Comparison of Pharmacokinetics and Hemostatic Efficacy of Red Cell Microparticles (RMP) in Rabbits Using Different Infusion Regimens

INTRODUCTION: Excessive bleeding is a life-threatening challenge in many areas of medical practice, especially in surgical procedures and trauma care. Few treatment options are available to meet the challenge of preventing or treating excessive bleeding, and none of them is satisfactory. The efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models, as summarized in a recent publication by Jy et al. [Thromb. & Haemost., 2013;110:751-60]. No adverse effects were noticed in short-term observation of either model with the effective dose used. The rate of clearance of RMP in vivo has not been analyzed systemically. The purpose of this study is to characterize the pharmacokinetics / rate of clearance of RMP in rabbit model by different infusion regimens and to establish the relationship between blood concentration and hemostatic efficacy of RMP.

METHODS: (i) RMP were produced by high pressure extrusion method [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 sedated non-bleeding rabbits. Blood samples (1 mL each) were collected at intervals: 5 min pre-injection, and at 1, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 5 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with dual labeling: anti-CD235a-PE and Annexin V-FITC. The former is specific for human RMP and will not label rabbit RMP. The latter is not species sensitive and labels both human and rabbit MP. (iii)The procoagulant activity of RMP in rabbit blood was assayed by thromboelastogram (TEG).

RESULTS:(1) BolusInfusion of RMP (3 x10⁹ counts/kg) resulted in a rapid rise of RMP levels peaking at 1 min post-infusion followed by rapid decline to baseline by 10 – 15 min. The half-life (T_1/2) in circulation was estimated to be ≈ 4 – 7 min. The peak RMP concentration reached 2.5 – 3.4 x10⁷ counts/mL. (2) The 2 markers used yielded small but significantly different rates of clearance after reaching peak concentrations: the T_1/2 by anti-CD235a was 6.2 ±1.0 min, and by annexin V was 4.4 ±0.7 min (p = 0.01). These results indicate that these two phenotypes of RMP were cleared by different mechanisms. RMP expressing phosphatidylserine (annexin V positive) seem to be cleared faster than those expressing CD235a. (3) Bolus followed by continuous infusion resulted in a smaller initial spike (0.7 – 1.0 x10⁷ counts/mL) followed by a rapid decline to ≈25-30%, then steady rise over the course of 30 min. infusion, finally reaching a steady-state level of 0.4 -0.6 x10⁷ counts/mL. RMP levels returned to baseline within 15 min after cessation of infusion. (4) The ex vivo TEG data revealed good correlation between rise and fall of circulating RMP and procoagulant activity. However, T_1/2 for procoagulant activity was longer (7.4 ±1.2 min.) compared to T_1/2of circulating RMP, whether measured by anti-CD235a or annexin V. On the other hand, bolus followed by continuous infusion resulted in steady elevation of procoagulant activity, with little fluctuation during the course of infusion.

CONCLUSIONS: These data demonstrate that bolus followed by continuous infusion of RMP is capable of maintaining almost steady-state levels for extended periods, with concomitantly increased procoagulant activity. Accordingly, this regimen is expected to be the optimum clinical treatment for excessive bleeding. This work also demonstrates the existence of different rate of clearance for different phenotypes of RMP, suggesting that multiple mechanisms are involved in the clearance of RMP.