Maternal human platelet antigen (HPA)-1a alloantibodies causing neonatal alloimmune thrombocytopenia can bind also to endothelium, via the β3-integrin (CD61). The aim of this study was to investigate the effect of HPA-1a Abs on endothelial cell function, with emphasis on monolayer integrity. We used a CD61 mAb as a model for the HPA-1a alloantibodies and confirmed the results with purified IgG fractions from HPA-1a alloimmunized women. The effect of these antibodies was examined by monitoring the adhesion, spreading, and monolayer integrity of primary HUVECs with conventional adhesion assays as well as electrical cell-substrate impedance sensing. We found that both the mAb CD61 and the HPA-1a antibodies caused a significant reduction in HUVEC spreading. Moreover, addition of the mAb CD61 and the HPA-1a antibodies prior to or following formation of a stable endothelial monolayer negatively affected endothelial monolayer integrity, which was accompanied by a redistribution of junctional proteins. Our data suggest that HPA-1a alloantibodies have a direct effect on endothelial cell spreading and monolayer integrity, which could contribute to the increased bleeding tendency in children with neonatal alloimmune thrombocytopenia.