Decreased Whole Blood Factor IX Activity Following Hemodilution with Hemoglobin A-Zero In-Vitro

Abstract

Previous studies of hemoglobin-based oxygen carriers and their effects on coagulation have shown conflicting results. This study re-examined the effect of hemoglobin solution on blood coagulation in whole blood using highly purified human hemoglobin Ao (HbAo). Citrated human whole blood samples were diluted 1:1 with HbAo (7gHb/dl) or human serum albumin (HSA; 5g/dl) as a protein control, and both activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured using a mechanically based whole blood coagulometer. Multiple runs were performed with the same volunteer donor sample. The mean aPTT time of HbAo-diluted samples (105.9±19.9 sec., n=41) was significantly longer than the undiluted controls (46.4±5.6 sec., n=54) or HSA-diluted blood (77.1±12.7 sec., n=41) indicating an abnormal intrinsic coagulation pathway. There was no significant difference between the PT times of the HbAo and HSA-diluted samples. To examine the cause of the prolonged aPTT times with HbAo dilution, we performed activity assays of intrinsic factors XII, XI, IX, and VIII in a citrated human whole blood system diluted 1/5 and 1/10 with normal saline solution (NSS), and then 1:1 with either HbAo, HSA, or NSS. Only the Factor IX activity was significantly depressed by hemodilution (1/5 HbAo 50.20±8.11; HSA 61.05±6.72; NSS 74.75±9.83. 1/10 HbAo 46.50±5.57; HSA 64.97±11.01; NSS 67.92±16.03). These results suggest that in-vitro hemodilution with HbAo causes a hypocoagulatory response through interference with Factor IX function.