Surface expression patterns in immature versus mature platelets of bleeding patients with abnormal light transmission aggregometry

Introduction: In patients with mild to moderate bleeding tendencies, platelet function defects (PFD) are often suspected based on abnormal findings in light transmission aggregometry (LTA) [1]. Aging is a crucial factor in hemostasis that unfolds at the individual level with older bleeding patients facing heightened bleeding risk, and the cellular level, resulting in changes of platelet count and reactivity [2] [3]. However, the influence of platelet aging on glycoprotein (GP) receptor expression remains widely unknown, both in PFD patients and healthy individuals.

Method: In this study, 22 patients from the Vienna bleeding biobank, [4] with impairments in LTA (at least with one agonist at two separate investigations) were compared to 19 age-and sex-matched healthy controls without a clinically relevant bleeding tendency. Platelets were prepared and stained according to previously established protocols [5]. The number of immature (RNA-rich) platelets was determined through flow cytometry by staining platelets with anti-CD61 antibodies and the Syto RNASelect dye.

Results: Patients (age 40±11.8 years, 63% female) had a mean platelet count of 241.5±57.0 x10⁹/L, immature platelet fraction of 8.8±4.7%, and mean platelet volume of 10.4±1.5 fL, which was comparable to healthy controls (age 35±12.0 years, 64% female). No differences in hemoglobin levels, global coagulation assays or von Willebrand factor levels, which were all within the normal range, were seen. As expected, patients had prolonged closure times in the whole-blood shear-depended assay PFA-100^®​ with epinephrine (168.0 (±53.8) vs. 122.0 (±30.4) seconds, p<0.001). Moderate associations between individual age and the PFA-100^®​ were observed in patients (Spearman r=0.479, p=0.024) and controls (r=0.356, p=0.022). Furthermore, age correlated positively with higher Vicenza bleeding scores (r=0.7428, p=0.001) in patients. Principal component analysis revealed a clear distinction in GP expression between immature and mature platelets, in both patients and controls, with immature platelets clustering tightly before and after activation ([Fig. 1]). [Fig. 2] highlights significant differences in GP expression on a cellular level between patients and controls. Patients&apos; immature platelets showed reduced degranulation (CD62P) as well as lower expression of CD36, CD31, toll-like receptor (TLR-)2, and TLR4, but higher TLR9 expression. Mature platelets showed no differences in GP expression between patients and controls. After activation with collagen and TRAP-6, immature platelets in patients exhibited increased CD9, CD36 and CD62P expression, while mature platelets had higher TLR4 and TLR9 expression (not shown).

Fig. 1 Principal component analysis of surface expression levels on immature and mature platelets. CD, cluster of differentiation; TLR, toll-like receptor; RNA, ribonucleic acid; HC, healthy controls

Fig. 2 Comparison of platelet receptor expression according to cellular age (immature and mature p. A: Experimental setup B: Heatmap illustration of surface receptor expression in immature and mature platelets in both patients (left) and healthy controls (right). C: Receptor abundance on the surface of immature (RNA+) and mature platelets (RNA-) in patients and healthy controls. CD, cluster of differentiation; TLR, toll-like receptor; RNA, ribonucleic acid; HC, healthy controls

Conclusion: Our study demonstrates that patients with suspected PFD exhibit significant alterations in GP and TLR expression on immature platelets, alongside reduced degranulation. These findings suggest that impaired platelet maturation might contribute to the bleeding phenotype.

• References

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