At Wednesday’s State-of-the-Art Session, “Platelets Beyond Thrombosis”, Wolfgang Bergmeier, Ph.D., presented ISTH attendees with a review of the literature on RAP1 signaling in platelets as well findings from his most recent research.
- His work identified the RAS-GAP, RASA3 (GAP1IP4bp, R-RAS-GAP) as a critical negative regulator of RAP1 signaling in platelets.
- An antagonistic balance between CalDAG-GEFI and RASA3 signaling in platelets has been found, critical for the fine-tuning of platelet adhesiveness, both in the circulation and at sites of vascular injury.
- Genetic and environmental factors that shift the balance towards RAP1 activation may lead to thrombocytopenia and thrombosis, while those that lead to impaired RAP1 activation may cause bleeding.
Platelets are critical in the body’s response to vascular injury. To form a hemostatic plug, platelets must respond to trace amounts of agonists produced at injury sites, and rapidly transition to an adhesive state (integrin inside-out activation) required for plug formation. However, if unbalanced, this highly sensitive signaling machinery can lead to premature platelet activation and thrombocytopenia and/or thrombosis.
The small GTPase RAP1 is a critical regulator of platelet adhesiveness. RAP1 cycles between a GDP-bound “off-state” and a GTP-bound “on-state”, the latter being required for integrin activation. In platelets, the GEF, CalDAG-GEFI (RasGRP2), is the primary GEF mediating RAP1 activation. CalDAG-GEFI activity is regulated by a pair of EF hand domains, which are sensitive to calcium (KD~80nM) and thus sense minor changes in the cytoplasmic calcium concentration. Platelets lacking functional CalDAG-GEFI show impaired sensitivity to threshold concentrations of agonists, both in vitro and in vivo. In addition, CalDAG-GEFI is also critical for the near-immediate activation of RAP1, triggered by rapid calcium mobilization from stores in the endoplasmic reticulum. Both RAP1 activation and integrin inside-out activation are delayed in platelets from mice and humans lacking functional CalDAG-GEFI.
Bergmeier’s recent work identified the RAS-GAP, RASA3 (GAP1IP4bp, R-RAS-GAP) as a critical negative regulator of RAP1 signaling in platelets. RASA3 is anchored to the plasma membrane by a unique PH/Btk domain and thus is perfectly positioned to prevent premature integrin activation in resting platelets. Mice lacking functional RASA3 exhibit severe thrombocytopenia due to premature platelet activation in circulation. Concomitant deletion of CalDAG-GEFI reversed this phenotype in Rasa3 mutant mice, strongly suggesting that RASA3 is critical to restrain the highly sensitive CalDAG-GEFI/RAP1 signaling pathway. Thus, RASA3 serves as a “hand brake” in circulating platelets, required to maintain these cells in a quiescent state. At sites of vascular injury, this brake must be released to allow for hemostatic plug formation. The signal for RASA3 inactivation is provided by PI3 kinase signaling, downstream of the platelet ADP receptor P2Y12.
This research identified an antagonistic balance between CalDAG-GEFI and RASA3 signaling in platelets, critical for the fine-tuning of platelet adhesiveness, both in the circulation and at sites of vascular injury. Genetic and environmental factors that shift the balance towards RAP1 activation may lead to thrombocytopenia and thrombosis, while those that lead to impaired RAP1 activation may cause bleeding. This bleeding risk is impressively shown by the marked platelet adhesion defect and the moderate to severe bleeding diathesis observed in the rare patients with mutations in the gene for CalDAG-GEFI.