A Third Generation Perfluorocarbon Causes Thrombocytopenia, Platelet Dysfunction and Changes In Blood Morphology in a Baboon Model Of Systemic Inflammation

A 3rd generation perfluorocarbon (PFC) is under development for the treatment of traumatic brain injury (TBI). PFCs have high gas solubility and can deliver oxygen to support ischemic brain tissue. This drug increased brain tissue oxygen consumption, decreased infarct size, and improved functional deficits in animal models of brain injury, but it also caused transient thrombocytopenia of unknown etiology. Acute inflammation also causes platelet deficits and is common in the setting of severe TBI. The interaction between the two could exacerbate thrombocytopenia. Here we studied PFC effects on platelet function, platelet morphology and global hemostasis in a lipopolysaccharide (LPS) baboon model of inflammation.

Perfluorocarbon (Oxycyte^”, Oxygen Biotherapeutics Inc, Morrisville, NC) infusion was performed with and without administration of LPS. The groups studied included:

1. Saline control (20 ml saline + 12 ml/kg saline: SALINE)

2. Saline and LPS control (0.3 mg/kg LPS in 20 ml saline + 12 ml/kg saline: SAL-LPS)

3. Medium dose PFC with saline vehicle (20 ml SALINE + 3 ml/kg PFC: SAL-PFC3)

4. High dose PFC with saline vehicle (20 ml SALINE + 12 ml/kg PFC: SAL-PFC12)

5. Medium dose PFC with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 3 ml/kg PFC: LPS -PFC3)

6. High dose PFC with with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 12 ml/kg PFC: LPS –PFC12)

Platelet count, transmission electron microscopy, and flow cytometry were performed at baseline (BL) and at 2, 24, 48, 72, and 96 hours after perfluorocarbon infusion. Microparticles were quantified and characterized by flow cytometry and morphology examined via transmission electron microscopy. Impedance aggregometry and thromboelastography (TEG) were quantified at baseline (BL) and at T2, T72, and T96 (hours). Statistical analysis was by ANOVA, followed by pairwise comparisons and a Bonferroni adjustment.

PFCs caused delayed thrombocytopenia which began 2 days post-infusion and persisted beyond the 5-day period of observation (nadirs≤50% of baseline at day 5, p<0.05). Platelets (PLTs) also decreased due to LPS administration, but effects began within 6 hours, were less severe, and resolved more quickly (nadirs≥50% of baseline at day 2, with normalization of platelet counts by day 4, p<0.05). Thrombocytopenia was additive in LPS+PFC groups, occurring within 2 hours of study start time, and persisting for more than 5 days (p<0.05). The decrease in platelet count was more profound in LPS+PFC groups (nadirs of ≤25% of baseline, p<0.05). LPS+PFC increased aggregation corrected for PLTs (p<0.05), but PFCs alone did not. LPS+PFC decreased TEG MA values, whereas PFCs alone had minimal effects. PFCs exacerbated adverse LPS effects, resulting in diffuse microvascular hemorrhage in 2 of 5 baboons in the LPS-PFC3 group and 2 of 2 in the LPS- PFC12 group. Transmission electron microscopy and histology were consistent with shock associated with hemorrhage in multiple organs and demonstrated abnormal morphology of platelets and red blood cells.

PFC infusion caused clinically significant thrombocytopenia, particularly after LPS administration, and exacerbated LPS-induced platelet activation. The interaction between the two resulted in decreased hemostatic capacity, diffuse bleeding, and shock.