We are developing RMP for use as an infusible hemostatic agent to treat bleeding conditions. Preliminary evaluations in vitro, such as by thromboelesatography (TEG), were very promising, and were supported by in vivo studies in a small animal (rat) [Haemophilia 2011; 17: 44]. It was shown that RMP not only accelerate coagulation and correct hemostatic aberration of factor-deficient plasma but also enhance platelet aggregation and adhesion, suggesting its efficacy in both primary and secondary hemostasis [Blood 2011; 118:3263]. Here we report results of initial studies in a large animal, the rabbit ear bleeding model.
RMP were produced by high-pressure extrusion of human RBC, converting >99% of RBC to RMP. Hemostatic efficacy was evaluated in a rabbit ear bleeding model. New Zealand male white rabbits of 2.5–3.5 kg were used. After sedation, the ear was prepared by chlorhexidime scrub, then a standard incision 6 mm wide was made to penetrate the ear (#11 scalpel blade), in a region of the ear devoid of macroscopic (visible) vessels. The ear was maintained at 37°C with a heating pad. Bleeding time (BT) was defined as the time required for the disappearance of blood stains on a filter paper blot. To reliably measure reduction of BT by the RMP product, bleeding was prolonged by administration of busulfan (20 mg/kg SC) twice each at days 1 and 3, to suppress bone marrow, resulting in thrombocytopenia after 7–12 days. BT was significantly prolonged in animals when platelet counts fell to <60,000/μL. This regimen allowed all animals to survive, but higher doses of busulfan was toxic and caused severe pancytopenia, unacceptable for experimentation.
In normal (untreated) rabbits, the BT was 90 ±20 seconds (n = 12). Injection of RMP (3×109/kg, IV) in untreated rabbits resulted in no significant changes in BT, and no major adverse events were seen (n = 6). In thrombocytopenic rabbits, the BT was prolonged to 620 ±130 sec., and prolongation of BT correlated with decrease in platelet counts, 30,000 – 60,000/μL (n = 12). In this range of platelet counts, injection of RMP (at either 1 × 109 or 3 × 109/kg, IV) induced significant shortening of BT, to 440 ±90 sec (n = 8) at the lower dose (p<0.01), and to 280 ±65 sec (n = 8), at the higher dose (p < 0.001). When platelet counts dropped below 10,000/μL and BT >15 min, RMP had no significant effect. The hemostatic effects disappeared if >30 min elapsed between RMP infusion and making the incision, indicating a short half-life of RMP in circulation.
These data confirm efficacy of RMP in thrombocytopenic rabbits. Hemostatic efficacy of RMP was evident in moderate thrombocytopenia, and was dose-dependent. However, efficacy fell off if platelets <10,000/μL, indicating that RMP augment platelet activity but do not replace platelets. In both thrombocytopenic and non-thrombocytopenic rabbits, no major adverse reactions were observed with infusion of RMP.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.