Running head: Hemolysis-associated platelet activation

Summary Background - Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias and other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets in vitro and in vivo and several mechanisms have been suggested to account for this including (1) direct activation by hemoglobin, (2) increase in reactive oxygen species (ROS), (3) scavenging of nitric oxide by released hemoglobin, and (4) release of intraerythrocytic ADP. Objective – The aim of the current study is to elucidate the mechanism of hemolysis-mediated platelet activation. Methods – We used flow cytometry to detect PAC-1 binding to activated platelets for in vitro experiments and a Siemens' Advia 120 hematology system to assess platelet aggregation using platelet counts from in vivo experiments in a rodent model. Results - We show that Hb does not directly activate platelets. However, ADP bound to Hb can cause platelet activation. Furthermore, platelet activation due to shearing of RBCs is reduced in the presence of apyrase which metabolizes ADP to AMP. Use of ROS scavengers did not affect platelet activation. We also show that cell free Hb does enhance platelet activation by abrogating the inhibitory effect of NO on platelet activation. In vivo infusions of ADP and purified (ADP-free) Hb as well as hemolysate result in platelet aggregation as evidenced by decreased platelet counts.