PART-1: A Critical Role Of Thrombin/PAR-1in ADP-induced Platelet Secretion And Second Wave Of Aggregation PART-2: Platelet-mediated Mesenchymal Stem Cells Homing To The Lung Reduces Monocrotaline-induced Rat Pulmonary Hypertension

The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with artificially lowered extracellular calcium level have been attributed to enhanced thromboxane A2(TXA2) generation and inhibition of ectonucleotidases activity. However, the role of thrombin in ADP-induced platelet secretion and second wave aggregation is unknown. We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and second wave aggregation. ADP induces a phosphoinositide3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulted from the cleavage of αⅡbβ3-associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1(PAR-1) and mediates dense granule secretion and second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and second wave of platelet aggregation in PRP were similarly and non-additively blocked by thrombin inhibitor hirudin, PAR-1antagonist SCH-79797, or PI3K inhibitor wortmannin. Moreover, ADP-stimulation caused the dissociation of prothrombin from αⅡbβ3and increased plasma thrombin level, both were prevented by wortmannin. Furthermore, wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1activating peptide SFLLRN. Blocking TXA2production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1-dependence. Therefore, we found that a PI3K-dependent thrombin generation and the resultant PAR-1activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP. Bone marrow mesenchymal stem cell (BM-MSC) transplantation has been suggested to be a promising method for the treatment of pulmonary arterial hypertension (PAH), a fatal disease currently without effective preventive/therapeutic strategies. However, the detailed mechanisms underlying BM-MSC therapy are largely unknown. We designed the present study to test the hypothesis that circulating platelets facilitate BM-MSC homing to the lung vasculature in a rat model of PAH induced by monocrotalin (MCT). A single subcutaneous administration of MCT induced a marked rise in right ventricular systolic pressure (RVSP) and the weight ratio of right to left ventricle plus septum (RV/LV+S)3weeks after injection. The injection of MSCs via tail vein3days after MCT significantly reduced the increase of RVSP and RV/LV+S. The fluorescence-labeled MSCs injected into the PAH rat circulation were found mostly distributed in the lungs, particularly on the pulmonary vascular wall, whereas cell homing was abolished by an anti-P-selectin antibody and the GPIIb/IIIa inhibitor tirofiban. Furthermore, using an in vitro flow chamber, we demonstrated that MSC adhesion to the major extracellular matrix collagen was facilitated by platelets and their P-selectin and GPIIb/IIIa. Therefore, the current study suggested that platelet-mediated MSC homing prevented the aggravation of MCT-induced rat PAH, via P-selectin and GPIIb/IIIa-mediated mechanisms.