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Therapeutic antibodies, including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), bi-specific antibodies and antibody fragments, have become the predominant class of new drugs being developed. Some mAbs have been shown to affect platelet function, via the FcyRIIa receptor expressed on platelets, increasing the risk of thrombotic side effects. Platelet testing in the early stages of antibodies discovery and development could reduce the risk of drug attrition due to safety concerns or unexpected toxicity. We compared several platelet function readouts and test conditions to identify optimal parameters for an in vitro evaluation of possible effects of antibodies on primary haemostasis. Blood samples were obtained from healthy human volunteers. Platelet aggregation was assessed in platelet rich plasma (PRP) or washed platelets (WP) by light absorbance; platelet activation in different matrices, including whole blood (WB), was assessed by flow cytometry. The effect of a half log concentration curve of anti-CD226 antibody on aggregation was assessed in PRP (n=30); the effect of anti-CD226 antibody at 0.1, 1 and 10 μg/mL in the presence of increasing concentrations of collagen related peptide (CRP) or adenosine diphosphate (ADP), known platelet agonists, was assessed using both activation and aggregation assays in different matrices (WB, PRP and WP; n=6). All assays were performed in a 96-well plate format in duplicates. Testing the effects of anti-CD226 in platelet aggregation assay showed that 2/3 of the volunteers were “high responders” to this activating mAb. The effects of anti-CD226 in 6 volunteers were most prominent in washed platelets, where 10 μg/mL induced maximal activation and aggregation response directly, without agonists. Similar effects of anti-CD226 at lower concentrations, alone and with agonists, was observed with both activation and aggregation in WP. In PRP the effect of anti-CD226 at 10 μg/mL reached maximum aggregation but required longer stimulation. In WB, anti-CD226 at 10 μg/mL did not reach maximum platelet activation. In both matrices the antibody showed additive effect in combination with platelet agonists. Two agonists, CRP and ADP, produced full concentration response curves in aggregation assay. In activation assay ADP, being a weaker agonist, produced concentration dependent response that plateaued at 71% after 20 minutes stimulation, but this allowed for the additive effect of the antibody to be observed. When assessing secondary pharmacology effects of antibodies on platelets in vitro it is beneficial to recruit donors known to be “high responders” and test the effects of antibodies in combination with platelet agonists. The most sensitive matrix for assessing the effects of anti-CD226 by both aggregation and activation was washed platelets; other matrices could be considered suitable based on the characteristics of antibodies’ in development.