This study showed that after ex vivo blood mixing, TLR4 expression levels were upregulated in platelets in the matched (M), uncross-matched ABO type-specific mixing (S), and ABO incompatible (I) groups. In addition to its crucial role in hemostasis and thrombosis, platelet TLR4 significantly contributes to amplifying inflammatory and immune responses . This may also be true in the platelet TLR4-related transfusion immune and inflammatory responses . Platelet TLR4 expression influences innate immunity [5, 7, 8, 10, 11], leading to an adaptive immune response [3–7] and significant inflammation  as observed in transfusion reactions. Thus, platelet TLR4 may serve as a pathophysiological link between innate immunity and transfusion reactions.
TLR4 recognizes DAMPs , which are associated with host cell components that are released upon cell damage [5, 14, 15]. We found that certain DAMPs, including HMGB1, S100A8, and S100A9, were elevated after blood mixing, which is corroborated by previous reports [16–20] on blood transfusions (Fig. 5). For example, first, red blood cell transfusion increases the susceptibility to lung inflammation through the release of HMGB1 and induces necroptosis in lung endothelial cells . Second, stored human red blood cells contain soluble HMGB1, the levels of which are elevated during storage . Third, salvaged blood analyses revealed sustained high levels of certain DAMPs, including S100A8 and S100A9. Above all, transfusion reactions may result from increased levels of TLR4 and certain DAMPs including HMGB1, S100A8, and S100A9 that bind with TLR4 (Fig. 5). Upregulation of TLR4 and certain DAMPs suggests that innate immunity through TLR4-mediated signaling can induce the transfusion reaction.
TLR4 expression upregulation and the elevated levels of some DAMPs (HMGB1, S100A8, and S100A9) were observed in response to blood mixing. However, this did not involve SAA and LPS-binding protein and did not lead to the release of pro-inflammatory cytokines downstream , such as TNF-α, IL-1β, and IL-6, after blood mixing (Fig. 6). These finding may be because of the following: first, LPS-binding proteins is a soluble protein that is synthesized by hepatocytes and found in the blood  that may not synthesized in ex vivo study and not to detected by ELISA. Second, negative regulators target multiple levels of TLR4 signaling, and several molecules, such as RP105 and SIGIRR, inhibit the initiation of this signaling cascade . Other factors target molecules further downstream of TLR4 signaling through different mechanisms . Third, these results may have occurred because of the short duration of blood mixing (30 min) during which time pro-inflammatory cytokines were not expressed; thus, the interaction between platelet TLR4 and certain DAMPs does not trigger this signaling pathway of pro-inflammatory cytokine release. Fourth, ex vivo blood mixing was not able to provide an adequate context in which to observe the complete range of platelet response to transfusion in vivo. Above all, although our results suggest that platelet TLR4 serves as a pathophysiological link between innate immunity and transfusion, the levels of pro-inflammatory cytokines downstream of TLR4 signaling, including TNF-α, IL-1β, and IL-6, were not increased remain unanswered.
Among the three groups (M, S, and I), we did not observe significant differences in TLR4 levels. The major variances among the three groups (M, S, and I) were the responses of antibodies recognizing transfused (foreign) antigens . Human red blood cell membranes containing ABO system components are important in most blood transfusions. Individuals often produce antibodies (alloantibodies) against the alleles they lack within each system. Such antibodies are responsible for the most serious reactions to transfusions. However, ABO systems are not present on platelets . In addition, surface antigens on red blood cells include Duffy, Kell, Kidd, MNS, and P systems, which are not present on platelets . We found no differences in platelet TLR4 expression among the three groups (M, S, and I), which implies that reactions between antibodies and antigens did not play a role in inducing TLR4 expression.
TLR4 ligands recognize not only DAMPs but also pathogen-associated molecular patterns (PAMPs) [5, 14, 15, 23], such as circulating LPS (endotoxin), which are associated with microbial pathogens . PAMP signaling was less likely to participate in this response for the following two reasons: first, during blood mixing, PAMPs such as circulating LPS from microbial pathogens were disregarded, as the patients did not have a pathogenic infection; second, after blood mixing, the levels of LPS-binding protein, which binds with PAMPs but not DAMPs, did not increase (Fig. 6) [14, 15]. Therefore, blood mixing may initiate platelet TLR4 expression, which may trigger the innate immune system, likely independent of PAMPs.
The rationale for selecting patients undergoing cardiac surgery in this study was as follows: first, blood transfusion is routinely required during cardiac surgery, and the preparation of surgery routinely requires cross-match testing of blood at least one day before surgery. Therefore, cross-matched red blood cells were available routinely and sent to operation room from the blood bank before anesthesia for us to obtain donor red blood cells. The red blood cells also used for heart-lung machine priming or stored until transfusion later; second, patients undergoing cardiac surgery require arterial catheterization for aggressive hemodynamic monitoring. The arterial catheterization also used for recipient blood sampling before anesthesia induction and operation of skin incision prevents contamination due to anesthetics and tissue damage, which may be a confounding factor affecting platelet function and activation (Fig. 2). Red blood cells from groups S and I were also obtained from the blood bank for other major surgeries in this hospital.
Our blood mixing procedure is clinically relevant. In this study, we performed ex vivo mixing of donor and recipient blood, which is similar to the cross-matching procedure. Cross-matching is performed prior to blood transfusion to determine whether donor blood is compatible (or incompatible) with recipient blood. The mean body weight of subjects in this study was 66.8 kg; thus, the calculated total blood volume was approximately 4620 mL (estimated 7% of body weight). We used mixtures of 1–10%, which were equivalent to approximately 46.8 to 467.6 mL, respectively; these volumes are commonly used in clinical transfusions.
This study had two limitations. First, the baseline levels of HMGB1, S100A8, S100A9, SAA, LPS-binding protein, TNF-α, IL-1β, and IL-6 were not detected in the donor red blood cells. However, donor red blood cells were added to recipient whole blood at 1%, 5%, or 10% (v/v). The donor red blood cells have a hematocrit level of 55–60% and were standardized by our blood bank center. Thus, they would have contained less plasma than recipient whole blood. Therefore, the plasma volume of our donor red blood cells was far smaller than that of recipient whole blood. Second, we primarily used an ex vivo model because we included group I (ABO incompatible blood mixing), which could be harmful to patients if conducted in vivo. Therefore, the in vivo host innate immune responses were not evaluated. Platelets are not only directly involved in immune defense, but also assist and regulate several functions of innate immune cells. Platelets have also been shown to participate directly in the modulation of immune cell function by physically tethering to them or releasing platelet-derived microvesicles, lipid mediators, nucleosides, mitochondrial DNA, growth factors, cytokines, and chemokines. Platelets and their releasates have broader effects on differentiation, migration, phagocytosis, microbicidal activity, formation of extracellular traps, pathogen clearance, and cytokine response of innate immune cells .