Pharmacokinetics of Red Cell-Derived Microparticles (RMP) in Rabbits and Rats: Effects of Administration Regimen and Labels for Assay

Background:  Circulating cell-derived microparticles (MP) are currently of interest in the fields of hemostasis, thrombosis, and inflammation. Administration of MP has been proposed for use as a hemostatic agent. Efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models [Thromb. & Haemost., 2013;110:751-60]. One of the challenges in developing MP as a hemostatic agent is uncertainty of half-life (T_1/2) in circulation, reportedly ranging from several minutes to hours. The purpose of this study was to assess the pharmacokinetics of RMP in rabbits vs. rats, and comparing alternative dosing regimens and assay markers. Although this work was aimed at optimizing the dosage regimen of RMP as a hemostatic agent, it has wider interest because very few studies have addressed the question of MP half-life in circulation.

Methods. (i) Human RMP were produced by high-pressure extrusion [Thromb. & Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x10⁹ counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the anesthetized rabbits or rats, via cannulated jugular vein. Blood samples (0.4 mL each) were collected from the cannulated carotid artery at intervals: 5 min pre-injection, and at 1, 2, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 8 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with 2 separated labels: anti-CD235a-PE (anti-glycophorin) and annexin V-FITC (AnV). The former is specific for human RMP and does not label rabbit or rat RMP. The latter is not species-sensitive, and labels both human and host MP. (iii) The procoagulant activity (PCA) of RMP was assayed by thromboelastogram (TEG) ex vivo.

Results: (1) Bolus Infusion of RMP (3 x10⁹ MP/kg) resulted in a rapid rise of RMP levels peaking at 1.0 min post-infusion, followed by slower decline to baseline by 10 – 15 min. The half-life (T_1/2) of clearance determined by anti-CD235a was 6.2 ±0.7 and was 4.2 ±0.5 min for rabbits and rats, respectively. In contrast, the T_1/2 measured by AnV was 4.4 ±1.0 and 2.6 ±0.4 min, respectively. These results indicate that these two MP phenotypes were cleared by different mechanisms, i.e., one or more mechanisms predominates in clearing CD235a(+) MP vs. AnV(+) MP. The RMP enriched in phosphatidylserine (AnV+) are cleared faster than those expressing CD235a. (2) Two-step administration was bolus (1/3 total dosage) followed by continuous infusion (2/3 total dosage), and resulted in a smaller initial spike followed by rapid decline to ≈25-30%, then recovering to a nearly steady-state level of 0.3 -0.6 x10⁷ MP/mL after 20 min. The RMP levels returned to baseline within 15 min after cessation of infusion. The area under the curve (AUC) of the PK curve for bolus infusion was about 40 -60% less than that of two-step administration of the same dose. (4) Procoagulant Activity (PCA): For bolus infusion, the ex vivo TEG data revealed some correlation between circulating RMP and PCA at the early phase (1 to 6 min). At later phase (>10 min), although RMP had dropped to almost baseline, the PCA remained active considerably longer than presence of RMP (T_1/2= 9.3±1.2 min for PCA). However, bolus followed by continuous infusion resulted in maintaining stable PCA over the course of infusion.

Conclusion / Discussion. (i) The clearance rate of RMP in rats is faster than that in rabbits. (ii) When T_1/2 is determined by the two different markers, we found significant differences in half-life, indicating differential of mechanisms of clearance. (iii) The TEG data show that the PCA of RMP persists after detectable RMP have entirely cleared. We interpret this to mean that RMP continue to exert PCA by being bound to blood cells, even after free circulating RMP have cleared. (iv) Continuous infusion is expected to perform better than bolus infusion in controlling excessive bleeding in clinical patients.