Ristocetin, protamine and Polybrene promote factor VIII:vWF binding and agglutination of formalinized platelets. It has been suggested that these polycations neutralize platelet negative surface charges and promote the attachment of VIII:vWF to platelets. Platelet factor 4 (PF4), protamine, and Polybrene inhibit heparin activity by neutralizing heparin negative charges. We tested the hypothesis that PF4, which is bound to the platelet surface after platelet activation and secretion, could promote the binding of VIII:vWF and subsequent platelet agglutination. Purified PF4 in concentrations comparable to those of ristocetin did not agglutinate formalinized platelets or induce the disappearance of VIII:vWF from the suspending plasma. Platelets were thrombin-treated in order to induce the release of PF4, and then formalinized and resuspended in normal plasma. These platelets did not agglutinate spontaneously, or at lower ristocetin concentrations than platelets that were not treated with thrombin before formalin fixation. Platelets were also activated by thrombin in the presence of EDTA to prevent surface binding of VIII:vWF or secreted PF4, and then formalinized. These platelets did not bind VIII:vWF in the presence of purified PF4. We conclude that even though PF4 binds to both polyanions and the platelet membrane, it does not promote the attachment of VIII:vWF.