HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR

1987 ◽  
Author(s):  
A KÖhlin ◽  
J Stenflo
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Plasma Proteins ◽  
Metal Ion ◽  
Protein C ◽  
Protein S ◽  
Protein Z ◽  
High Affinity ◽  
Epidermal Growth

In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

1991 ◽  
Vol 66 (01) ◽  
pp. 049-061 ◽  
Author(s):  
Björn Dahlbäck
Keyword(s):  
Vitamin K ◽  
Complement System ◽  
Protein C ◽  
Protein S ◽  
High Affinity ◽  
Anticoagulant System ◽  
Dependent Protein ◽  
The Complement System ◽  
Β Chain ◽  
Dependent Domain

SummaryThe protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF)epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 γ-carboxy glutamic acid residues in the vitamin K-dependent domain, a β-hydroxylated aspartic acid in the first EGF-like domain and a β-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical α-chains and one β-chain. The α-and β-chains are linked by disulphide bridges. The cDNA cloning of the β-chain showed the α- and β-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the β-chain to contain the single protein S binding site on C4BP, whereas each of the α-chains contains a binding site for the complement protein, C4b. As C4BP lacking the β-chain is unable to bind protein S, the β-chain is required for protein S binding, but not for the assembly of the α-chains during biosynthesis. Protein S has a high affinity for negatively charged phospholipid membranes, and is instrumental in binding C4BP to negatively charged phospholipid. This constitutes a novel mechanism for control of the complement system on phospholipid surfaces. Recent findings have shown circulating C4BP to be involved in yet another calcium-dependent protein-protein interaction with a protein known as the serum amyloid P-component (SAP). The binding sites on C4BP for protein S and SAP are independent. SAP, which is a normal constituent in plasma and in tissue, is a so-called pentraxin being composed of 5 non-covalently bound 25 kDa subunits. It is homologous to C reactive protein (CRP) but its function is not yet known. The specific high affinity interactions between protein S, C4BP and SAP suggest the regulation of blood coagulation and that of the complement system to be closely linked.

scholarly journals Vitamin K-Dependent Coagulation Factors That May be Responsible for Both Bleeding and Thrombosis (FII, FVII, and FIX)

2018 ◽  
Vol 24 (9_suppl) ◽  
pp. 42S-47S ◽  
Author(s):  
Antonio Girolami ◽  
Silvia Ferrari ◽  
Elisabetta Cosi ◽  
Claudia Santarossa ◽  
Maria Luigia Randi
Keyword(s):  
Venous Thrombosis ◽  
Vitamin K ◽  
Protein C ◽  
Protein S ◽  
Coagulation Factors ◽  
Bleeding Tendency ◽  
Clotting Factors ◽  
Protein Z ◽  
Clinical Observations

Vitamin K-dependent clotting factors are commonly divided into prohemorrhagic (FII, FVII, FIX, and FX) and antithrombotic (protein C and protein S). Furthermore, another protein (protein Z) does not seem strictly correlated with blood clotting. As a consequence of this assumption, vitamin K-dependent defects were considered as hemorrhagic or thrombotic disorders. Recent clinical observations, and especially, recent advances in molecular biology investigations, have demonstrated that this was incorrect. In 2009, it was demonstrated that the mutation Arg338Leu in exon 8 of FIX was associated with the appearance of a thrombophilic state and venous thrombosis. The defect was characterized by a 10-fold increased activity in FIX activity, while FIX antigen was only slightly increased (FIX Padua). On the other hand, it was noted on clinical grounds that the thrombosis, mainly venous, was present in about 2% to 3% of patients with FVII deficiency. It was subsequently demonstrated that 2 mutations in FVII, namely, Arg304Gln and Ala294Val, were particularly affected. Both these mutations are type 2 defects, namely, they show low activity but normal or near-normal FVII antigen. More recently, in 2011-2012, it was noted that prothrombin defects due to mutations of Arg596 to Leu, Gln, or Trp in exon 15 cause the appearance of a dysprothrombinemia that shows no bleeding tendency but instead a prothrombotic state with venous thrombosis. On the contrary, no abnormality of protein C or protein S has been shown to be associated with bleeding rather than with thrombosis. These studies have considerably widened the spectrum and significance of blood coagulation studies.

Binding of calcium ions to bovine αsl-casein and precipitability of the protein–calcium ion complexes

1980 ◽  
Vol 47 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Douglas G. Dalgleish ◽  
Thomas G. Parker
Keyword(s):  
Ionic Strength ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Binding Constants ◽  
Substitution Effects ◽  
Binding Isotherms ◽  
Decreasing Ph ◽  
Increasing Temperature

SummaryBinding isotherms for the calcium ion–αsl-casein system have been measured, as functions of ionic strength, temperature, and pH, and the isotherms have been analysed in terms of binding constants modified by substitution effects. The results demonstrate that the strength of binding is increased with increasing temperature and decreased by increasing ionic strength or decreasing pH, all of which may be explained semi-quantitatively. Parallel studies on the precipitability of the αsl-casein–Ca2+ complexes showed that there is considerable variation in the extent of calcium binding required to initiate precipitation of the protein, and in the calcium concentration necessary to achieve the required extent of ligand binding.

A study of the Ca2+ – Arsenazo III system and its application to the spectrophotometric determination of free calcium in solution

1990 ◽  
Vol 68 (11) ◽  
pp. 1932-1936 ◽  
Author(s):  
Byron Kratochvil ◽  
Xi-Wen He
Keyword(s):  
Ionic Strength ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Metal Ion ◽  
Arsenazo Iii ◽  
Free Calcium ◽  
Ionic Calcium ◽  
Free Metal ◽  
Free Metal Ion

A two-wavelength method was applied to the determination of free, ionic calcium with Arsenazo III in solutions containing calcium-binding ligands. By this procedure impurities in the indicator can be corrected for, thereby allowing the use of commercial indicator preparations with purities as low as 80%. Only a 1:1 complex with a conditional log stability constant of 4.28 ± 0.13 at pH 4.6 and ionic strength 0.1 was found under the conditions studied. Key words: free metal ion determination, calcium ion speciation, spectrophotometry, arsenazo III, ion increment method for speciation.

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