Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2

Abstract

Summary. Background: Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)‐bound protein C is activated by thrombomodulin‐bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease‐activated receptor‐1 (PAR‐1). Binding of APC to EPCR both modifies and induces PAR‐1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. Aim: To determine whether platelets possess receptors for protein C that can generate intracellular signals. Results: Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca2+. Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2’ (ApoER2′) and by receptor‐associated protein (RAP), an inhibitor of the low‐density lipoprotein receptor family. Under shear, surface‐bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibα‐dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2′ or RAP. APC bound to purified recombinant ApoER2′ or GPIbα. Conclusions: Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbα and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbα. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbα.

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

“…As the assay for cell binding to immobilized APC involved vigorous washing steps, these data do not reflect a simple equilibrium binding reaction, and no definitive conclusion can be drawn regarding receptor-receptor interactions or regarding the energetic contributions of either individual receptor binding to APC. Although no data directly indicate that APC simultaneously binds to both ApoER2 and EPCR, such a situation is not inconsistent with data that suggest that EPCR binds to only APC's N-terminal Gla-domain (46,47) whereas sApoER2 binds to Gla-domain-less APC (19). One possibility is that the distinct APC binding characteristics for Gla-domain-dependent EPCR binding (i.e., fast association) combined with Gla-domain-independent ApoER2 binding (i.e., slow dissociation) allow for the temporal retention of APC on the cell membrane beyond what can be accomplished by either receptor alone.…”

“…Previously, by using a non-equilibrium binding method, we showed that APC binds to sApoER2 immobilized on a microtiter plate (19). Here we used the equilibrium binding method of surface plasmon resonance (SPR) analysis to compare parameters for APC binding to sApoER2 and to sEPCR.…”

“…First, for a few in vitro assays of APC's effects on certain cells, it appeared that APC-initiated cell signaling did not require PAR1 (15,16) or EPCR (17). Second, in a report of APC-platelet interactions, an important role was posited for APC binding to apolipoprotein E receptor-2 [ApoER2; aka LDL receptor (LDLR)-related protein 8; LRP8] (18) and to glycoprotein Ib␣ (19). ApoER2 is a member of the LDLR family that includes, among others, the LDLR, the LDLR related protein (LRP), ApoER2, and the very low density lipoprotein receptor (VLDLR) (20)(21)(22)(23).…”

Redistribution and Hemostatic Action of Recombinant Activated Factor VII Associated with Platelets

“…26,27 Although platelets do not possess an equivalent for the EPCR, a recent report indicated that immobilized protein C (PC) or activated protein C (APC) support platelet adhesion and activation through mechanisms that could involve GPIb␣. 48 The latter finding indirectly suggests that platelets may at least express receptors that could mediate interactions with APC. In our studies, an excess of recombinant human APC in the incubation media inhibited up to 30%, but never completely pageed the rFVIIa association with platelets.…”

Activated protein C: biased for translation

“…Thus, other PAR3-effector interactions are required for signal induction, diversification, and regulation ( Figure 3). PAR-effector complexes are hypothesized to involve the formation of PAR-PAR heterodimers and homodimers, 43 which may enable PARinduced transactivation of other PARs, integrate the transactivation of other GPCRs such as S1P1, 18,25,26 and incorporate cooperative cross talk with integrins such as Mac1 44,45 or other receptors such as ApoER2 46,47 or Tie2. 20,42,48 Formation of these complexes may achieve a signaling bias by promoting or discouraging the association of particular G-protein ensembles, 49,50 by recruiting b-arrestins, 30 or by incorporating nontraditional PAR signaling pathways via transactivations such as the activation of Tie2 by noncanonical activation of PAR3.…”