In this study we conducted a comprehensive platelet proteomic analysis from patients with compensated and decompensated alcohol induced liver cirrhosis and non-cirrhotic controls.
The comparative analysis identified four platelet proteins, namely Ras-related protein Rab-7a (Rab-7a), Ran-specific binding protein 1 (RANBP1), Rho GDP-dissociation inhibitor 1 (RhoGDI1), and 14-3-3 gamma with progressively decreased protein expression in patients with liver cirrhosis. This indicates that alcoholic liver cirrhosis is associated with changes in protein expression in platelets These proteins are either directly and or indirectly linked to pathways associated with the haemostatic and possibly immunoregulatory function of platelets. As a consequence there might be altered haemostatic function of platelets in cirrhosis(i.e., thrombocytopathy) and a change in immunoregulatory function of platelets might play a role in cirrhosis progression.
Rab-7a is part of the most abundant family of small GTPases. Originally described as Ras-like proteins in the brain (Rab) these GTPases are often referred to as switches and regulators of vesicle trafficking in both endocytic and exocytic pathways. Rab-7a specifically plays a role in advanced stages of endosomal vesicle trafficking in platelets and is associated with advanced endocytic compartments and transport to lysosomes (17). Consequently, GTPases are an important component of platelet function in both haemostasis and immunological regulation (18). Although the precise process of Rab7a in platelet function and membrane trafficking remains unclear. Furthermore, the pathways of interaction and influence of Rab7a in regard to the platelet granule secretion warrant further research. But the consequently influenced process of platelet granule secretion and the composition of its granules play a role in haemostatic and possible immunological pathways (19).
The function of RANBP1 in platelets is currently unclear but the protein itself was identified in platelets in the past (20). It is known that RANBP1 is a Ran/TC4-binding protein and interacts especially with GTP-charged Ras-like nuclear (RAN) (21). RAN is the most abundant intracellular small GTPase. RAN acts as a molecular switch that alternates between an active GTP-bound and an inactive GDP-bound state. As already mentioned with Rab-7a the specific pathways of regulation are unclear.
The third protein with progressively decreased protein expression Rho GDP-dissociation inhibitor 1 is one of two Rho GDI dissociation proteins (RhoGDIs) expressed in platelets (22). These RhoGDIs are responsible for tuning and adjusting Rho GTPase activity (23). Due to its high expression (approximately 21000 copies per platelet) Rho GDP-dissociation inhibitor 1 provides a plausible candidate for the inhibitory regulation of Rho GTPases (22, 24). These GTPases are part of the Rho family and are small signalling G proteins and a subfamily of the Ras (Rat sarcoma virus) superfamily. In the last decades members of the RhoGTPase family were associated with changes in platelet physiology, the regulation of granule secretion as well as aggregation. Rho GTPases are known key players in cytoskeletal dynamics and therefore serve a critical role in platelets physiology and subsequently vital interactions like haemostasis and inflammation via modulation of platelet activation programs (24). A down regulation of RhoGDI1 is also seen in patients with hepatocellular carcinoma and is used as a prognostic factor (25). Through its inhibitory regulation Rho GDI-dissociation inhibitor 1 likely affects the various roles of platelets.
Lastly 14-3-3 gamma is part of the 14-3-3 family of proteins which play a substantial role in signal transduction pathways of eukaryotic cells. Members of the 14-3-3 protein family were previously described in platelets (26). They play a variety of regulatory roles in various phosphorylation-dependent signalling pathways, among others G-protein signalling (27). More specifically 14-3-3 proteins interact with several phosphoserine-dependent binding sites in glycoprotein Ib-IX complex (GPIb-IX), the major platelet adhesion receptor, thus affecting and possibly indirectly regulating its interplay with von Willebrand factor (VWF) and mediating platelet activation (28). One of these interplays is the platelet activation-dependent fibrin formation which can be relevant in haemostasis (29). Although 14-3-3 zeta is the main part of the 14-3-3 protein family interacting, all six 14-3-3 isotypes are able to bind to GPIb-IX (30). The interaction of members of the 14-3-3 protein family with GPIb-IX furthermore plays a critical role in enabling the platelet response to low concentrations of thrombin (31).
Thrombocytopenia is one of the most common haematological abnormalities in patients with chronic liver disease, especially in patients with liver cirrhosis. It was long believed that the role of platelets in patients with liver cirrhosis was solely to promote aggregation and form the primary haemostatic plug after adhering to the damaged vessel walls through an interaction with von Willebrand factor. (32).
Nowadays, there is growing evidence that platelets, however, play an important role in progression of liver fibrosis through cellular interaction with HSC as well. (10). For example, increasing numbers of platelets as well as platelet-derived chemokine CXCL4 were seen in the immediate vicinity of fibrotic areas in the liver (11). Platelets were shown to stimulate hepatocyte proliferation in vitro through secretion of hepatic – and insulin like growth factor (33). Clinical studies have shown that administration of antithrombotic medication such as aspirin leads to a slowing of the development of fibrosis (34). Interestingly it seems that platelets also may play a role in suppressing HSC activation (35). Therefore, suppressing the activation from a quiescent state to stellate cells producing extracellular matrix proteins that lead to scar formation (36). The regulatory role of platelets in liver fibrosis progression is a subject of ongoing research and the cellular mechanisms are still not fully understood.
The changes in the platelet proteome identified in our study might be associated with an altered platelet response, potentially affecting their role in haemostasis or fibrosis progression. Likely by indirectly influencing pathways affecting the platelet response. Such as the previously mentioned 14-3-3 gamma which interacts with the glycoprotein Ib-IX complex which in turn interplays with VWF or Rho GDI 1 which adjusts Rho GTPase activity and affects platelet physiology and subsequent interactions.
In the past, platelet proteomics were successfully performed in a variety of diseases and in many cases identify molecular and functional platelet changes compared to healthy donors. Yet, a more precise assessment of the consequences of platelet proteome alteration is lacking in many cases.
As with other platelet proteome studies, this analysis is potentially influenced or limited by several factors. Collection and preparation of samples as well as use of non-antithrombotic medications like anti-depressants might have influenced platelet activation. Standardization of collection and preparation as well as rigid exclusion criteria were applied to minimize such influences. Due to the numerically very low platelet count in patients with advanced cirrhosis, we were able to extract a limited total amount of platelets from these patients. This limited further evaluation of findings in subsequent analysis such as immunoassays or alternative MS-based methods due to our focus of running each sample both in a technical and biological duplicate. Lastly, a prospective longitudinal approach would have been more conclusive to show changes throughout disease progression.
In conclusion, this study is to our knowledge the first to investigate platelet proteome changes in patients with alcohol induced liver cirrhosis. We identified several down-regulated proteins thus providing novel information regarding the platelet proteome and its changes in liver cirrhosis patients. Future studies are needed to further clarify the role and significance of the described proteins due to the many pathways and interactions still unclear in platelet regulation. Additionally future research is now needed to determine the clinical significance for haemostatic or immune regulatory function of platelets in cirrhosis.