Time-dependent cleavage of HK in Lewis, Fischer, and Buffalo rat plasma. (A) Citrated plasmas were activated with dextran sulfate (40 μg/mL) and examined for HK cleavage by the Western blot technique. At the time points indicated an aliquot was removed from the reaction mixture and added to a customized LDS-PAGE plasma sample buffer containing reducing agent and subjected to Western Blot as described in Figure 2 legend. A specific band at 110 kDa was observed representing rat HK in the plasma protein mix. Disappearance of the band represents cleavage of the rat HK to lower protein forms (not shown). (B) Quantitative densitometric analysis of the substrate stained bands above was performed to determine the rate of HK cleavage with time. The data are graphed indicating the percentage of cleavage of original material (110 kDa) with time. ○ indicates Lewis rat plasma; □, Fischer rat plasma; and ▿, Buffalo rat plasma. The differences between Lewis and Buffalo rats as well as Lewis and Fischer rats at all points between 5 and 30 minutes were highly significant (P < .001). No statistical difference was found at all points between Fischer and Buffalo rats. Mean ± SEM, n = 3.