The mediation of platelet quiescence by NO-releasing polymers via cGMP-induced serine 239 phosphorylation of vasodilator-stimulated phosphoprotein

Abstract

Nitric oxide (NO) releasing (NORel) materials have been shown to create localized increases in NO concentration by the release of NO from a diazeniumdiolate-containing or S-nitrosothiol-containing polymer coating and the improvement of extracorporeal circulation (ECC) hemocompatibility. However, the mechanism and, in particular, the platelet upregulation of the NO/cGMP signaling protein, vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP (ser 239)), for the improved ECC hemocompatibility via NO release still needs elucidation. In this work, two NORel polymeric coatings were evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism investigated for rabbit platelet P-VASP upregulation. Polymer films containing 25 wt% diazeniumdiolated dibutylhexanediamine (DBHD) or 5 wt% S-nitroso-N-acetylpenicillamine (SNAP) coated on the inner walls of ECC circuits yielded significantly reduced ECC thrombus formation and maintained normal platelet aggregation compared to polymer controls after 4 h of blood exposure. Platelet P-VASP (ser 239), a useful tool to monitor NO/cGMP signaling, was upregulated after 4 h on ECC and markedly increased after ex vivo sodium nitroprusside (SNP) stimulation. Interestingly, in the rabbit platelet, NO did not upregulate the cAMP P-VASP phosphoprotein P-VASP (ser 157) as previously shown in human platelets. These results suggest that NORel polymers preserve rabbit platelet quiescence by sustaining a level of cGMP signaling as monitored by P-VASP (ser 239) upregulation. The upregulation of this NO-mediated platelet signaling mechanism in this rabbit thrombogenicity model indicates the potential for improved thromboresistance of any NORel-coated medical device.

Keywords: DBHD; Extracorporeal circulation (ECC); Hemocompatibility; NO releasing; NORel; Nitric oxide (NO); P-VASP (ser 157); P-VASP (ser 239); Platelets; RSNO; S-nitrosothiol; SNP; Vasodilator-stimulated phosphoprotein (VASP); cGKI; cGMP; cGMP-dependent protein kinase types I β subunit; dibutylhexanediamine; sGC; serine 157 phosphorylation of VASP; serine 239 phosphorylation of VASP; sodium nitroprusside; soluble guanylate cyclase.