Platelets Form Aggregates on the Surface of AML Cells Via CD62P/PSGL-1 and Release TGF-β1 to Promote Drug Resistance
Authors: Liang Xue , Wang Huiping , Zhou Ji , Zhu Jinli , Jiao Xunyi , Zhai Zhimin 
Published: December 2024

Drug resistance remains a major challenge in the treatment of acute myeloid leukemia (AML). Our recent findings indicate that AML patients with elevated platelet levels have lower remission rates and shorter survival, significantly correlated with higher levels of transforming growth factor-beta 1 (TGF-β1). TGF-β1 levels are particularly elevated in relapsed/refractory AML patients, and exogenous TGF-β1 can promote drug resistance in AML cells. Although platelets are known to be primary carriers of TGF-β1, the synergistic role of platelets and TGF-β1 in inducing AML drug resistance has not been well documented. Peripheral blood smears from newly diagnosed and relapsed AML patients were stained with Wright-Giemsa stain and analyzed morphologically. We observed varying numbers of platelet-leukemia cell aggregates in AML patient samples (there are no established standards for identifying platelet-leukemia cell aggregates, In this study, we define platelet-leukemia cell aggregates as leukemia cells with one or more platelets adhered to their surface). To quantitatively assess platelet-leukemia cell aggregates, we developed a flow cytometry protocol specifically designed for their detection. AML cells that are double-positive for CD42a and CD62P are classified as platelet-leukemia cell aggregates. We analyzed samples from several patients, finding that the proportion of these aggregates was higher in relapsed patients (n=6) compared to newly diagnosed patients (n=8) (8.00±3.25% vs 4.46±2.85%, p=0.093). Correlation analysis revealed a significant positive relationship between platelet counts and platelet-leukemia cell aggregates (R2=0.674, p＜0.001). Among newly diagnosed patients, those who did not achieve remission after initial treatment had higher proportions of these aggregates compared to the remission group (6.91±2.98% vs 2.99±1.85%, p=0.057). We further explored ligand-receptor interactions between platelets and AML cells. Comparing the mean immunofluorescence intensity of PSGL-1 on platelet-leukemia cell aggregates and leukemia cells without platelet adhesion, we found higher PSGL-1 intensity on aggregates. Confocal microscopy of immunofluorescently labeled peripheral blood smears showed that platelet CD62P primarily adhered to regions of high PSGL-1 expression on leukemia cells. These findings suggest that platelet-leukemia cell aggregates form through CD62P and PSGL-1 interactions. In vitro co-culture experiments demonstrated that platelets adhered to AML cells, forming aggregates, and increased phosphorylation of SMAD2. This was accompanied by a decrease in the proportion of leukemia cells in the S phase and an increase in the G0/G1 phase, along with elevated stemness markers (CD34, CD44, CD133, CD150, LGR5) and colony formation capacity, indicating a tendency towards stemness. Additionally, co-cultured cells exhibited increased resistance to doxorubicin, suggesting that direct platelet-leukemia cell interaction enhances drug resistance. The addition of the TGF-β receptor inhibitor SD-208 during co-culture with KG-1A cells blocked TGF-β signaling activation, reducing leukemia cell stemness and anti-apoptotic capabilities, indicating that platelets may exert their effects on AML cells through TGF-β1. Our study demonstrates that platelets are readily activated in AML patients, forming platelet-leukemia cell aggregates that generate and retain high concentrations of TGF-β1 around leukemia cells. This activates the TGF-β signaling pathway, endowing AML cells with stem cell-like properties, facilitating drug resistance, and enabling rapid proliferation upon re-entry into the cell cycle, ultimately leading to treatment failure or relapse.