In-vivo model to assess platelet age in humans: during increased platelet turnover large and small platelets are produced simultaneously

Objective Human platelets vary in size, function, and age. Large platelets are often considered to be young platelets. Two situations have to be distinguished, normal steady state platelet production and increased platelet turnover. Here we focused on large and small platelets in humans during increased platelet turnover. To avoid artefacts by interfering factors (medication, comorbidities), we established a platelet apheresis model to deplete platelets from healthy volunteers with subsequent increased platelet production.

Material and Methods Platelets were isolated from ACD-A anticoagulated blood obtained from healthy donors. Then their platelet count was depleted by platelet apheresis from 227 to 186 Gpt/l median range. At day 3 and 10 after apheresis blood was obtained again. Large and small platelets were assessed by gating using 15 defined forward scatter gates in flow cytometry (gate 1 = smallest; gate 15 = largest) and after separation by differential centrifugation. In flow cytometry platelet activation was measured by CD62P expression and PAC-1 binding and the relative proportion of RNA-positive (reticulated) platelets by Cy5-labeled oligo-dT and oligo-dA.

Results The proportion of reticulated platelets increased on day 3 after platelet apheresis from 18 % to 55 % (p = 0.031, Fig. 1A). Unexpectedly, this was much more pronounced in small platelets. RNA content rose nearly 5 times in smaller platelet fractions (gates 1-5), while the increase in larger platelet fractions (gates 10-15) was only 1.5-2 (Fig. 1B). At day 10 proportion of reticulated platelets was still much more increased in small compared to large platelets (Fig. 1B). After separation of large and small platelets by differential centrifugation, the persistence of reticulated platelets in the small platelet fraction until day 10 was confirmed (Fig. 1C). The response to agonists under increased turnover was not different from steady state and differences between large and small platelets remained at the same level.

Conclusion The assumption that in humans large platelets are young platelets has to be revised. During increased platelet turnover the proportion of reticulated platelets increases in small and large platelets, indicating simultaneous synthesis of both subpopulations. Thereby, the RNA-content as marker for young platelets, increases much more pronounced and persists longer in small platelets compared to large platelets.

Fig. 1 The proportion of reticulated platelets was increased 3 days after apheresis [A]. The increase was more pronounced and long-lasting in the smaller platelet fractions (gates 1-5) than in the larger platelet fractions (gates 10-15) [B]. Similar effects were observed when large and small platelets were separated by differential centrifugation [C]. Statistical significance was tested by Wilcoxon test (*=p<0.05)

Publication History

Article published online:
18 June 2021