Abstract
Platelet adhesion and activation through the interaction of von Willebrand factor (VWF) with platelet glycoprotein (GP) Ibα are the early key events in hemostasis and thrombosis especially under high blood shear stress. P-selectin translocation from α granule to the cell surface is a typical platelet function phenotype, which makes the platelet-induced inflammatory response of flowing leukocytes possible and can be induced by either chemical agonists (thrombin, ADP, etc.) or high blood shear stress, but regulations of VWF mutation and blood shear stress on VWF-induced P-selectin translocation remain unclear. With flow cytometry, parallel plate flow chamber, and immunofluorescence staining techniques, we examined the P-selectin translocation of platelets on immobilized wild-type (WT) VWF-A1 domain and its two mutants, the gain-of-function (GOF) mutant R1308L and the loss-of-function (LOF) mutant G1324S, respectively. The results showed that the VWF-A1-induced platelet P-selectin translocation was triggered, accelerated, and enhanced by fluid shear stress and could be correlated with shear stress accumulation (SSA, the product of fluid shear stress and mechanical stimulus time), and the PI3K/Akt axis was involved in the platelet P-selectin translocation. The force-triggered P-selectin translocation occurred quickly on partial platelet surface first and then extended gradually to the whole platelet surface as SSA increased. The P-selectin translocation process would be promoted by the GOF mutation (R1308L) but slowed down by the LOF mutation (G1324S). These findings demonstrated a force-enhanced regulation mechanism for the VWF-induced platelet P-selectin translocation through the PI3K/Akt pathway and provided a novel insight into the mechano-chemical regulation mechanism for the key events, such as platelet activation and functional phenotype change in hemostasis and thrombosis.
Highlights
Platelet activation is essential for thrombosis and hemostasis (Semple et al, 2011)
It is demonstrated that inhibiting von Willebrand factor (VWF)-GPIbα interaction can reduce effectively the force-induced platelet P-selectin translocation (Goto et al, 2000; Rahman and Hlady, 2021), while VWF-GPIbα signaling through PI3K/Akt participated in platelet integrin activation and high shear stress-induced platelet P-selectin translocation (Spater et al, 2018; Chen et al, 2019; Kral-Pointner et al, 2019)
To confirm the role of shear stress in platelet P-selectin translocation induced by VWF-A1, the washed platelets were incubated in five distinct solutions with (i) phosphate-buffered saline (PBS) which served as a blank control, (ii) 1 μM of phorbol 12-myristate 13-acetate (PMA) as a positive control, (iii) 200 μg/ml of WTA1, (iv) 200 μg/ml of the mutant R1308L, and (v) 200 μg/ml of the mutant G1324S for 10 min at room temperature and centrifuged at 1,000 × g for 5 min to remove excessive agonists
Summary
Platelet activation is essential for thrombosis and hemostasis (Semple et al, 2011). P-selectin translocation is a key event that occurs in platelet activation and contributes to subsequent platelet aggregation and neutrophil recruitment (Ludwig et al, 2007; Korporaal et al, 2019; Tseng et al, 2019). The platelet P-selectin translocation would increase with applied shear stress and exposure time using a centrifugal flow-through Couette device (Chen et al, 2015). High shear stress of 150 dyn/cm induced 12.8% P-selectin translocation after 5 min with a rotational viscometer and was enhanced with the increase of exposure time (Konstantopoulos et al, 1998; Merten et al, 2000). It is demonstrated that inhibiting VWF-GPIbα interaction can reduce effectively the force-induced platelet P-selectin translocation (Goto et al, 2000; Rahman and Hlady, 2021), while VWF-GPIbα signaling through PI3K/Akt participated in platelet integrin activation and high shear stress-induced platelet P-selectin translocation (Spater et al, 2018; Chen et al, 2019; Kral-Pointner et al, 2019). This work might provide a novel insight into the mechano-chemical regulation mechanism for the key early events in hemostasis and thrombosis
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