Thrombus Formation with Rehydrated, Lyophilized Platelets

Abstract

Stored human platelets are frequently used in hemorrhagic emergencies, but have limited immediate utility for controlling bleeding due to storage lesion and are frequently contaminated with microorganisms. The development of paraformaldehyde-treated, lyophilized and rehydrated (RL) platelets, which are sterile and have a prolonged shelf life (years), ameliorate the efficacy and sterility problems with stored platelets. RL platelets have been shown to have many native functions of fresh platelets in vitro and to mediate hemostasis in vivo in large animal models of hemorrhagic shock and cardiopulmonary bypass induced platelet dysfunction. To further evaluate the functional properties of this transfusion product, we studied the role of RL platelets in three aspects of thrombus formation and lysis. First, the interaction between RL platelets and fibrinogen was investigated. The surface density of unligated GPIIb-IIIa on RL and fresh platelets were, respectively 30,000 and 70,000 molecules per cell as detected with the monoclonal antibody 10E-5. Freezing, lyophilization and rehydration steps in the preparation of RL platelets resulted in the surface presentation of 120,000 molecules of fibrinogen per cell from alpha granule sources. After ADP activation, RL platelets bound exogenous ¹²⁵I-labeled fibrinogen in a dose-dependent manner with an affinity that is similar to that of fresh platelets and was inhibited by RGD peptides. ¹²⁵I-Labeled fibrinogen binding to RL and fresh platelets, respectively, saturated at 14,000 and 32,000 molecules per cell. Scanning electron microscopic ultrastructural analysis showed that fibrin strands interacted with the surface of RL platelets in a normal manner. The second set of studies investigated the ability of RL platelets to catalyze and amplify the clot formation process in an activation-dependent manner. We showed that RL platelets undergo degranulation in fibrin in clots and functioned as thrombogenic surfaces for the generation of activated coagulation factors and fibrin generation. A final set of studies was performed to investigate fibrin of clots that contained RL platelets. RL platelet clots were lysed in the presence of tissue plasminogen activator with a similar time course as clots without platelets, and lysis occurred faster than when fresh platelets were included in the fibrin mass. The results of these three studies demonstrate that RL platelets are capable of mediating thrombus formation and do not inhibit lysis. Our results help explain how RL platelets restore hemostasis in vivo, and indicate that these cells might be a viable alternative to fresh stored platelets in transfusion medicine.

Keywords:

• Platelets
• Lyophilization
• Thrombus
• Fibrinogen
• Degranulation
• Fibrin