Brain-Derived Neurotrophic Factor Induces Platelet Aggregation

Background: A large extra-cerebral pool of Brain-Derived Neurotrophic Factor (BDNF) is found in platelets. With concentrations reaching 100-1000 fold those of neurons, platelet-stored BDNF is hypothesized to play an important role in the vasculature.

Aims: To investigate the effect of BDNF on platelets, alone and synergistically with traditional agonists.

Methods: The presence of BDNF in platelets was confirmed by Western Blotting and confocal microscopy. The ability of platelets to release BDNF upon stimulation was assessed by ELISA on platelet releasates following stimulation by maximal concentrations of arachidonic acid, adenosine diphosphate (ADP), epinephrine, collagen, or thrombin-receptor activating peptide (TRAP). To investigate the effect of BDNF on platelet function, we used a recombinant BDNF protein (rBDNF, Peprotech) added exogenously to a preparation of washed platelets obtained from healthy volunteers. Aggregation was recorded by light transmission aggregometry over 20 minutes. Synergistic assays were used with priming (sub-threshold) concentrations of either collagen or TRAP. The dependence on secondary mediators was assessed by blocking either COX-1-dependent thromboxane formation with aspirin or the ADP-P2Y12 receptor with AR-C66096. Platelet aggregation was blocked with a GPIIbIIIa inhibitor (abciximab) to assess the contribution of outside-in signalling.

Results: Western blotting showed a 14kD band for the washed platelet protein preparations and for rBDNF used as a positive control. BDNF appeared stored in granules on confocal microscopy, consistent with previous reports. BDNF in the platelet releasate was increased 5-fold following platelet aggregation induced by traditional agonists, as compared with baseline. Exogenous rBDNF had no effect on platelet aggregation at low doses (1 and 3 µg/ml), but induced complete biphasic platelet aggregation at a high dose (10 µg/ml). The secondary wave of platelet aggregation was abrogated in the presence of aspirin and AR-C66096, but they had no effect on the primary wave. Platelet aggregation was completely abolished in the presence of the GPIIbIIIa inhibitor abciximab. Synergistic studies were conducted with priming concentrations of rBDNF, collagen and TRAP which were insufficient to elicit platelet aggregation on their own. Incubation of platelets with rBDNF (3 µg/ml) potentiated the platelet responses to priming concentrations of both collagen and TRAP. Indeed, in the absence of BDNF, no platelet aggregation was seen at the concentrations used, whereas incubation with BDNF led to complete and irreversible platelet aggregation to priming concentrations of collagen and TRAP.

Conclusion: Platelets contain BDNF within their granules, which they release upon platelet activation with traditional agonists. Stimulation of washed platelets with high concentrations of rBDNF induced platelets to aggregate; in the presence of low concentrations of rBDNF, platelet aggregation was potentiated in response to priming concentrations of traditional agonists. Platelet responses to rBDNF were partly dependent on secondary mediators, as inhibition of COX-1 and the ADP-P2Y12 receptor inhibited the secondary wave of aggregation. However, the primary wave of aggregation was unaltered, thereby suggesting that platelets either possess a BDNF receptor or a traditional receptor that can signal in the presence of BDNF. Further studies are needed to uncover which receptor is implicated in platelet responses to BDNF, and the physiological role of BDNF-induced platelet responses.