FACTOR-XII DOES NOT INITIATE PREKALLIKREIN ACTIVATION ON ENDOTHELIAL-CELLS

It is well known that on artificial surfaces, binding and autoactivati on of factor XII (FXII) is the initiating event of plasma prekallikrei n (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEV). Activation of PK on HUVEC required an optimal substrate and Zn2+ conce ntration, the latter of which varied with the buffer's carrier protein , Maximal PK activation required the addition of 250 mu M or 10 mu M Z n2+ to buffers containing bovine serum albumin (BSA) or gelatin, respe ctively. However, the actual free Zn2+ concentration in these buffers was the same at 8 mu M. In both BSA- and gelatin-containing buffers an d using two different chromogenic substrates for FXII, no autoactivati on of FXII on HUVEC was seen when incubated for up to 60 min. Rather. initiation of FXII enzymatic activity required the presence of PK. FXI I activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not ab olish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively. soybean try psin inhibitor abolished the proteolytic activity associated with PK a nd FXII activation on HUVEC. Further, only normal human and FXII-defic ient plasmas, not PK-deficient plasma, had the ability to generate pro teolystic activity when incubated over endothelial cells. In a purifie d system, maximal PK activation was measured after a 10-15 min incubat ion depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma. maxi mal activation was seen in 4 min. These data indicate that on HUVEC, u nlike artificial surfaces, PK activation when bound to HK is the initi ating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic act ivity. These data challenge the accepted dogmas of ''contact activatio n'' and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.