Abstract Number: OC 37.1
Meeting: ISTH 2021 Congress
Background: Circulating platelet counts are tightly regulated by a delicate balance between production of new platelets and clearance of aged platelets. The small GTPase Rap1 plays a central role in platelet activation signaling during hemostasis and thrombosis mainly by mediating activation of αIIbβ3 integrin, and deficiency in Rap1 or its activator CalDAG-GEFI causes impaired platelet function. Conversely, mice harboring a missense mutation in Rasa3 (Rasa3hlb/hlb), the major negative regulator of Rap1, are severely thrombocytopenic with substantially reduced platelet lifespan due to platelet pre-activation. However, the mechanisms underlying the rapid clearance of Rasa3hlb/hlb platelets from circulation remain incompletely understood.
Aims: To determine the cellular and molecular mechanisms leading to enhanced platelet clearance and thrombocytopenia in Rasa3hlb/hlb mice.
Methods: Platelet counts were monitored in whole blood using flow cytometry. Inhibition of αIIbβ3 in vivo was achieved by administration of an anti-αIIbβ3 F(ab’)2 antibody. Platelets were visualized in organs by immunofluorescence on frozen tissue sections, and in vivo by 2-photon intravital microscopy (2P-IVM).
Results: Both concurrent loss of Talin1 in MKs/platelets and administration of anti-αIIbβ3 F(ab’)2 antibody significantly restored platelet counts in Rasa3hlb/hlb mice. Platelets accumulated in the peri-marginal zone in spleens of Rasa3hlb/hlb mice in an integrin-dependent manner, visualized by tissue immunofluorescence and 2P-IVM. Interactions between sequestered Rasa3hlb/hlb platelets and spleen-resident immune cells were assessed by 2P-IVM. However, splenectomy had no impact on platelet counts, and clearance of platelets in liver and lung were increased in the absence of the spleen, suggesting a hierarchy of platelet clearance mechanisms in Rasa3hlb/hlb mice. Finally, administration of an activating anti-αIIbβ3 IgG to wild-type mice induced thrombocytopenia with a similar pattern of splenic platelet clearance.
Conclusions: Unrestrained Rap1 activation caused by loss of Rasa3 leads to severe thrombocytopenia driven primarily by αIIbβ3-mediated platelet clearance, which is also observed in a model of immune thrombocytopenia induced by anti-αIIbβ3 antibody.
To cite this abstract in AMA style:Lee R, Ghalloussi D, Kramer P, Kenny J, Nieswandt B, Bergmeier W. Loss of the Rap-GAP, Rasa3 Leads to Severe Thrombocytopenia Primarily due to αIIbβ3 Integrin-dependent Platelet Clearance in the Spleen [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/loss-of-the-rap-gap-rasa3-leads-to-severe-thrombocytopenia-primarily-due-to-%ce%b1iib%ce%b23-integrin-dependent-platelet-clearance-in-the-spleen/. Accessed December 5, 2021.
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