Introduction

Vincristine sulphate, a widely used antineoplastic drug, is applied in the treatment of immunomediated or idiopathic thrombocytopenia with satisfactory results (Ganti et al. 2006; Weigert et al. 2008). Owing to the cytostatic effects of this agent, demonstrated in various anticancer protocols, and to the contradictory results regarding the effect of vincristine in the elements of the megakaryocyte-platelet system, we performed the present study.

Materials and Methods

Our experimental protocol was designed aiming at analyzing the morphological ultrastructural changes which could be induced in the above mentioned elements by the administration, in dogs, of vincristine in two distinct dosages. The experimental groups were constituted as follows: G1--composed of five dogs that received one intravenous injection of vincristine sulphate at the dose of 0.010 mg/kg every week, for a period of eight weeks; G2--composed of five dogs that received one intravenous injection of vincristine sulphate at the dose of 0.025 mg/kg every week, for a period of eight weeks. The experimental protocol, lasting 24 weeks, was subdivided in three phases of eight weeks duration: P1--with the purpose of obtaining control results; P2--period during which the animals were exposed to vincristine sulphate; and P3--post-treatment period to study the reversibility of the changes. Peripheral blood and bone marrow were collected in distinct time points. Laboratory studies included ultrastructural analysis of the platelets and megakaryocytes that were conducted according to Benke (1968) and Miyazaki et al. (2000). The experimental design is in the accordance with the principles of guidance for the care and use of animals at the Faculdade de Ciências Agrárias e Veterinárias/UNESP/ Jaboticabal (SP).

Results and Discussion

In this study, we have shown that injection of vincristine sulphate, a microtubule disrupting drug, into normal dogs causes time-dependent morphological changes in the platelets and megakaryocytes. There were no significant differences between the two dosages applied. In both groups, the morphological alterations intensified with time during the injection period. All dogs receiving vincristine exhibited some or most of the following changes in the platelets: 1) appearance of pseudopods; 2) modification of the normal discoid shape into a spherical shape; 3) development of membrane blebs; 4) formation of membrane complexes; 5) loss of the circumferential bundle of the microtubules; 6) and dilation of the opened canalicular system. These results confirm the important role of the microtubules in the maintenance of the discoid shape of the platelet described by White & Rao (1998). In the megakaryocytes, the ultrastructural alterations observed were: 1) presence of blebs on the cell surface; 2) increased amount of dense compartments; 3) dilation, derangement and expansion of the dense tubular system; and 4) formation of membrane complexes. The membrane complexes observed in megakaryocytes from vincristine treated dogs were composed of demarcation membrane systems and smooth endoplasmic reticulum. This suggests that the membrane complexes are the result of collapse of the demarcation membrane system and smooth endoplasmic reticulum, implying that microtubules are necessary for the normal organization of these structures. Although the effect of in vivo administration of vincristine sulphate on megakaryocytopoiesis in Wistar rats have been well characterized, the consequences of the administration of this drug in dogs had not been described. Our findings corroborate the essential role of the ultrastructure, more precisely of the microtubules, in preserving platelet and megakaryocyte shape. The membrane blebs observed in megakaryocytes and platelets after vincristine administration are indicative of a defect in cytoskeleton-membrane interaction. These changes were more pronounced in megakaryocytes than in platelets, which may be related to a more dynamic state of the megakaryocyte cytoskeleton as compared to that of the platelet. The most extensive analysis of the effects of vincristine sulphate on platelet ultrastructure was performed by White & Krumwiede (1999), who studied platelets of rats after 30 minutes of incubation with various concentrations of the drug. He noted only minimal changes in platelets. Our observation of significant alterations in platelet ultrastructure after injection of vincristine sulphate in vivo suggests that the changes occur some hours after vincristine administration, rather than minutes. An increase in the frequency of dense compartments in megakaryocyte cytoplasm has been noted under conditions in which megakaryocytopoiesis is stimulated (Bentfeld-Barker & Bainton 1977).

Conclusion

The present study indicates that in vivo vincristine sulphate administration in dogs, under the conditions here experimented, results in important changes of the ultrastructural morphology in megakaryocytes and platelets, related with disruption of microtubules.

References

1.  Ganti AK, Landmark JD, Kessinger A, Smith LM, Tarantolo SR. 2006. Vincristine-laden platelet transfusion for patients with refractory thrombocytopenia. In Vivo. 20(4):559-63.

2.  Weigert O, Wittmann G, Gruetzner S, Cnossen J, Becker MC, Dreyling M, Ostermann H. 2008. Successful salvage treatment of chronic refractory immune thrombocytopenia using a simplified method to generate vincristine-loaded platelets. Thromb. Haemost. 100(4):705-707.

3.  Benke O. 1968. An electron microscope study of the megakaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J. Ultr. Res. 24:412-433.

4.  Miyazaki R, Ogata H, Iguchi T, Sogo S, Kushida T, Ito T, Inaba M, Ikehara S, Kobayashi Y. 2000. Comparative analysis of megakaryocytes derived from cord blood and bone marrow. Brit. J. Haematol. 108:602-609.

5.  White JG, Rao GH. 1998. Microtubule coils versus the surface membrane cytoskeleton in maintenance and restoration of platelet discoid shape. Am. J. Pathol. 152(2):597-609.

6.  White JG, Krumwiede MD. 1999. Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets. Platelets. 10(2-3):159-68.

7.  Bentfeld-Barker ME, Bainton DF. 1977. Ultrastructure of rat megakaryocytes after prolonged thrombocytopenia. J. Clin. Invest. 56(6):1635-1649.