Abstract
The light chain of kallikrein-cleaved human high molecular weight kininogen is solely responsible for its cofactor activity in blood clotting. Sequencing of the NH2-terminal region of the light chain reported herein identified the third kallikrein cleavage site of high molecular weight kininogen as Arg-437. The co-factor activity of high molecular weight kininogen consists of the capacity to bind to negatively charged surfaces and to factor XI or prekallikrein. Chemical modification of the histidines by either photooxidation or ethoxyformic anhydride affected the equivalent of 14-16 of 23 histidines available and resulted in over 90% loss in procoagulant activity. The modified protein had drastically reduced surface- and zinc-binding capacity, but it bound successfully to either factor XI or prekallikrein. In contrast, modification of two carboxyl groups, which led to approximately 80-90% loss of procoagulant activity, seriously compromised protein binding but left surface binding unaffected. All 3 tryptophans were modified at pH 4.0 with N-bromosuccinimide with a 70% reduction in procoagulant activity, but only 1 tryptophan was available for reaction at pH 7.35, resulting in a 50% loss in activity. Tryptophan modification at acidic pH affected protein binding but did not modify surface or zinc binding. Modification of both available tyrosine and 9 of 18 available lysine residues did not have a significant effect on the procoagulant activity of the light chain. These studies indicate that histidines participate in surface binding and that free carboxyl groups and tryptophan side chains are involved in binding of high molecular weight kininogen to other clotting factors.
Highlights
Procoagulant activity, seriously compromised protein The procoagulant activity of human HMWKresides exclubinding but left surface binding unaffectAedll.3 tryp- sively in the light chain (6, 7, 16, 17)
Residues did not have a significant effect on the pro- Efforts to determine the location of sites of High molecular weight kininogen (HMWK) that coagulantactivity of thelightchain.Thesestudies are responsible foreither surface or protein bindinghave been indicate that histidines participate in surface binding limited to thebovine molecule
Whereas at a ratio of prekallikrein to L-chain of 1.0, almost all of the L-chain was found in complexeswith the proenzyme, thesameratio of subunit factor XI to L-chainfailed to produce significant amounts of factor XI-L-chain complex
Summary
Procoagulant activity, seriously compromised protein The procoagulant activity of human HMWKresides exclubinding but left surface binding unaffectAedll.3 tryp- sively in the light chain (6, 7 , 16, 17). The effects of modifications which affected procoagulant activity were evaluated on surface, zinc, and protein binding.
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