C-Type Lectin-like Receptor 2 Expression Is Decreased upon Platelet Activation and Is Lower in Most Tumor Entities Compared to Healthy Controls

Abstract

The C-type lectin-like receptor 2 (CLEC-2) is expressed on platelets and mediates binding to podoplanin (PDPN) on various cell types. The binding to circulating tumor cells (CTCs) leads to platelet activation and promotes metastatic spread. An increased level of soluble CLEC-2 (sCLEC-2), presumably released from activated platelets, was shown in patients with thromboinflammatory and malignant disease. However, the functional role of sCLEC-2 and the mechanism of sCLEC-2 release are not known. In this study, we focused on the effect of platelet activation on CLEC-2 expression and the sCLEC-2 plasma level in patients with cancer. First, citrated blood from healthy volunteer donors (n = 20) was used to measure the effect of platelet stimulation by classical agonists and PDPN on aggregation, CLEC-2 expression on platelets with flow cytometry, sCLEC-2 release to the plasma with ELISA and total CLEC-2 expression with Western blot analysis. Second, sCLEC-2 was determined in plasma samples from healthy donors (285) and patients with colorectal carcinoma (CRC; 194), melanoma (160), breast cancer (BC; 99) or glioblastoma (49). PDPN caused a significant increase in the aggregation response induced by classical agonists. ADP or PDPN stimulation of platelets caused a significant decrease in CLEC-2 on platelets and sCLEC-2 in the plasma, whereas total CLEC-2 in platelet lysates remained the same. Thus, the increased plasma level of sCLEC-2 is not a suitable biomarker of platelet activation. In patients with CRC (median 0.9 ng/mL), melanoma (0.9 ng/mL) or BC (0.7 ng/mL), we found significantly lower sCLEC-2 levels (p < 0.0001), whereas patients with glioblastoma displayed higher levels (2.6 ng/mL; p = 0.0233) compared to healthy controls (2.1 ng/mL). The low sCLEC-2 plasma level observed in most of the tumor entities of our study presumably results from the internalization of sCLEC-2 by activated platelets or binding of sCLEC-2 to CTCs.

Keywords: cancer thrombosis; platelet function; podoplanin; soluble CLEC-2.

Figure 1

Aggregation response of healthy donors (n = 20) upon stimulation of platelets with the classical agonists ADP, AA and COL and PDPN as the co-agonist. The primary aggregation (PA) is given in % and corresponded to the maximum aggregation in all measurements. Box-whisker plots show the 25th–75th percentile (boxes) with the median indicated by the line inside each box, and the 1.5 interquartile range by vertical lines (whiskers). The use of 5 μg PDPN caused a significant (* p < 0.05) increase in the aggregation response (n = 10).

Figure 2

Results from flow cytometry for the detection of CLEC-2 and CD62P expression on platelets: (a) Representative result from one donor, indicating a decrease in CLEC-2 and increase in CD62P expression upon ADP and PDPN stimulation; (b) Summary of the results from 20 healthy donors. The decrease in CLEC-2 expression and the increase in CD62P expression were significant (*; p < 0.001) for ADP and PDPN stimulation. For further decription of the box-whisker plots see legend to Figure 1.

Figure 3

Measurement of sCLEC-2 in plasma (a) and total CLEC-2 in platelet lysates (b) after ADP and PDPN stimulation. (a) sCLEC-2 levels in unstimulated (w/o) and ADP- and-PDPN stimulated PRP by using ELISA; (b) Relative quantification of total CLEC-2 in platelet lysates without activation and ADP and PDPN stimulation with Western blot analysis and actin as the reference protein. The decrease in sCLEC-2 upon stimulation was significant (*; p < 0.001), whereas the total CLEC-2 protein concentration was comparable. For further decription of the box-whisker plots see legend to Figure 1.

Figure 4

The plasma level of sCLEC-2 in healthy controls (HC; n = 285) and patients with colorectal cancer (CRC; n = 194), melanoma (n = 160), breast cancer (BC; n = 99) and glioblastoma (n = 49). For further decription of the box-whisker ploets see legend to Figure 1. Circles indicate outliers.

Figure 5

The plasma level of sCLEC-2 in patients with CRC and melanoma stages I to VI compared to healthy controls (HC). N in brackets. Box plots show the 25th–75th percentile with the median indicated by the line inside each box. Whiskers represent the 1.5 interquartile range (circles indicate outliers).