Insights into platelet pharmacology from a cryo-EM structure of the ABCC4 transporter

Abstract

Structural determination of the ABCC4 transporter is a major first step in providing crucial molecular insights into the transport of platelet substrates into granules, as well as drug transport from platelets. The findings provide a framework for understanding platelet interactions and potential design of specific platelet antagonists.

The use of low-dose aspirin as an antiplatelet agent in the treatment and secondary prevention of adverse thrombotic cardiovascular events (such as acute coronary syndromes and strokes) was one of the most important public health achievements of the twentieth century¹. Development of other antiplatelet agents quickly followed, including phosphodiesterase and adenine deaminase inhibitors (such as dipyridamole), ADP-receptor P2Y₁₂ antagonists (ticlopidine and clopidogrel), PAR1 antagonists (vorapaxor), and GPIIb/IIIa inhibitors (abciximab and eftifibatide)². With the increasing repertoire of antiplatelet agents, issues have emerged including excessive bleeding, drug–drug interactions, and pharmacological resistance or insensitivity^3,4. A molecular understanding of platelet pharmacodynamics and pharmacokinetics for platelet substrates and targeted drugs is needed.

The ATP binding cassette (ABC) proteins are an important class of transporters that hydrolyze ATP to move small molecules (such as inorganic ions, metals, peptides, fatty acids, sugars and nucleotides) against their electrochemical gradient, across cell membranes⁵. The core tertiary structure of a typical ABC polypeptide includes two transmembrane domains and two nucleotide-binding domains⁶. The human ABC family is arranged in seven subfamilies, A–G, consisting of at least 49 ABCs⁵. Among these, the ABC subfamily C (ABCC) has 12 members (ABCC1–ABCC12), all of which adopt the typical core structure of ABC pumps⁶. The better characterized members of this family, ABCC1–ABCC6 and ABCC10–ABCC12, also referred to as multidrug resistance proteins (MRP1–MRP9, respectively), export various chemotherapeutic agents extracellularly, resulting in drug resistance⁶. ABCC4, which is expressed in liver, brain, kidney, pancreas and erythrocytes⁶, is also highly concentrated in human platelets, where it localizes on dense granules as well as the plasma membrane and is crucial for normal platelet function⁷. The essential platelet agonist ADP is transported into dense granules for storage via ABCC4 and subsequently released after platelet activation⁷. Once extracellular, ADP can bind to and activate its receptors (P2Y₁ and P2Y₁₂)⁸. The platelet agonist thromboxane A₂ (TXA₂), an end-product of the cyclooxygenase 1 (COX1) pathway and a target of aspirin, is another substrate for the ABCC4 transporter⁹. Like ADP, once exported out of platelets, TXA₂ activates the thromboxane receptor to stimulate activation of new platelets in a positive feedback loop. Other endogenous substrates of ABCC4 include leukotriene B4 and prostaglandin E₂ (ref. 10). Aspirin has also been reported as a putative substrate of the ABCC4 transporter¹¹.

Among the known inhibitors of ABCC4 that are in clinical use are probenecid, sildenafil, indomethacin, prazosin, doxazosin, tyrosine kinase inhibitors such as erlotinib and gefitinib, and cilostazole¹². Most of these inhibitors are promiscuous and inhibit other ABCCs, and some also interact with other non-ABCC receptors. In the context of antiplatelet therapy, dipyridamole, an analogue of ADP, is a known weak inhibitor of ABCC4 (ref. 12). Given how important ABCC4 is in the transport of numerous endogenous and exogenous substrates, precise determination of its structure–function mechanism may offer opportunities for modulation of platelet activity in the treatment of cardiovascular disease.

In this issue of Nature Cardiovascular Research, Chen et al.¹³ provide important insights into the structural mechanism of ABCC4-mediated transport in relation to platelet biology. The study focuses on the structural biochemistry of ABCC4 and investigates its interactions with important modulators of platelet function that are in current use. Using cryo-electron microscopy (cryo-EM) analysis, the researchers determined the three-dimensional structures of human ABCC4 both in its apo form and when bound to platelet agonists and antagonists, including aspirin.

The authors made a notable discovery that U46619, a stable analog of TXA₂, directly binds within the ABCC4 transmembrane cavity¹³. In the presence of ATP, a dynamic conformational change occurs, with the receptor transitioning from an inward-facing to a more outward-facing occluded conformation, which facilitates substrate release (Fig. 1). Functional assays demonstrated enhanced ATPase activity of ABCC4 upon U46619 binding, and site-directed mutagenesis of several highly conserved amino acid residues in ABCC4 provided insights into the specific structural mechanisms that underlie this transport process.

Fig. 1 |. Proposed mechanism of ABCC4-mediated transport of platelet substrates and inhibitors.

Binding of substrates (such as U46696) to ABCC4 in the presence of ATP causes a conformational change, from an inward-facing to a more outward-facing occluded form, which facilitates substrate release. NBD1 and NBD2, nucleotide-binding domains 1 and 2; TMD1 and TMD2, transmembrane domains 1 and 2.

In addition to solving the cryo-EM structure of ABCC4 bound to dipyridamole, its known inhibitor, Chen et al.¹³ showed that aspirin is also a substrate of ABCC4. By cryo-EM structural analysis, dipyridamole and aspirin were shown to complex with ABCC4 in the same substrate-binding cavity as U46619, stabilized by hydrophobic residues that are highly conserved among ABCC4 homologs. Biochemical assays revealed that dipyridamole binding, but not aspirin, inhibited the ATPase activity of ABCC4. Interestingly, surface plasmon resonance (SPR) experiments demonstrated that dipyridamole possesses a lower dissociation constant (K_d) for ABCC4 than aspirin or U46619, which suggests a greater binding affinity of the transporter for dipyridamole. Because aspirin and dipyridamole can be used clinically as a combination therapy, the authors surmise that dipyridamole with its higher binding affinity to ABCC4 may inhibit the export of aspirin, retaining aspirin intracellularly and causing greater inhibition of the COX1 cyclooxygenase (Fig. 2).

Fig. 2 |. Competitive inhibition of ABCC4-mediated aspirin transport by dipyridamole.

Dipyridamole has a high binding affinity for ABCC4 and may inhibit the export of aspirin through the receptor, thus retaining aspirin on the intracellular side of the membrane and causing increased COX1 inhibition.

After this initial important step, improved resolution with multiple conformations is now needed to capture crucial structural changes associated with binding and transport, particularly in the presence of several substrates. Analysis of this very dynamic protein will require other structural techniques such as nuclear magnetic resonance. Platelet biology in the presence and absence of ABCC4 and substrates requires further investigations including ABCC4 genetic variants, using conventional and cutting edge platelet biology approaches¹⁴.

The findings of Chen et al.¹³ serve as a crucial foundation in enhancing our understanding of precise structure–function relationship of ABCC4 with its substrates, especially in the context of platelet physiology and pharmacology. The authors lay the fundamental groundwork for the development of modulators of ABCC4 to enhance efficacy and possibly reduce side effects. Moreover, given the role of ABCC4 in the transport of other endogenous and exogenous substrates, particularly as a multidrug-resistance protein, precise knowledge of the ABCC4 structure has broader implications in other physiological and pathological processes such as chemotherapeutic agents.