Platelets are small disc-shaped cell fragments that undergo a rapid activation when they encounter exposed subendothelial matrix caused by vascular damage. Platelets adhere to subendothelial surfaces and become activated, causing them to aggregate and form a plug, which is responsible for primary haemostasis. Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. The most potent activator of platelets is thrombin. Thrombin induces platelet activation through a unique family of G protein-coupled receptors, namely protease-activated receptors (PARs). Two of these receptors (PAR1 and PAR4) are present in human platelets. PAR1 is a high-affinity receptor that mediates the activation of human platelets at low thrombin concentrations. In the absence of PAR1 function, PAR4 can mediate platelet activation, but only at high thrombin concentrations. The inhibition of the thrombin-stimulated PAR1 signaling pathway in platelets has emerged as a target for clinical development, but specificity of this type of treatment is a major problem because PAR1 is widely expressed in various cell types.
In the previous works, Dr. ZHANG Yun (Kunming Institute of Zoology, the CAS) and his colleagues found a frog trefoil factor, Bm-TFF2, from the frog Bombina maxima skin secretion is able to bind and activate PAR1 to induce platelet activation. In the present study, using Bm-TFF2 as molecule probes, Dr. ZHANG Yong and Ph.D candidate WANG Yanjie in the Dr. ZHANG’s team identified PHBs as regulators of PAR1-stimulated platelet aggregation. Targeting PHBs might be a useful therapeutic approach for anti-platelet therapy.
PHBs, with two homologous members PHB1 (32 kDa) and PHB2 (37 kDa), comprise a highly evolutionary conserved and ubiquitously expressed family of proteins. They have been implicated in the stabilization of mitochondrial proteins, transcriptional regulation, the regulation of sister chromatid cohesion and cellular signaling. The membrane PHBs are reported to be involved in typhoid fever, obesity and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown. PHB1 and PHB2 were detected on the surface of human platelets using flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts along with PAR1, as determined by sucrose density centrifugation. In addition, the PHBs were associated with PAR1, as determined by Bm-TFF2 (a PAR1 agonist)-affinity chromatography, co-immunoprecipitation and confocal microscopy.
Moreover, the platelet aggregation, aIIbb3activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin or PAR1-activating peptide were reduced or abolished as a result of the blockade of PHBs by anti-PHB antibodies or their Fab fragments; however, the same results were not observed when induced by high concentrations of thrombin or PAR4-AP. Also, the calcium mobilization in MEG-01 megakaryocytes stimulated by PAR1-AP was significantly suppressed by PHB depletion using RNA interference against PHB1 and PHB2.
Dr. ZHANG’s results showed that PHBs are localized to the platelet membrane and are involved in PAR1-mediated platelet aggregation. PHBs were identified as regulators of PAR1-stimulated platelet aggregation. Targeting PHBs might be a useful therapeutic approach for anti-platelet therapy. These findings offer new opportunities for studies on the regulation of thrombin-induced signaling pathways in platelet activation.
The results have been published in the Journal of Thrombosis and Haemostasis. http://onlinelibrary.wiley.com/doi/10.1111/j.1538-7836.2011.04607.x/abstract
