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. These proteins are involved in pathways associated with the haemostatic and possibly immunoregulatory function of platelets. This indicates that alcoholic liver cirrhosis is associated with changes in protein expression in platelets and thus possibly with the haemostatic function of platelets (i.e., thrombocatopathy). A change in immunoregulatory function of platelets might as well 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 specific mechanisms and pathways are still unknown and research especially with human platelets is still ongoing.
The function of RANBP1 in platelets is currently unclear but the protein itself was identified in platelets in the past (19). It is known that RANBP1 is a Ran/TC4-binding protein and interacts especially with GTP-charged Ras-like nuclear (RAN) (20). 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 (21). These RhoGDIs are responsible for tuning and adjusting Rho GTPase activity (22). 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 (23).
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 (24). They play a variety of regulatory roles in various phosphorylation-dependent signalling pathways, among others G-protein signalling (25). 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 regulating its interplay with von Willebrand factor (VWF) and mediating platelet activation (26). The interaction of members of the 14-3-3 protein family with GPIb-IX also plays a critical role in enabling the platelet response to low concentrations of thrombin (27). The various functions of 14-3-3 in platelets suggest that it is a possible target for the treatment of thrombosis and inflammation.
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. (28).
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 (29). Clinical studies have shown that administration of antithrombotic medication such as aspirin leads to a slowing of the development of fibrosis (30). Interestingly it seems that platelets also may play a role in suppressing HSC activation (31). 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 indicate an altered platelet response, potentially affecting their role in haemostasis or fibrosis progression. 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. Further studies are now needed to determine the clinical significance for haemostatic or immune regulatory function of platelets in cirrhosis.