Solution Property and Blood Compatibility of Hemoglobin-Albumin Cluster 'HemoAct™'

摘要

In the last few decades, hemoglobin (Hb)-based O_2 carriers have been developed as a red blood cell (RBC) substitute. However, none satisfies all requirements for clinical use. An important concern (side effect) is increased blood pressure which is caused by NO scavenging by Hb diffused into extra vascular space. More recently, we have synthesized covalent core-shell structured protein clusters of Hb and human serum albumin (HSA) (Hb-HSA_m, m=2-4) by linkage of Hb surface lysines to HSA cysteine-34 via an a-succinimidyl-ε-maleimide crosslinker. The obtained Hb-HSA_m cluster solution ([Hb]=5 g/dL), -HemoAct™", could become a new type of RBC alternative and O_2-therapeutic reagent in transfusion medicine. This paper presents the structure, solution property, and blood compatibility of the -HemoAct™" artificial O_2-carrier. HPLC and Native-PAGE measurements revealed that the major component of cluster is Hb-HSA_3 heterotetramer. The average number of HSA binding per Hb was 2.8 (the diameter: 8.9±2.5 nm). The isoelectric point (pI=5.1) was almost identical to the value of HSA. This means that the surface net charge of the cluster is satisfactorily negative to be used as a transfusion medicine. HemoAct™ showed a moderately high O_2-affmity compared to native Hb. The P_(50) value (O_2 partial pressure where Hb is half-saturated with O_2) is 9 Torr. The viscosity of 2.8 cP at 230 s~(-1) is slightly lower than that of whole blood. After the addition of HemoAct™ into whole blood at 10, 20, and 40 vol%, no significant time dependence was observed in the numbers of RBCs, white blood cells, or platelets for 6 h at 37 ℃. The blood coagulation parameters were also unaltered. These results suggest that the HemoAct™ has no effect on blood cell components in vitro. All rats (Wister rats, male) given HemoAct™ have been alive for 7 days after the infusion. During the observation period, no remarkable reaction was seen in the appearance of the animals. Parameters of the clinical blood serum chemistry were all in the normal ranges.