Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage

Abstract

Objectives The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor GPIbα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed. Approach and Results Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα (hTg) were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored hTg platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently 5G6 Fab-stored 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. Conclusions Specific inhibition of GPIbα shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be utilized to optimize platelet storage conditions.

Comparison between manufacturing sites shows differential adhesion, activation, and GPIbα expression of cryopreserved platelets

“…If GPIbα shedding is secondary to a damaging event, then shedding inhibitors will not help but in the case that shedding sparks downstream lesions, inhibitors may be able to mitigate damage. For age‐related storage lesion of liquid PLTs, specific inhibition of GPIbα shedding rescued the rapid clearance of aged PLTs to some extent . Future research can always exploit the GPIbα‐dim subpopulation as a biomarker for the extent of damage.…”

“…For age-related storage lesion of liquid PLTs, specific inhibition of GPIbα shedding rescued the rapid clearance of aged PLTs to some extent. 34 Future research can always exploit the GPIbα-dim subpopulation as a biomarker for the extent of damage. The impact of directed modifications in the cryoprocess can be easily monitored using this particular marker in flow cytometry.…”

Protein kinase A determines platelet life span and survival by regulating apoptosis

“…Forskolin also significantly inhibited ΔΨ m depolariza- vation of p38 MAPK and p38 MAPK-mediated cytosolic phospholipase A2, resulting in release of arachidonic acid from membrane phospholipids, which transfers 14-3-3 to the cytoplasmic domain of GPIbα (48,49). In the meantime, p38 MAPK activates ADAM17, leading to GPIbα ectodomain shedding (47), which enhances GPIbα clustering and association of 14-3-3 with the cytoplasmic domain of GPIbα (50). 14-3-3 competes with BCL-XL to bind BAD, thus increasing 14-3-3-GP1ba interaction (38,39).…”

“…However, although the enzymes were inhibited, the alleviation for storage lesion was not significant. It is noteworthy that, despite the mechanism remaining unclear, inhibition of p38 MAPK (46) or p38 MAPK-mediated GPIbα shedding (46,47) significantly improves the quality and posttransfusion survival and function of stored platelets. We and others previously reported that GPIbα-dependent signaling could induce platelet apoptosis and that association of 14-3-3 with the cytoplasmic domain of GPIbα is essential for apoptotic signaling (7, 48, 49).…”

Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets

“…Shedding of GPIbα in platelets during storage at RT is well documented. Inhibition of GPIbα shedding by inhibitors of ADAM17 activity or an anti-GPIbα antibody improves the post-transfusion recovery of RT-stored platelets 38–40 . Little GPIbα shedding was observed after refrigeration of Adam17 ΔZn/ΔZn murine platelets that express only an inactive ADAM17, but refrigerated Adam17 ΔZn/ΔZn platelets were cleared as rapidly as the WT control 5 .…”