ABSTRACT
Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.
Subject(s)
Factor VIIIa/antagonists & inhibitors , Factor V/pharmacology , Integrin alphaXbeta2/pharmacology , Protein C/pharmacology , Protein S/antagonists & inhibitors , Anticoagulants/antagonists & inhibitors , Anticoagulants/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Factor VIIIa/metabolism , Humans , Integrin alphaXbeta2/metabolism , Kinetics , Membranes, Artificial , Phospholipids/metabolism , Protein Binding , Protein C/metabolism , Protein S/drug effects , Protein S/metabolism , Protein S/pharmacologyABSTRACT
We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.
Subject(s)
Complement C4b/metabolism , Complement Inactivator Proteins , Factor Va/antagonists & inhibitors , Factor Va/metabolism , Glycoproteins , Protein C/physiology , Receptors, Complement/physiology , Animals , Antibodies, Monoclonal/metabolism , Arginine/genetics , Binding Sites, Antibody/genetics , Binding, Competitive/genetics , Binding, Competitive/immunology , Cell Line , Cricetinae , Enzyme Activation , Factor Va/genetics , Glutamine/genetics , Humans , Kidney , Protein Binding/genetics , Protein C/antagonists & inhibitors , Protein C/metabolism , Protein S/physiology , Receptors, Complement/genetics , Receptors, Complement/immunology , Receptors, Complement/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence DeletionABSTRACT
An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory C4b-binding protein (C4BP). The sites of interaction of protein S and C4BP are discussed.
Subject(s)
Anticoagulants/blood , Complement C4b/metabolism , Integrin alphaXbeta2/blood , Protein S/metabolism , Animals , Binding Sites , Blood Coagulation/physiology , Drug Interactions , Humans , Protein C/metabolism , Protein S/antagonists & inhibitorsABSTRACT
Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.
Subject(s)
Complement C4b/physiology , Complement Inactivator Proteins , Glycoproteins , Protein S/physiology , Receptors, Complement/physiology , Consensus Sequence , Humans , Microsatellite Repeats , Recombinant Fusion ProteinsABSTRACT
C4b-binding protein (C4BP) regulates the complement system and the anticoagulant activity of protein S. Protein S can bind to C4BP, resulting in a decreased cofactor activity of protein S for anticoagulant activated protein C. C4BP contains several identical a-chains and a single 3-chain. Each chain contains Short Consensus Repeats (SCRs). By making chimeras of 13-chain SCRs fused to tissue-type plasminogen activator (tPA chimeras), we found that 13-chain SCR-2 contributed to the interaction of 13-chain SCR-1 with protein S (van de Poel RHL, Meijers JCM, Bouma BN. J Biol Chem 274:15144-15150, 1999). Chimeras containing C4BP a-chains with SCR-1, SCR-l +2 or SCR-l +2+3 replaced by their 13-chain counterpart had affinities for protein S similar to C4BP (Hardig Y, Dahlb¿ck B. J Biol Chem 271:20861-20867, 1996). This was not in agreement with the finding that Beta-chain SCR-2 contributed to the interaction and could be explained by the possibility that alpha-chain SCR-2 in the alpha-chain chimeras contributed comparable with Beta-chain SCR-2 in the tPA chimeras. To investigate this we constructed a tPA chimera containing Beta-chain SCR-1 and alpha-chain SCR-2 (Beta1alpha2). Binding studies showed that Beta1alpha2 had a lower affinity compared with SCR-1 +2, indicating that alpha-chain SCR-2 did not contribute to the interaction. The difference with the alpha-chain chimeras may be explained by the fact that the alpha-chain chimeras were linked by their C-terminal cysteines, resulting in multiple binding sites in a single molecule. Thereby, the effect of a lower affinity of each alpha-chain chimera may have been masked. The studies performed here help to clarify the apparent inconsistencies in two previous reports about the contribution of the SCR-2 domain in C4BP to protein S binding. In conclusion, Beta-chain SCR-2 specifically contributes to the interaction of SCR-1 with protein S.
