Cryopreserved platelets augment the inflammatory response: role of phosphatidylserine‐ and P‐selectin–mediated platelet phagocytosis in macrophages

Abstract

BACKGROUND Cryopreservation in dimethyl sulfoxide and storage at −80 °C extends the shelf life of platelets to at least 2 years, allowing greater availability in rural and military areas. While cryopreserved platelets (CPPs) have been extensively characterized for coagulation and thrombin generation, reports on the mechanism of adverse reactions to CPPs transfusion are scarce. Here, we tested the hypothesis that CPPs facilitate phagocytosis by Kupffer cells and subsequently promote the inflammatory response in Kupffer cells. STUDY DESIGN AND METHODS P‐selectin expression, glycoprotein Ibα clustering and phosphatidylserine (PS) surface exposure on platelets stored at 22 °C, 4 °C and − 80 °C for 3 days were examined by flow cytometry. The phagocytosis of mepacrine‐labeled platelets coincubated with THP‐1 cells was examined by flow cytometry and confocal microscopy, and the release of cytokines from THP‐1 cells was measured by enzyme‐linked immunosorbent assay. RESULTS CPPs showed a marked enhancement of exposed PS but dramatically reduced glycoprotein Ibα expression and clustering compared with platelets stored at 4 °C. Activation of THP‐1 cells was stronger by CPPs than by platelets stored at 22 °C and 4 °C. CPP interference tests using annexin V and anti–P‐selectin showed that CPPs induced increases in PS‐ and P‐selectin–mediated phagocytosis, as well as secretion of the proinflammatory cytokine tumor necrosis factor‐α, and interleukins IL‐1β and IL‐6, but a decrease in transforming growth factor‐β production in THP‐1 cells. Surface‐exposed PS was more effective than P‐selectin for the activation of THP‐1 cells. CONCLUSION CPPs triggered PS and P‐selectin–mediated phagocytosis by macrophages and stimulated the inflammatory response of macrophages.

Platelet Biochemistry and Morphology after Cryopreservation

“…Cold storage (4 • C) of platelets may cause GPIbα receptor clustering and deglycosylation of N-glycans, a phenomenon that leads β-N-acetylglucosamine (β-GlcNAc) exposure and accelerated clearance from circulation [31][32][33]. These characteristic changes to GPIbα hydrocarbon sidechains are however not necessarily applicable to cryopreserved platelets because Waters et al could not find significant GPIbα clustering and Zhao et al found no increased exposure of β-GlcNAc after cryopreservation [28,34].…”

The immune potential of ex vivo stored platelets: a review

“…The initial response of platelets to tissue damage is the initiation of haemostasis via adhesion to sub‐endothelial structures and subsequent activation of the coagulation cascade; however, they also play a key role in recruitment and activation of leucocytes in order to contain and eliminate any potential infiltrating pathogens [3]. Attachment to leucocytes requires platelet activation and degranulation, which can be induced by stimulatory signals, including haemostatic agonists (thrombin, collagen and calcium), PAMPs/DAMPs or ex vivo storage [4,8,15,39,40]. Specifically, platelet activation results in α‐granule release, and the consequent localization of P‐selectin, CD40L and phosphatidylserine on the platelet surface, which facilitates leucocyte attachment (Figure 1 & Table 1) [3,41].…”

“…1) [3,30] or through fibrinogen bound to platelet GPIIb/IIIa [42]. Activated platelets also exhibit externalized phosphatidylserine on the surface membrane, which has been shown to facilitate platelet attachment to macrophages and neutrophils through a range of potential surface receptors [39,43]. Notably, the formation of platelet–leucocyte aggregates results in the activation and degranulation of both cell types, increasing the local haemostatic and inflammatory effects [3].…”

“…The cryopreservation process alters the surface phenotype and increases degranulation and release of phosphatidylserine‐positive EVs (Table 2) [26,72]. In terms of membrane changes, 50–70% of cryopreserved platelets externalize phosphatidylserine (Table 1) [39,73,74]. There is some evidence that phosphatidylserine on cryopreserved platelets may be pro‐inflammatory, mediating interactions with macrophages [39,75].…”

“…In terms of membrane changes, 50–70% of cryopreserved platelets externalize phosphatidylserine (Table 1) [39,73,74]. There is some evidence that phosphatidylserine on cryopreserved platelets may be pro‐inflammatory, mediating interactions with macrophages [39,75]. Similarly, GPIbα expression is reduced on up to 50% of cryopreserved platelets (Table 1) [39,68,74,76], which may affect their ability to bind to leucocytes.…”