The spontaneous anticoagulant-dependent platelet activation in vitro may potentially interfere with the determination of haemostatic parameters. The effects of various blood anticoagulants on platelet activation were monitored using flow cytometry. Regardless of a blood anticoagulant used (EDTAK 2, heparin, citrate or PPACK), platelet activation began immediately after blood withdrawal and was most pronounced in the EDTAK 2-anticoagulated blood samples. The progressing expression of GMP-140 antigen was accompanied by the enhanced abundance of the subunit β 3 of the platelet membrane integrin α IIbβ 3 without parallel changes in the fluorescence attributed to the complex form of the integrin α IIbβ 3. The increased expression of GMP-140 was paralleled by the enhanced platelet clumping in the samples anticoagulated with either EDTAK 2 or heparin, and the raised platelet microparticles in blood withdrawn into citrate. The EDTAK 2-induced platelet activation was markedly reduced by methyl 2,5-dihydroxycinnamate, tyrosine kinase inhibitor. The influence of disodium EDTA on platelet membrane dynamics closely mimicked the alterations induced upon the interaction of fibrinogen with platelet GPIIb-IIIa. Thus, the EDTAK 2-induced platelet activation might result from an interference with platelet membrane protein structure and conformation and possibly relate to an “unspecific” trigerring of a signal transduction pathway. Overall, EDTAK 2 and heparin appeared the least suitable anticoagulants, particularly with the regard to the expression of GMP-140 antigen. The failure to recognize the importance of a spontaneous anticoagulant-induced platelet activation may result in misdiagnoses during the monitoring of coagulation parameters.