The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin αIIbβ3 Outside-in Signaling

Abstract

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading.

Integrin αIIbβ3 outside-in signaling

“…An a IIb b 3 -expressing CHO cell model has also proved to be a valuable tool, although inevitably some differences exist in the integrin-signaling machinery used by these cells and platelets. [46][47][48][49][50] Hence, there is now considerable evidence that activated a IIb b 3 can act as a signal transducer to the cell interior, coupling to intracellular effectors to drive a multitude of outside-in signaling events, ultimately leading to changes in cellular behavior. Platelet spreading, stable thrombus formation, and clot retraction involve significant contributions from the cytoskeletal machinery, and hence outside-in signaling commonly couples a IIb b 3 to actin polymerization, cytoskeletal reorganization, and force sensing/transmission.…”

“…95,100 Similarly, platelets deficient in phosphoinositide-dependent protein kinase 1 (PDK1), a kinase upstream of Akt, show reduced spreading on fibrinogen and clot retraction through dysregulation of Akt and GSK3. 101 The PtdIns(3,4,5)P 3 binder Ras/Rap GTPase-activating protein (Ras/Rap GAP) Ras GTPase-activating protein 3 (Rasa3) plays a key role in integrin a IIb b 3 -mediated cell spreading via regulation of Rap1, 47 and other PtdIns(3,4,5)P 3 -binding small GTPase regulators might also play roles in outside-in signaling through the regulation of Arf and Rho family members (Figure 3). …”

PI3K inhibitors in thrombosis and cardiovascular disease

“…PtdIns(3,4,5)P 3 (and PtdIns(3,4)P 2 ) effectors commonly possess a subtype of the pleckstrin homology (PH) domain, which interacts with the phosphorylated headgroup of this phosphoinositide via a conserved network of basic residues, although other protein domains can also interact with PtdIns(3,4,5)P 3 , including the DHR‐1 domain of DOCK‐family GEFs and the PX domain of sorting nexins [54–57]. While AKT (also known as Protein Kinase B) is the best characterised PtdIns(3,4,5)P 3 effector and has received considerable attention in platelets [58], a range of others have key roles in this cell type, including RASA3 [59,60], DAPP1 [13] and ELMO1 [61]. In highly dynamic cells like platelets, which utilise major cytoskeletal reorganisation events and protein trafficking during activation and thrombus formation, the ability of Class I PI3Ks to control small GTPases via a range of PtdIns(3,4,5)P 3 ‐regulated GAPs and GEFs is particularly interesting and warrants further investigation.…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…The active GTP‐bound Gαi‐subunit dissociates from the β and γ subunits and binds to AC leading to diminished cAMP synthesis (Figure 3 and Table 1). Simultaneously Gi triggers the phosphatidylinositol 3‐kinase (PI3K) pathway, which converts phosphatidylinositol 4,5‐bisphosphate to phosphatidylinositol 3,4,5‐trisphosphate at the plasma membrane leading to the activation of the kinase Akt and inhibition of the Rap1‐GAP RASA3, key activating pathways in platelets 20, 21, 22. The synergism between P2Y12 function and blockage of cyclic nucleotide pathways has been shown for both, cAMP and cGMP components, although cGMP regulation by P2Y12 is not well understood 9, 23.…”

“…During platelet activation Rap1GAP2 S9 phosphorylation enhances 14‐3‐3 binding, whereas PKA/PKG‐mediated S7 phosphorylation does the opposite (Figure 7). 92 Platelet activation has also been shown to inhibit the function of RASA3, a highly expressed GAP of Rap1 in platelets, in a PI3K‐dependent manner 21, 22. Vasodilator‐stimulated phosphoprotein (VASP) is an actin filament and focal adhesion binding protein and was one of the first substrates of PKA/PKG to be identified in platelets.…”