The use of the synthetic peptide, Gly-Pro-Arg-Pro, in the preparation of thrombin-degranulated rabbit platelets

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.