Since the concept of TEPs was developed, the collection of platelets from peripheral blood was important in clinical and basic science studies. As reported, the widely used method for collecting platelets from peripheral blood was the PRP method, while the TEPs sequenced and uploaded in GSE datasets were also collected with the PRP method (5)(13). PRP method was a centrifugation method, which distinguished peripheral blood into a red blood cell layer and a plasma layer containing most of the platelets(14). The PRP technique was the basis for earlier platelet research, and the platelets found in the erythrocyte layer were alien platelets that had not yet been removed, analyzed, or sequenced. In this research, to collect the alien platelets, a second centrifugation step was used to establish a modified approach, which could collect all platelets from peripheral blood.
As we have reported PRR to present the platelets collection rate from whole blood, our modified approach has improved PRR almost to 100%. This shows that our method is capable of obtaining almost all platelets in peripheral blood, and it also suggests that this fraction of platelets cannot be extracted using the PRP method. Our modified approach offers technical assistance for the application of alien platelets for cancer early diagnosis. However, the relationship between PRR2 with bMPV and bPLT suggests that platelets with larger size and number are more likely to be deposited in the RBCL layer. As previous research proved that larger size platelets might correlate with lung cancer progression, the alien platelets attracted us for their possible role in cancer progression(12)(15)(16). Interestingly, the platelets in the erythrocyte layer retain not only a greater number of functional and unknown noncoding areas but also an amount of totally different DEGs. This implies that dPLTs may have further unidentified roles in the development of tumors in addition to being an entirely distinct type of platelet from those found in earlier research, which were exactly alien platelets.
Further to investigate the reasons behind the large platelets that are deposited in erythrocytes and determine whether any of these may be connected to tumor stimulation, we first performed a correlation analysis to identify potential genes associated with large platelets. The four genes, ACSL1, FAM120A, HIST1H2BC, and GABARAPL2, had a negative correlation with MPV in dPLT, might be essential for the development of large platelets. What mechanism do tumor cells use to change the expression of these four genes to cause platelets to enlarge, given that these genes are not important in normal and uPLT populations? From the perspective of tumor progression, we discovered that the dPLT DEGs were potentially enriched in the tumor-associated MAPK pathway. Subsequently, the String platform was used to investigate whether the four genes ACSL1, FAM120A, HIST1H2BC, and GABARAPL2 might be connected to the activation of the MAPK pathway in the tumor cells via specific pathways, resulting in modifications to the expression of the genes and the production of large, easily activated platelets. Consequently, HIST1H2BC may interact with MYC, JUN, and other proteins in the MAPK signaling pathway. Hence, we were interested in learning more about the potential relationship between platelet HIST1H2BC and the MAPK pathway in tumor cells. It was observed that HIST1H2BC was highly expressed in LUAD patient tissues, whereas JUN and FGFR1 in the MAPK signaling pathway were lowly expressed in LUAD patient tissues. Conversely, HIST1H2BC was lowly expressed in peripheral blood dPLT, whereas JUN and FGFR1 were highly expressed. Following searching through the literature, we discovered that tumor cells may intercept platelets in the tumor microenvironment in quest of proteins, nucleic acids, and other substances (17)(18). Based on this, we conjectured that platelets exhibiting elevated HIST1H2BC expression could be seized in LUAD tumor tissues, resulting in elevated HIST1H2BC expression in tumor tissues concurrent with platelet HIST1H2BC depletion. The hijacked HIST1H2BC may further activate the tumor cells' MAPK signaling pathway while also transmitting to peripheral platelets, causing the latter to highly express genes linked to the MAPK signaling pathway and become readily activated. This allows the peripheral platelets to enter peripheral blood and form a significant volume of dPLT. This precisely implies that alien platelets might come into direct contact with tumor cells and then be the source of dPLT, which is more valuable for diagnostic purposes. Next vivo tests will confirm this notion, providing a theoretical basis for the use of dPLT for early LUAD detection (supplementary Fig. 3).
Limitation
Due to the lack of a standard method for extracting peripheral blood platelets, its consistency was only assessed by comparing it with the commonly used PRP method, following which a more comprehensive methodological evaluation through standard samples would be established. In this investigation, a direct comparison of the MPV in dPLT and uPLT was absent to dependably affirm that the volume of dPLT is more than that of uPLT, due to processes like centrifugation, transferring plasma, and mixing blood activating platelets further to influence the results of pMPV. The manipulating restrictions resulted in PRR2 greater than 100%. Not all the PRP might have been pipetted after the first centrifugation, and the remained PRP might have stayed with red blood cells and mixed with PPP to participate in the second centrifugation. Furthermore, our hypothesis has yet to be validated by additional clinical and basic studies, which we will do in the future.