Impaired Platelet Function in Sept8-Deficient Mice In Vitro

Abstract

Septins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

Role of Septins in Endothelial Cells and Platelets

“…SEPT5 rings have also been detected in human fibrinogen-adhered platelets ( Martinez et al, 2006 ). Sept8 displays rings in fibrinogen- and in collagen-adhered mouse platelets ( Neubauer et al, 2021 ). Rings are the most often observed higher-order structure of septins in various organisms and cell-types.…”

“…There is ample evidence that microtubules form a closed circular bundle running near the cell periphery, known as marginal band, which maintains the characteristic discoid shape of resting platelets ( Kowit et al, 1988 ) suggesting that septin-rings may play a role in ensuring shape stability of resting platelets. Furthermore, SEPT5, SEPT6, and Sept8 display a punctate localization in the cytoplasm ( Martinez et al, 2006 ; Neubauer et al, 2021 ). In platelets, septins demonstrate strong affinity to each other in forming complexes composed of multiple septin proteins ( Sandrock et al, 2011 ).…”

“…After platelet activation, SEPT4 and SEPT8 move to the platelet surface, suggesting a direct role in exocytosis ( Blaser et al, 2004 ). Indeed, Sept8-deficient mouse platelets exhibit remarkably reduced α-granule secretion, but not of δ- or lysosomal granules ( Neubauer et al, 2021 ). Transgenic overexpression of Sept5 in mice is associated with fewer and larger α-granules, indicating that Sept5 supports normally sized α-granules ( Kato et al, 2004 ).…”

“…In addition to the degranulation described above, Sept8 is known to be involved in platelet functions in primary hemostasis through comprehensive analyses using a Sept8-knockout mouse model ( Neubauer et al, 2021 ). Loss of Sept8 in mouse platelets caused a pronounced defect in activation, adherence, and aggregation.…”

“…In contrast to the reduced aggregation caused by the loss of Sept8, a Sept5 deletion leads to an enhanced aggregation with an agonist requiring a platelet-secretory response ( Dent et al, 2002 ). Sept8-deficient platelets exhibited delayed spreading on fibrinogen, a process by which adherent platelets flatten at sites of vascular damage and expand their contact area by deforming of the plasma membrane ( Neubauer et al, 2021 ). Reorganization of the cytoskeleton network is essential for all these processes and for degranulation.…”