Background: Upon vascular injury, platelets are activated and release molecules that affect the vascular microenvironment, promoting coagulation, wound healing, and clot architecture. Previously, we have shown that serglycin (SRGN), an intra-granular proteoglycan, plays multiple roles during platelet granule cargo packaging, retention and release, and in the extracellular environment by affecting receptor shedding.

Aims: To investigate SRGN’s function in platelet cargo packaging and release and granule-plasma membrane pore dynamics upon platelet activation. Second, to define SRGN’s role in platelet surface proteins shedding and downstream signaling.

Methods: Anti-SRGN nanobodies were produced in alpacas using recombinant, unglycosylated SRGN. These were used in pulldown assays to study the interaction of SRGN with platelet releasate proteins, to identify interacting partners. Serial block face EM was used to study how SRGN affects granule-plasma membrane pore dynamics and release kinetics upon activation. Multiplex, western blotting, and proteomics were used to determine how SRGN affects membrane protein shedding and downstream signaling.

Results: Anti-Serglycin Nanobody Production. (A). Nanobody production in Alpaca. (B) Recombinant, unglycosylated SRGN protein (black arrow). C) ELISA measure of anti-SRGN response using sera from immunized alpaca.

Platelets from SRGN^-/- showed reduced 𝝰-granule decondensation and swelling upon stimulation. We have generated platelets from SRGN^-/- and wild-type control mice to examine fusion pore expansion by 3D EM analysis. Recombinant SRGN protein and its N- and C-terminal domains have been produced and used as antigens and for screening our cDNA library. Sera from immunized alpaca was screened by ELISA to confirm the immune response. The initial panning of the libraries shows promising clones that recognize the full-length SRGN.  GPVI shedding increased in SRGN^-/- platelets after convulxin treatment, but GP1b was unaffected compared to SRGN^+/+ controls suggesting different roles of SRGN in receptor shedding and downstream signaling.

Conclusions: SRGN regulates 𝝰-granule decondensation and swelling after stimulation, affects convulxin-induced GPVI shedding in platelets, and influences cargo packaging and release in both megakaryocytes and platelets.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/serglycin-an-intragranular-proteoglycan-with-many-effects-on-platelet-function/