Regulation of monocyte anti-thrombotic gene and protein expression through platelet-derived material

Activated platelets can recruit monocytes into thrombi through the P-selectin*PSGL-1 interaction. Cross-talk between the cells may regulate monocyte gene and protein expression. A previous genome-wide transcriptomic study identified >3000 genes upregulated in monocytes in response to activated platelets, including a number of antithrombotic genes; in particular tfpi and procr. This thesis aimed to establish the mechanisms by which platelets regulate these genes, and if this was through the nuclear receptor PPARγ. Using the same experimental model as in previous studies, in which platelets were activated in whole blood with the platelet-specific GPVI agonist CRP-XL, monocyte tfpi and procr expression was confirmed, shown to be affected by inhibitors of COX-1 (aspirin) and 12-LOX (esculetin and baicalein), and regulated through soluble factors released from platelets, which were shown to be oxylipins for tfpi and proteins ~10kDa for procr. Expression of pparγ was also increased and largely regulated through direct platelet-monocyte contact, although oxylipins potentiated expression. An associated increase in protein expression was partially confirmed for all three genes. Mass spectrometry (MS) of platelet-derived releasate measured 386 proteins and identified platelet factor 4 (PF4) and RANTES (CCL5) as likely candidates for procr regulation. Expression was attenuated with releasate incubated with heparin agarose (HA) but this was not rescued with exogenous PF4. LC-MS/MS measured no change in PF4 levels in the releasate incubated with HA but complete removal of RANTES. MS identified 10 oxylipins released from platelets (AA, 8-, 9-, 11-, 12-, 15-HETE, 9-, 13-HODE, PGD/E2 and TxA2) of which four (11-, 15-HETE, 9-, 13-HODE) were significantly, and TxA2 and PGD/E2 completely reduced by aspirin, and all but AA and 9-HETE reduced with 12-LOX inhibitors. These oxylipins included known PPARγ agonists. Incubation with 15d-PGJ2 and rosiglitazone confirmed potentiation of tfpi expression in monocytes. Using a transactivation assay 12- and 15-HETE were shown to activate PPARγ expression in vitro, while X-ray crystallography indicated, for the first time, interaction of both with PPARγ. These results are the first to show regulation of antithrombotic genes in monocytes by factors released from platelets. It is the first to profile oxylipins released from GPVI-activated platelets and identify PPARγ as a regulator of tfpi, and RANTES as a potential regulator of procr expression in monocytes. The observation that aspirin attenuates expression of both these genes raises issues with its use in the treatment of cardiovascular disease, for which it is relied on heavily.