OC 03.2 - The Prekallikrein and Factor XII Heavy Chains in Surface-Independent Reactions.

Background: The plasma zymogens prekallikrein and factor XII (FXII) are reciprocally converted to their protease forms, kallikrein and FXIIa, when blood is exposed to surfaces by a process called contact activation. During contact activation the non-catalytic heavy chains of prekallikrein and FXII mediate surface-binding. Prekallikrein and FXII also undergo reciprocal activation in a surface-independent manner. The roles of the heavy chains of these proteins in this setting are not well understood.

Aims: Determine the roles of the heavy chains and catalytic light chains of PK and Factor XII in surface-independent reciprocal activation, and in PKa and FXIIa-mediated proteolytic reactions.

Methods: Cysteines that connect the heavy chains and catalytic domains (CD) of prekallikrein (Cys-364 and Cys-484) and FXII (Cys-340 and Cys467) were changed to serine. The resulting zymogen proteins, PK-Ser-364,484 and FXII-Ser-340,467, were compared to wild-type FXII and PK in reciprocal activation reactions using chromogenic substrates for detection. When PK-Ser-364,484 and FXII-Ser-340,467 are activated the heavy chains and catalytic domains dissociate. Isolated catalytic domains (PKa-CD and FXIIa-CD) were prepared from PK-Ser-364,484 and FXII-Ser-340,467.

Results: In prekallikrein-FXII reciprocal activation assays, replacing PK with PK-Ser-364,484 markedly slowed reactions, while replacing FXII with FXII-Ser-340,467 accelerated reactions. Kallikrein and PKa-CD activated FXII comparably. Similarly, FXIIa and FXIIa-CD activated PK comparably. Cleavage of the cofactor high-molecular-weight kininogen (HK) by PKa-CD was significantly reduced when compared to PKa.

Conclusion(s): Our data indicate that during surface-independent reciprocal activation of PK and FXII, the PK heavy chain is required, probably for the proper interaction with FXIIa. The FXII heavy chain, in contrast, is not required for surface-independent FXII activation, and may actually slow the reaction by obscuring the FXII activation cleavage site. The kallikrein heavy chain is required for proper cleavage of HK, consistent with an exosite on the kallikrein heavy chain required for HK binding.