Mechanisms of platelet clearance and translation to improve platelet storage

Abstract

Hundreds of billions of platelets are cleared daily from circulation via efficient and highly regulated mechanisms. These mechanisms may be stimulated by exogenous reagents or environmental changes to accelerate platelet clearance, leading to thrombocytopenia. The interplay between antiapoptotic Bcl-x_L and proapoptotic molecules Bax and Bak sets an internal clock for the platelet lifespan, and BH3-only proteins, mitochondrial permeabilization, and phosphatidylserine (PS) exposure may also contribute to apoptosis-induced platelet clearance. Binding of plasma von Willebrand factor or antibodies to the ligand-binding domain of glycoprotein Ibα (GPIbα) on platelets can activate GPIb-IX in a shear-dependent manner by inducing unfolding of the mechanosensory domain therein, and trigger downstream signaling in the platelet including desialylation and PS exposure. Deglycosylated platelets are recognized by the Ashwell-Morell receptor and potentially other scavenger receptors, and are rapidly cleared by hepatocytes and/or macrophages. Inhibitors of platelet clearance pathways, including inhibitors of GPIbα shedding, neuraminidases, and platelet signaling, are efficacious at preserving the viability of platelets during storage and improving their recovery and survival in vivo. Overall, common mechanisms of platelet clearance have begun to emerge, suggesting potential strategies to extend the shelf-life of platelets stored at room temperature or to enable refrigerated storage.

Figure 1.

Measurement of platelet clearance kinetics. (A) Endogenous platelet count is monitored over time following the injection of a reagent to assess its effect on platelet clearance. (B) A radioisotopic or fluorescent compound is administered into human or mice. Thereafter, the percentage or radioactivity of labeled platelets in the whole platelet population is measured over time. (C) Exogenous platelets are labeled with radioisotopes or chromophores, and transfused into a host. The percentage of these exogenous labeled platelets is measured over time. The recovery indicates the initial appearance of transfused platelet in the circulation, and the survival means the time that the transfused platelets stay in the circulation.

Figure 2.

Apoptotic machinery in platelet clearance and lifespan. The anti-apoptotic Bcl-xL restrains the proapoptotic Bax/Bak in platelets. Mitochondrial damage induced by CCCP, an ionophore, leads to robust ectodomain shedding of GPIbα. If inhibition by Bcl-xL is blocked pharmacologically, Bax/Bak will induce mitochondrial damage, leading to the apoptotic cascade. The BH3-only initiator of apoptosis Bad may also affect platelet lifespan, though further study would help to elucidate its role. Apoptotic cells redistribute PS from the inner to the outer leaflet of their plasma membranes. One calcium-independent pathway may involve Xkr8. Another pathway present in platelets is facilitated by TMEM16F, a calcium-activated phospholipid scramblase.

Figure 3.

Protein desialylation as a clear-me sign in platelets. Over the platelet lifespan, surface glycoproteins lose the terminal sialic acid residues in their glycans, a process associated with clearance. Neuraminidases are glycoside hydrolases that can remove terminal sialic acid from glycans. Neuraminidases are found in platelets, which present neuraminidase on their surface downstream of GPIb-IX complex signaling. In many glycans, desialylation leads to exposure of the penultimate galactose residues on glycans. These can in turn be recognized by the AMR. Further deglycosylation leads to exposed GlcNAc residues, which may be recognized by other carbohydrate receptors and potentially mediate their uptake by macrophages.

Figure 4.

The trigger model of GPIb-IX-mediated signaling that leads to platelet clearance. A soluble multimeric ligand, such as plasma VWF or anti-LBD antibodies, can bind to the LBD of GPIbα and crosslink platelets. Under physiological shear, the crosslinking can generate a pulling force on GPIbα and induce unfolding of the MSD therein. Consequently, it induces platelet signaling as illustrated, including desialylation (the exposure of β-gal), leading to rapid clearance of platelets. Adapted from Deng et al with permission.

Figure 5.

Platelet storage at room temperature. At room temperature, platelets can only be stored for up to 5 days, which is mainly due to the risk of bacteria growth. In addition, GPIbα shedding is also tightly correlated to platelet storage lesion. Inhibiting GPIbα shedding by using GM6001 or 5G6 significantly improves the posttransfusion recovery and survival of room temperature–stored platelets.

Figure 6.

Platelet storage by refrigeration. (A) Desialylation-mediated clearance. Sialic acid is removed by Neu1 from platelet glycoproteins following refrigeration. The exposed β-gal is recognized by the AMR, and the platelets are cleared by hepatocytes. The utility of neuraminidase inhibitors such as DANA or the AMR inhibitor asialofetuin can impede the clearance of desialylated platelets. (B) GPIbα clustering–mediated clearance. GPIbα clusters on platelet surface, and14-3-3ζ dissociates from Bad and associates with GPIbα after refrigeration. This induces the platelet apoptosis process. A broad caspase inhibitor Q-VD-Oph or arachidonic acid depletion can inhibit the apoptosis process of refrigerated platelets and improve the posttransfusion recovery and survival. (C) VWF binding–mediated clearance. Refrigeration leads to binding of plasma VWF to GPIbα. Upon transfusion and thus exposure to the shear flow, VWF binding may generate a pulling force and induces MSD unfolding, leading to rapid platelet clearance. OGE cleaves off the LBD of GPIbα, therefore precludes the VWF-GPIbα interaction and subsequently platelet clearance.