Model 490

Human blood from healthy volunteers without using medications interfering with platelet activity for at least 10 days prior to testing was collected in 3.8% sodium citrate (1:9). Platelet aggregation using plasma rich in platelets (PRP) was performed as previously described [85 (link)]. For the aggregation assay, 7.5 μg of L. gaucho, L. laeta and L. intermedia venoms were pre-incubated or not with 0.1, 0.3 or 0.6 μg/μL of anti-LgRec1ALP1 IgGs in a final volume of 100 μL. The reaction was incubated for 60 min at 37 °C and then centrifuged for 5 min at 10,000 g before use. Platelet-poor plasma (PPP) was used as blank and 0.6 μg/μL of IgG anti-EGFP pre-incubated with 7.5 μg of L. gaucho, L. laeta or L. intermedia venoms in a final volume of 100 μL were used as a negative control. The agonist ADP (final concentration of 10 μM) was used as positive control for platelet aggregation. The experiments were performed in triplicate (n = 3) on a Chrono-Log Model 490 aggregator (Chrono-Log Corporation, Havertown, PA, USA) and reported as the mean ± SEM.

Light transmission aggregometry (a schematic example is shown in Figure 2) was performed according to standard protocols (13 (link)). Briefly, platelet aggregation was assessed using platelet-rich plasma (PRP) and platelet-poor plasma (PPP) by the turbidometric method in a two-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp., Havertown, PA, USA). PRP was obtained as a supernatant after centrifugation of citrated blood at 100 g for 10 min and PPP was obtained by a second centrifugation of the blood fraction at 1500 g for 15 min. Light transmission was adjusted to 0% for PRP and to 100% for PPP for each measurement. Maximal platelet aggregation (MPA) was stimulated by 20 and 5 μmol/L adenosine diphosphate (ADP) as agonists. High on-treatment platelet reactivity (HPR) was defined as MPA > 64.5% and >42.9% with ADP 20 and 5 μmol/L, respectively (14 (link)).