Disarrangement of Platelet Cytoskeleton might contribute to Hemorrhagic Diathesis in Scurvy

A previously heathy 4-year-old boy presented with hemorrhagic symptoms (ISTH-BAT:4) including gum bleeding, petechiae, subperiosteal hemorrhages, positive tourniquet test indicating capillary fragility, and pseudoparalysis (refusal to walk due to pain). Based on the diagnosis of malnutrition-induced scurvy (patient's diet was completely free from fruits or vegetables) with vitamin C levels <0.5 mg/L (normal range: 5–15 mg/L), oral substitution of ascorbic acid was started with prompt improvement of bleeding manifestations.

At the peak of symptoms, coagulation studies showed normal international normalized ratio (INR), slightly decreased activated partial thromboplastin time (aPTT), elevated D-dimers, and mild thrombocytosis (platelet count: 720 × 10⁹/L) with a mean platelet volume (MPV) of 9 fL (normal range: 9–13 fL). The patient also displayed a mild microcytic anemia (Hb: 8.5 g/dL; mean corpuscular volume [MCV]: 68.9 fL) due to concomitant iron deficiency. White blood cell count was normal. Upon treatment, platelet count (334 × 10⁹/L, MPV: 11 fL), hemoglobin concentration (13.5 g/dL), and MCV (75 fL) normalized.

By light microscopy, platelet morphology was normal ([Fig. 1A]), whereas by immunofluorescence microscopy,[1] a defect of the platelet cytoskeleton was apparent with peculiar disarrangement of α-tubulin in 60% of the platelets ([Fig. 1B, C]), while other cytoskeletal proteins (filamin A, nonmuscular myosin IIA, β1-tubulin, [Fig. 1D–F]) appeared normal.

Fig. 1 (A) By light-microscopy, a heterogeneous platelet population with some mildly enlarged platelets was apparent. (B, C) By immunofluorescence-microscopy, the cytoskeletal protein α-tubulin displayed a substantial heterogeneity of expression. Compared to the ring-shaped expression found in healthy controls, who were stained in parallel (insets), the protein appeared mostly diffused throughout the platelets (B, arrow), disarranged and curled-up. The latter appears more clearly using the overlay with the counterstaining against von Willebrand factor (magenta) by confocal microscopy (C). When α-tubulin rings were still formed, their structure was often twisted (B, asterisk). The other cytoskeletal proteins filamin A (D), non-muscular myosin IIA (NMMIIA) (E), and β1-tubulin (F) showed milder if any alterations compared to the expression observed in the corresponding healthy controls (insets). Upon vitamin supplementation (G, H), a considerable recovery of the α-tubulin ring structure was observed. Scale bars correspond to 5µm.

Interaction between ascorbic acid and cytoskeletal physiology have been reported. In 1987, Nath and colleagues showed that vitamin C influenced tubulin tyrosination and promoted microtubule assembly in granulocytes.[2] In 2016, Parker et al demonstrated ascorbic acid improved endothelial tightness by enhancing the microtubule network via specific stabilization of α-tubulin.[3] More recently, the role of vitamin C in cytoskeletal organization was corroborated by experiments on porcine oocytes.[4]

Here, we show that also a cytoskeletal platelet defect is associated with vitamin C deficiency. α-Tubulin is not expressed in the typical ring form but appears tangled or twisted within the platelets. These changes are compatible with the predicted major role of α-tubulin in cytoskeleton stabilization exerted by vitamin C.[3] Re-assessment after >12 months of supplementation (vitamin C level: 9.8 mg/L) showed a remarkable normalization of the α-tubulin structure ([Fig. 1G, H]).

α-Tubulin is a component of microtubule cytoskeleton, which plays a major role at different stages of platelet function. Cytoskeletal defects also impair clot retraction, thus making blood clots more prone to fibrinolysis.[5] The resulting bleeding tendency is usually mild, but can worsen the symptoms in the presence of a concomitant hemostatic defect. Our findings suggest that the alteration of the platelet cytoskeleton likely contributes to the bleeding alongside the scurvy-related impairment of collagen synthesis.

Authors' Contribution

C.Z. and A.G. designed the research. J.V. and M.D. collected clinical information. C.Z. and A.G. performed and interpreted blood smear immunofluorescence. C.Z. and M.D. drafted the manuscript. All authors helped in data interpretation and critically reviewed the manuscript.

^* These authors equally contributed to this study.

Publikationsverlauf

Eingereicht: 07. Mai 2024

Angenommen: 18. September 2024

Accepted Manuscript online:
19. September 2024

Artikel online veröffentlicht:
08. Oktober 2024

• References

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• 3 Parker WH, Rhea EM, Qu ZC, Hecker MR, May JM. Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton. Am J Physiol Cell Physiol 2016; 311 (04) C652-C662
• 4 Zhang X, Li W, Sun X, Li J, Wu W, Liu H. Vitamin C protects against defects induced by juglone during porcine oocyte maturation. J Cell Physiol 2019; 234 (11) 19574-19581
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