*2.2.2. Platelets*

Bovine platelet activation and microaggregates have been studied using various markers in calves implanted with CF-VADs. Baker et al. developed a method using the antibodies BAQ56, BAQ125 and GC5A (platelet antigen, CD equivalent unknown, available from Washington State University) to measure platelet activation, and CAPP2A (anti-CD41/CD61) to measure platelet microaggregates in calves implanted with CF-LVADS (the Sun Medical centrifugal pump or the HeartMate II) [23]. Platelet microaggregates, i.e. platelet positive events (binding anti-bovine CD41/61 antibody) with forward scatter larger than single platelets, increased post-operatively and then showed some decline in calves implanted with Sun Medical whereas they remained elevated in the calves implanted with HeartMate II. Platelet activation increased post-operatively for both implants and remained elevated. BAQ56 provided the strongest signal and GC5 the weakest signal of the three antibodies, but BAQ125 and GC5 exhibited the strongest agreement with one another, and with the circulating microaggregates [23].

Snyder et al. used a modified version of Baker's methodology in two studies of calves implanted with the CF-LVADs—HeartMate II [24] and the EVAHEART [25]. Snyder showed that the microaggregate levels in surgical sham controls remained at pre-operative levels, confirming that the CF-LVAD and not the cardiac surgery cause elevated levels [24]. Microaggregate levels increased post-implantation, and decreased within the first week in all animals implanted with the EVAHEART [25], and in those animals implanted with the HeartMate II who had an uneventful post-operative course [24]. In calves who suffered adherent thrombi in the outflow region of the pumps, the microaggregate levels either remained elevated or increased before conclusion of the study [24].

Part of Snyder's modification was the inclusion of additional markers for platelet activation to address the limitations BAQ56, BAQ125, and GC5 with their unknown target antigens, namely Annexin V binding [24], anti-CD62P and anti-CD63 [25]. All platelet activation/apoptosis markers tested (BAQ125, GC5, Annexin V, CD62P, CD63) increased immediately after implantation and remained significantly elevated in CF-LVAD animals versus the pre-operative control [24, 25]. Those tested in sham animals (BAQ125, GC5, Annexin-V) decreased around 2 weeks post-operatively [24]. Although CD62P and CD63 successfully identified activated platelets in CF-LVAD calves, the signal was weaker than that of the BAQ125 and GC5 antibodies [25].

CAPP2A also binds to ovine platelets whereas anti-bovine platelet activation antibodies BAQ125 and GC5 do not. Hence, Johnson et al. used CAPP2A as a platelet lineage marker along with cross-reactive anti-human CD62P antibodies (clones Psel.KO.2.7 and Psel.KO.2.12) to assess platelet activation in sheep implanted with paediatric CF-LVADs [26–28]. Johnson showed that, similarly to calves, platelet activation increases post-operatively in surgical sham control sheep and returns to pre-operative levels at around 2 weeks. The implanted sheep did not demonstrate a common pattern [28]. However, a finding also consistent with previous work was that platelet activation spiked in animals that suffered complications [26–28].
