Page 208 - Haematologica Vol. 107 - September 2022

ARTICLE - Effects of specific inhibition of platelet MRP4
R. Wolf et al.
calization of the major collagen receptor GPVI and in- hibition of collagen-induced platelet aggregation in their knockout mouse model.10 While the role of MRP4 in cAMP signaling is well established, its role in platelet cGMP ho- meostasis and other cAMP-independent pathways is less clear. MRP4 also transports lipid mediators such as eico- sanoids and may directly mediate the export of thrombo- xane from platelets.12,13 In addition, we showed that MRP4 is also involved in the release of sphingosine-1-phosphate (S1P), a potent pro-inflammatory mediator, from platelets.14 Thus, MRP4 appears to be an essential factor in the para- crine function of platelets. Based on these findings, the transporter has emerged as a potential target to interfere with platelet function.1,3,6,9-11,14 MRP4 inhibitors may comple- ment the currently used aggregation inhibitors, whereby especially platelet hyperreactivity as well as platelet-in- duced inflammatory processes may be reduced. Enhanced platelet reactivity has been linked to MRP4 overexpression in cases of aspirin resistance3,15,16 as well as in patients in- fected with the human immunodeficiency virus (HIV).17 The present study aimed to comprehensively characterize the effect of a selective MRP4 inhibitor on different medi- ators and signaling pathways in platelets, thereby evalu- ating the impact of a short-term pharmacological MRP4 inhibition on the function of human platelets. In previous studies, often rather unspecific inhibitors such as the leu- kotriene receptor antagonist MK57118 were used to inhibit MRP4.19,20 Meanwhile, more selective inhibitors of MRP4, namely Ceefourin-1,21 have become available. Effects on thrombus formation were also studied in a microfluidic flow chamber model to mimic physiological shear stress in both whole human blood and in samples from MRP4-defi- cient compared to control mice.
Methods
More details are provided in the Online Supplementary Ap- pendix.
Human blood samples and animals
Human venous blood was taken from healthy volunteers after written informed consent according to the Declaration of Helsinki and approval from the Institutional Ethics Com- mittee. Mrp4-deficient (Mrp4 (-/-)) mice were kindly pro- vided by the late Dr. Gary D. Kruh (Cancer Center, University of Illinois, Chicago, IL, USA) and were maintained and back- crossed to C57BL/6 wild-type (WT) animals at the animal facility of the University Medicine Greifswald. Murine blood was obtained by right ventricular heart puncture.
Light transmission aggregometry and platelet thromboxane release
For aggregometry, platelet-rich plasma (PRP) was pre-
pared from human citrate blood as described14 and pre- incubated with Ceefourin-1, aspirin or cinaciguat as stated in the figure legends. For measurement of thromboxane release, washed platelets were pre-incubated with Cee- fourin-1 and activated with collagen-related peptide CRP- XL and thrombin receptor-activating peptide PAR1-AP (15 minutes [min]). Concentrations of TxB2 in separated pla- telets and supernatants were determined by liquid chro- matography-tandem mass spectrometry (LC-MS/MS).
Flow cytometric analyses
Fibrinogen binding to integrin aIIbb3 - PRP was diluted in HEPES-buffered saline (HBS), pre-incubated with Cee- fourin-1 (10-50 mM) (15 min) and stimulated with either CRP-XL or ADP (10 min), before FITC-labeled human fibri- nogen and R-phycoerythrin anti-human-CD62P were added (for supplier see the Online Supplementary Appen- dix). After 20 min, samples were fixed in 0.2% formalde- hyde and subjected to flow cytometric acquisition.
VASP phosphorylation - Washed platelets were resus- pended in HBS and pre-incubated with either Ceefourin- 1 (50 mM) alone or with Ceefourin-1 added to PGE1 or cinaciguat (0.5-1 mM). Platelets were fixed with formalde- hyde and permeabilized with Triton-X100. Phospho-spe- cific antibodies either against serine residue 157 or serine residue 239 of vasodilator-stimulated phosphoprotein (VASP) and the respective Alexa Fluor 488-conjugated secondary antibodies were added for flow cytometric analysis.
Calcium measurements
Washed platelets were incubated with Fura-2 AM and subjected to ratiometric calcium analysis using a fluor- escence spectrophotometer (excitation 340/380 nm, emission 510 nm).
Flow chamber experiments
Parallel channel flow chambers (ibidi m-slide VI 0.1, Grä- feling, Germany) were coated with Horm collagen (Takeda Pharmaceutical, Berlin, Germany). Human or murine blood was anticoagulated with hirudin (525 ATU/mL) and heparin (5 IU/mL) or with PPACK (Cayman Chemical, Ann Arbor, MI, USA) (400 mM) and hirudin, respectively. Platelet specific antibodies (FITC-labeled anti-human CD42a or DyLight 488-conjugated anti-mouse GPIbb) (for supplier see the Online Supplementary Appendix) were added and blood was incubated with Ceefourin-1 or the respective solvent at 37°C. Blood was perfused through the microchannels under high arterial shear conditions (1,800-1) for 5 min. After completion of the experiment, two images at the be- ginning, two at the end and one in the center of the chan- nel were obtained, using a confocal laser scanning microscope (Carl Zeiss LSM 780, Oberkochen, Germany) (40x objective). Size of thrombi and surface area coverage
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