Rationale:
Platelet signalling pathways linking inflammatory and thrombotic circuits play a key role in atherosclerosis-caused thrombotic events. However, the underlying mechanisms are only poorly understood. Here, we tested the role of the inflammatory signalling adapters TRAF-1 and TRAF-5 in freshly prepared washed platelets (WP) and platelet-rich plasma (PRP) from wild type (WT), TRAF-1 (TRAF-1^-/-) and TRAF-5 (TRAF-5^-/-) deficient C57BL/6 mice.

Methods and Results:
Animal experiments were performed according to the German legislation (“Tierschutzgesetz”). Platelet aggregation was evaluated using a Multiple Analyzer equipment (Roche) in freshly prepared whole blood from mice. After stimulation with ADP, samples from both, TRAF-1^-/- and TRAF-5^-/- mice, showed a 36% (p<0.05) and 30% (p<0.05) increase in the area under the curve (AUC), respectively, compared to WT, indicating an overall enhanced platelet reactivity. While collagen-induced aggregation resulted in a 23% decrease (p<0.05) for TRAF-1^-/-, stimulation with PAR-4 induced a 25% (p<0.05) reduction for TRAF-5^-/-. Flow cytometry data presented complementary results for PRP stimulated with ADP in TRAF-1^-/-, with a 68% (p<0.05) higher expression of P-selectin compared to WT. No differences were detected in mRNA levels of coagulation factors in the liver. However, plasma fibrinogen showed a 43% decrease in TRAF-1^-/- (p<0.001) and a 43% in TRAF-5^-/- (p<0.001) compared to WT. Plasma concentration of von-Willebrand-Factor (vWF) in TRAF-1^-/- was also decreased by 19% (p<0.01). We detected expression of TRAF-associated receptors TNFR-1 and CD40 on WT platelet surface by flow cytometry. Stimulation with recombinant TNF-α or CD40-stimulating antibodies resulted in significantly higher expressions of platelet-specific activation markers compared to unstimulated controls in WT mice. Platelet count showed no changes among the genotypes, although megakaryocytes in the bone marrow were 42% lower in TRAF-1^-/- (p<0.01) and 47% higher in TRAF-5^-/- (p<0.001) compared to WT.

Conclusions:
These findings underline the connection of inflammatory signalling and haemostasis driven by TRAFs. Overlaps between conventional platelet activation pathways and TRAFs and the specific role of TRAF-1 and TRAF-5 needs to be further investigated.