Drug Type: Small Molecule; Approved
Other Brand Names containing Anagrelide: Agrylin; Xagrid;
Absorption: Not Available
Toxicity (Overdose): There are no reports of overdosage with anagrelide, however thrombocytopenia, which can
potentially cause bleeding, is expected from overdosage. Single oral doses of anagrelide at 2,500, 1,500 and 200
mg/kg in mice, rats and monkeys, respectively, were not lethal. Symptoms of acute toxicity were: decreased motor
activity in mice and rats and softened stools and decreased appetite in monkeys.
Protein Binding: Not Available
Biotransformation: Extensive, with < 1% recovered unchanged in the urine. Metabolized primarily in the liver by
cytochrome P450 1A2 (CYP1A2). Recently, it was found that anagrelide is bio-transformed in humans into two
major metabolites (6,7-dichloro-3-hydroxy-1,5 dihydro-imidazo[2,1-b]quinazolin-2-one (BCH24426) and 2-amino-
5,6-dichloro-3,4,-dihydroquinazoline (RL603). Whether these metabolites have biological activities that may
underlie the mode of action of the parent drug is presently unclear.
Half Life: At fasting and at a dose of 0.5 mg of anagrelide, the plasma half-life is 1.3 hours.
Dosage Forms of Xagrid: Capsule Oral
Chemical IUPAC Name: 6,7-dichloro-5,10-dihydro-3H-imidazo[2,1-b]quinazolin-2-one
Chemical Formula: C10H7Cl2N3O
Anagrelide on Wikipedia: http://en.wikipedia.org/wiki/Anagrelide
Organisms Affected: Humans and other mammals