Cocaine-Induced Hemolysis Activates Platelets in Whole Blood

The clinical phenotype of sickle cell disease (SCD) is primarily a consequence of two processes: chronic hemolysis and vaso-occlusive phenomena, the latter a result of the enhanced adhesiveness of erythrocytes, leukocytes, and platelets. These processes produce the myriad clinical manifestations of the disease, which include endothelial dysfunction, chronic inflammation, episodic ischemia/reperfusion injury, tissue infarction, and oxidative injury. The proximate cause of SCD is a mutation in the gene encoding the beta-globin chain of hemoglobin, converting glutamate at the sixth position of the polypeptide chain to valine. This seemingly innocuous change results in a hemoglobin molecule prone to polymerize under low oxygen tension, altering the shape of the erythrocyte, its mechanical stability, and its adhesiveness. Very little is known about the mechanisms that render some patients more susceptible to the consequences of chronic hemolysis or vaso-occlusion. Cocaine abuse is known to trigger pathways consistent with both increased adhesivity (vaso-occlusive pain crises) and hemolysis (priapism) in SCD. Cocaine use has also been associated with a 29% increased risk of death/year following even a single documented cocaine exposure in SCD patients. It is also noteworthy that cocaine is associated with thrombotic processes that suggest both endothelial and platelet activation, both of which have been implicated in SCD pathophysiology. We hypothesized that cocaine activates platelets, which would be augmented in sickle cell disease. We assessed the activation of platelets exposed in vitro to cocaine by assaying two markers of platelet activation:

1. surface P-selectin exposure reflecting activation-dependent release of platelet a granules, and

2. exposure of an activation-dependent epitope in the integrin a_IIbb₃ recognized by the antibody PAC-1.

Surprisingly, neither washed platelets nor platelets in plasma exposed to cocaine in vitro showed evidence of activation above background. In fact, treatment of platelets with cocaine under these conditions rendered them hyporesponsive to subsequent activation by ADP, epinephrine, or thrombin with similar dose–response curves. In contrast, when whole blood was exposed to cocaine, platelet activation was observed by both increased platelet P-selectin exposure and PAC-1 binding. When individual blood components were then mixed with washed platelets, only addition of erythrocytes resulted in platelet activation to cocaine. We explored the mechanism of this response using isolated erythrocytes. By flow cytometry, we observed accumulation of erythrocyte-derived microparticles within 10–20 min of cocaine exposure, which also occurred in cocaine treated whole blood. Erythrocytes from patients with SCD were more sensitive to cocaine, producing microparticles at lower concentrations of the drug than non-SCD erythrocytes. Using DIC microscopy and video recording, we found that erythrocytes (both SCD and non-SCD) underwent rapid morphologic changes upon cocaine exposure, changing from discocytes to echinocytes then rapidly to stomatocytes. The echinocytes appeared within 15–30 sec of cocaine exposure, then transformed into stomatocytes within the next 45 sec to 2 min. We did not observe reversal of this morphologic transition, with 90–100% of erythrocytes undergoing the full transformation into stomatocytes and retaining this morphology through 60 minutes of observation. We also observed release of small membrane vesicles from the erythrocytes. A fraction of the cells undergoing this transformation lysed after reaching the stomatocyte phase, with increased hemolysis observed with sickle erythrocytes compared to erythrocytes from ethnically matched non-sickle controls. These results suggest that cocaine activates platelets indirectly by a mechanism that involves erythrocyte lysis. Other aspects of cocaine’s effects on erythrocytes would be expected to potentiate a prothrombotic phenotype, including microparticle release and nitric oxide scavenging resulting from hemolysis. Because of the increased fragility of their erythrocytes, individuals with sickle cell anemia would be expected to be especially susceptible to the cardiovascular complications of cocaine use.