Full Length ArticleA complementary role for tetraspanin superfamily member TSSC6 and ADP purinergic P2Y12 receptor in platelets
Introduction
Platelets play a pivotal role in haemostasis and thrombosis, by limiting blood loss following vascular injury or in the context of pathological conditions such as atherosclerosis [1]. There are various receptors and signalling molecules in platelets responsible for signalling pathways involved in platelet thrombus formation [2]. Among them, the tetraspanin superfamily, TSSC6, also termed as Tspan32 or Pan hematopoietic expression (Phemx), was confirmed as a member of this family by Robb et al. [3]. In their study, they identified the presence of the four transmembrane domains and the highly conserved cysteines in the large extracellular loop which is a characteristic of all other tetraspanin members [3]. However, the expression of TSSC6 appears to be restricted to the hematopoietic cells and organs [3] unlike other tetraspanin superfamily members where their expression is more broadly distributed.
In mice, TSSC6 was mapped to chromosome 7 in a region syntenic with human chromosome 11p15 [4], [5] and found to be present on the surface and intracellular pools in both human and mouse platelets [6]. Similar to other tetraspanins, TSSC6 contains two extracellular loops and another two intracellular domains which consist of N- and C-terminal domains. The latter is relatively large compared to other tetraspanin superfamily members.
There are at least 10 tetraspanins present in platelets and among them CD151, CD9, and CD63 have been well-studied. In 2006, TSSC6 protein was found to be present on the surface of murine platelets as well as in intracellular pools [7]. The study demonstrated a constitutive physical relationship of TSSC6 with integrin αIIbβ3 in resting wild-type mouse platelets [7]. The same study showed that ‘outside-in’ signalling of integrin αIIbβ3 in TSSC6 KO platelets was impaired causing a delay in kinetics of clot retraction, a defect in platelet aggregation and reduced ability of platelets to spread on fibrinogen. These data suggested a functional relationship between integrin αIIbβ3 and tetraspanin superfamily member, TSSC6 [7]. Unstable haemostasis in TSSC6 KO mice was also observed as indicated by a prolonged bleeding time as well as an increased tendency for rebleeds. Additionally, thrombus formation and stability in vivo in TSSC6 KO mice was clearly affected indicating that this tetraspanin superfamily member is important for regulation of platelet thrombus formation [7].
The ADP purinergic receptor P2Y12 has attracted a lot of attention due to its limited distribution on tissues and selective expression in platelets [8]. P2Y12 receptor is a member of the G protein-coupled receptor family that is linked to the αi2 subunit of G protein. In contrast, to the ADP purinergic receptor P2Y1, P2Y12 distribution appears to be restricted to the brain and platelets [8], [9]. Recent studies reported that P2Y12 receptor is additionally expressed by glial cells [10] and smooth muscle cells [11]. P2Y12 receptor is expressed on platelets at a moderate level (600 copies/platelet) when compared to ADP purinergic receptor P2Y1 which is expressed only at 150 copies/platelet [12]. However, the expression of both purinergic receptors is relatively low when compared to other platelet glycoproteins [12]. P2Y12 receptor plays a pivotal role in amplifying platelet aggregation through the major platelet integrin, αIIbβ3 [13]. In addition, P2Y12 receptor contributes to all signalling pathways in platelets. This is clear by its critical role in amplification of platelet aggregation via all known platelet agonists that activate different signalling pathways [14], [15]. Humans [16] or mice [8], [17] deficient in ADP purinergic receptor, P2Y12, display a significant increase in bleeding time. This increase was even more obvious in Clopidogrel treated mice [18]. P2Y12 receptor is involved in mechanisms including granule secretion [19] and stabilisation of thrombus formation [18]. Application of FeCl3 induced injury in mesenteric arterioles in P2Y12 receptor deficient mice revealed delayed thrombus formation, smaller blood clots, clot instability and embolisation [19]. Deficiency of P2Y12 receptor in humans has also been reported and resulted in platelet dysfunction and bleeding diathesis similar to platelet profiles after administration of thienopyridines (e.g. Clopidogrel) to humans [20], [21]. All these characteristics make P2Y12 receptor a good target for anti-thrombotic therapy.
We have recently reported the first detected physical interaction between tetraspanin superfamily and G-protein-coupled receptor family in platelets [22]. More specifically, the physical interaction showed that CD151 forms a lateral association with P2Y12 oligomers and monomers on the platelet membrane while TSSC6 forms a lateral association with P2Y12 oligomers, monomers as well as dimers. We also demonstrated that tetraspanin superfamily member, CD151 forms a functional contribution with the purinergic ADP receptors, P2Y12 receptor in the regulation of ‘outside-in’ integrin αIIbβ3–mediated platelet aggregation, clot retraction and cytoskeletal reorganisation [22]. In addition, the complementary role was found to be required in the regulation of the in vitro (through examining thrombus growth under physiological arterial flow conditions) or in vivo (through evaluating thrombus and stability using FeCl3–induced vascular injury of mesenteric arterioles). Our findings excluded the role of this co-operative interaction in the regulation of granule secretion or ‘inside-out’ integrin αIIbβ3–mediated signalling properties [22]. In the current study, we aimed to examine whether the complementary role for tetraspanin superfamily member TSSC6 and ADP purinergic P2Y12 receptor in platelets does exist since we have previously shown that they are physically associated [22].
Section snippets
Materials
Prostaglandin E1 (PGE1), was purchased from Sigma Aldrich (Castle Hill, NSW). Bovine serum albumin (BSA) was obtained from Bovogen Biologicals (Keilor East, Victoria). Adenosine diphosphate (ADP) was obtained from Chrono-log Corporation (Havertown, PA). Phycoerythrin (PE)-conjugated anti-rat Ab and fluorescein isothiocyanate (FITC)–conjugated anti–mouse P-selectin mAb were purchased from BD Pharmingen (San Diego, CA). FITC-conjugated fibrinogen was generated as previously described [17].
Results
TSSC6 KO platelets with P2Y12 blockade display better synergy in the inhibition of platelet aggregation.
In order to establish whether P2Y12 receptor blockade in mice was achieved using oral administration of Clopidogrel (50 mg/kg), we first confirmed > 90% inhibition of ADP–mediated platelet aggregation profiles in all cohorts as ADP can activate platelets specifically through P2Y1 and P2Y12 purinergic receptors (Fig. 1A, B). In addition, in order to determine if combined TSSC6 deficiency with P2Y
Discussion
Recent studies suggest there are up to 19 tetraspanins in megakaryocytes and likely to be expressed in platelets [35]. Platelet tetraspanins are thought to be important for fine tuning platelet function rather than having an essential role like the major platelet receptors, integrin αIIbβ3, glycoprotein GPVI/FcR gamma chain and glycoprotein GPIb/IX/V complex. While CD9, CD151, CD63, Tspan9 and TSSC6 (Tspan32) are more well characterised, other platelet tetraspanins are poorly defined. However,
Acknowledgements
This work was supported by grants from NHF (G09M 4360) and NHMRC (#603812) of Australia (to Denise Jackson). The author (Mohammed Makkawi) would like also to thank King Khalid University in Saudi Arabia for providing a PhD scholarship.
Authorship contributions
Mohammed Makkawi performed the experiments, analysed the data and wrote the manuscript; David Howells revised the manuscript; Mark Wright revised the manuscript and provide the TSSC6 KO mice; and Denise Jackson directed the project and revised the manuscript.
Disclosure of conflicts of interest
The authors state that they have no conflict of interest.
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