Platelet reactivity with liposome-encapsulated hemoglobin in the rat

Infusion of liposome-encapsulated hemoglobin (LEH) induces a transient thrombocytopenia in rats (Rabinovici R, Rudolph AS, Ligler FS, Smith EF, III, Feuerstein G [1992] Biological responses to exchange transfusion with liposome-encapsulated hemoglobin. Circ Shock 37:124). A specific mechanism such as a localization of platelets during this transient LEH-induced thrombocytopenia has not been reported previously in the literature. In this study, platelets were isolated and labeled with indium-111 (111In), then retransfused into the same animal. Fifteen minutes after the 111In platelets were retransfused and allowed to equilibrate with the blood pool, a 10% top load volume of either LEH (1.8 mL, 262 mg phospholipid/kg body weight, 92 mg hemoglobin (Hb)/kg body weight), liposome vehicle (LV) (1.8 mL, 262 mg phospholipid/kg body weight), or free bovine Hb (1.8 mL, 92 mg Hb/kg body weight) (n=6 per group) was infused. Serial blood samples were drawn to determine platelet counts by radioactivity and light scattering methods. LV and Hb demonstrated no significant changes from baseline levels in circulating platelet levels, whereas LEH caused a transient 50% decrease in 111In platelet activity 2-5 minutes postinfusion, which returned to baseline levels by 15 minutes. Platelet counts based on traditional light scattering methods were not significantly different among the three treatment groups over this same time course. Localization of these 111In platelets was monitored with a gamma scintigraphic camera. After infusion of LEH, 111In platelets rapidly moved out of the circulation and sequestered in the lungs and liver with subsequent return to the circulation by 15 minutes. In contrast, no significant changes in 111In platelet activity were noted in the lungs and liver after infusion of LV and Hb. 111In platelet activity in the spleen nearly doubled 30 minutes after Hb infusion and was significantly different than spleen 111In platelet activity in the LEH and LV groups. In vitro platelet aggregation studies were also performed to determine the direct effect of LEH, LV, and Hb on platelet aggregation. The rate of thrombin-induced aggregation did not differ among control samples and samples containing LEH, LV, or Hb; none of these agents induced platelet aggregation. We conclude that LEH-induced thrombocytopenia is associated with a transient (within minutes) sequestration of platelets in the lungs and liver, with subsequent release to the circulation within 15 minutes. Tetrameric bovine Hb is associated with increased platelet accumulation in the spleen. Light scattering methods for measuring platelet levels in blood samples containing LEH are unreliable because of particulate interference.