Do methodological differences account for the current controversy on tissue factor expression in platelets?

Abstract

Tissue factor (TF), the key activator of the blood coagulation cascade and of thrombus formation, is also expressed by circulating human platelets. Despite the documented in-depth characterization of platelet TF carried out in the past 15 years, some authors still fail to identify TF in platelets, especially when assessment in platelet-rich plasma (PRP) or washed platelets is carried out. This study aims to extend the characterization of the subset of TF-positive platelets in PRP from healthy subjects and to verify how different centrifugation forces, used to prepare the PRP, could affect the analysis of TF-positive platelets. Data indicate that large-size platelets express significantly higher amount of TF compared to small-size cells, in terms of both TF protein and TF mRNA. Upon stimulation, large platelets readily expose on the cell membrane TF, which is functionally active, i.e., able to generate factor Xa (FXa) as well as thrombin. By contrast, TF activity in small platelets is almost completely quenched by tissue factor pathway inhibitor (TFPI), becoming indeed detectable only after treatment with an anti-TFPI antibody. Our data highlight that particular attention must be paid to the preparation and collection of the PRP since such preanalytical variables may influence the platelet recovery and in turn affect subsequent analysis, whether it is flow cytometry, functional activity tests, proteome, or transcriptome analysis. Indeed, the TF-positive subset of large platelets can easily be lost if centrifugation protocols are not optimized, thus erroneously leading to a false-negative result.

Detection of tissue factor in platelets: why is it so troublesome?

“…In a recent study showing the presence of substantial amounts of TF in both resting and activated platelets, the authors discuss the controversy around TF expression in platelets due to methodological differences in platelet isolation [32]. The authors argue that only large platelets express functionally active TF antigen, whereas TF activity in small platelets is quenched by TFPI and therefore undetectable.…”

Toward the Relevance of Platelet Subpopulations for Transfusion Medicine

“…Brambilla et al found that large platelets express not only significantly higher amounts of tissue factor and tissue factor mRNA compared to small platelets. Large platelets also expose functionally active tissue factor on their cell membranes whereas the activity of tissue factor in small platelets is almost completely quenched by tissue factor pathway inhibitor ( 48 ). These results extent previous findings showing that platelets translate tissue factor ( 52 ) and point toward specific roles of large and small platelets in hemostasis.…”

Expression of flTF and asTF splice variants in various cell strains and tissues

“…Since asTF and flTF serve important and often distinct roles in various biological processes, it is appropriate to study them separately. However, as shown in Table III (15–52), total TF, including asTF and flTF, or flTF alone, has been investigated in studies over the last decade. Few studies have been conducted that have detected asTF and flTF levels separately, according to the primer sequences that have been designed.…”