title: Mechanisms of hemolysis-associated platelet activation.
abstract: BACKGROUND
  Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias, and in 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: (i) direct activation by hemoglobin (Hb); (ii) increase in reactive oxygen species (ROS); (iii) scavenging of nitric oxide (NO) by released Hb; and (iv) release of intraerythrocytic ADP.
  
  
  OBJECTIVE
  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 by using platelet counts from in vivo experiments in a rodent model.
  
  
  RESULTS
  We found that Hb did not directly activate platelets. However, ADP bound to Hb could cause platelet activation. Furthermore, platelet activation caused by shearing of red blood cells (RBCs) was reduced in the presence of apyrase, which metabolizes ADP to AMP. The use of ROS scavengers did not affect platelet activation. We also found that cell-free Hb enhanced 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, resulted in platelet aggregation, as shown by decreased platelet counts.
  
  
  CONCLUSIONS
  Two primary mechanisms account for RBC hemolysis-associated platelet activation: ADP release, which activates platelets; and cell-free Hb release, which enhances platelet activation by lowering NO bioavailability.
authors.affiliation: Department of Physics, University of Richmond, Richmond, VA, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Helms
authors.forename: M
authors.initials: M
authors.lastname: Marvel
authors.forename: W
authors.initials: W
authors.lastname: Zhao
authors.forename: M
authors.initials: M
authors.lastname: Stahle
authors.forename: R
authors.initials: R
authors.lastname: Vest
authors.forename: G J
authors.initials: GJ
authors.lastname: Kato
authors.forename: J S
authors.initials: JS
authors.lastname: Lee
authors.forename: G
authors.initials: G
authors.lastname: Christ
authors.forename: M T
authors.initials: MT
authors.lastname: Gladwin
authors.forename: R R
authors.initials: RR
authors.lastname: Hantgan
authors.forename: D B
authors.initials: DB
authors.lastname: Kim-Shapiro
mesh_terms: D004912:Erythrocytes / Q000378:metabolism; D006454:Hemoglobins / Q000502:physiology; D006461:Hemolysis / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000502:physiology; D015539:Platelet Activation / Q000502:physiology*
chemical_list: D006454:Hemoglobins; D009569:Nitric Oxide
keywords: hemoglobin; hemolysis; nitric oxide; platelets; red blood cells