W. J. DODDS AND L. W. HOYER 
465 
stage assay as previously described. ^ Some sam- 
ples were also assayed with human deficient 
substrate in a similar one-stage assay. i° There 
was good agreement between the activities 
measured in perfusates by these two methods. 
However, because the addition of human 
plasma was studied in a rabbit perfusion sys- 
tem, the relative coagulation activities of rabbit 
and human factors VIII and IX were also deter- 
mined. 
Coagulation activity of perfusates as shown 
in Figures 1-6 of "Results" refers to the per- 
cent activity compared to that of a standard 
pool of normal rabbit plasma which was as- 
signed a value of 100%. All coagulation data 
were computed and the best fit plot was ob- 
tained after fifth order regression analysis ; the 
standard deviation at each sample interval was 
also determined. 
RESULTS 
Figure 1 illustrates the control data for fac- 
tor VIII and IX activities produced by livers 
and speens perfused without additives. The ac- 
tivity present at the end of the initial hour of 
perfusion, labeled "flush," has been attributed 
primarily to release of tissue stores.^ Factor 
VIII activity (Figure la) is more active during 
splenic perfusion although perfused livers sus- 
tained their ability to increase perfusate factor 
VIII activity throughout the 4 hr perfusion pe- 
riod. In contrast to factor VIII, there was no 
difference between splenic and hepatic factor 
IX production (Fig. lb). The activities gener- 
ated in the experiments shown in Figure 1 were 
efficiently blocked by puromycin, cycloheximide, 
and actinomycin D.^-^ The data shown in sub- 
sequent figures include only the perfusion pe- 
riod following addition of plasma (the interval 
enclosed within the stippled areas on Figure 1) . 
Data for splenic and hepatic factor VIII and 
IX activities after addition of fresh normal rab- 
bit plasma are shown in Figure 2. Perfusate 
factor VIII and IX activities were increased 
after addition of normal plasma and both factor 
VIII (Figure 2a) and factor IX (Figure 2b) 
production ceased. As might be expected by the 
CONTROL RABBIT ORGAN PERFUSION 
Figure 1. — a. Factor VIII activities of 20 control rabbit spleen and 24 control rabbit liver perfusions, b. Factor IX 
activities of the same experiments shown in la. Areas enclosed by stippling represent the period of perfusion 
shown on subsequent figures. "Flush" values are activities after the initial hour of perfusion. Data computed and 
best fit curve obtained by fifth order regression analysis; limits shown represent the standard deviation at each 
sample point. 
