Calcium involvement in the protein-membrane systems of blood coagulation

Abstract

Blood coagulation is an extracellular process that occurs in a medium containing millimolar concentrations of calcium and utilizes this ion in several ways. One major utilization is by the vitamin K-dependent, γ-carboxyglutamate-containing proteins. Four of these proteins are known to function as enzymes and/or substrates for membrane-bound enzyme-catalyzed reactions of the blood-clotting casade. Three additional vitamin K-dependent proteins are known in the plasma, one of which probably acts as an enzyme in a membrane requiring reaction that decreases coagulation. All of these plasma proteins have homologous amino terminal sequences of about 40 residues which contain 10 to 12 γ-carboxyglutamic acid residues. Calcium functions to aid binding of these vitamin. K-dependent proteins to membranes containing acidic phospholipids. Acidic phospholipids are found primarily in cytosolic membranes and are nearly absent on the external surface of the plasma membranes. Exposure of cytosolic membranes through cell damage may therefore constitute an important signal enabling blood coagulation to occur. Calcium binding to γ-carboxyglutamic acid-containing plasma proteins causes a protein conformational change which is prerequisite to protein-membrane binding. In the case of prothrombin, the protein conformational change apparently involves a trans to cis proline isomerization (Pro 22) which has a characteristic reaction half-time of 90 minutes at O °C and an activation energy of 21 kcal/mol. The conformational changes for all of these proteins result in changes in intrinsic protein fluorescence as well as other spectral properties and are cooperative with respect to calcium. Very little selectivity for multivalent metal ions is displayed by the proteins and the sites have average K D of 10 −4 to 10 −3 M calcium which varies slightly for the different proteins. Prothrombin, the most thoroughly studied protein, binds 6 calcium ions in parallel with the protein conformation change with an average K D of 0.25 m M calcium. Many other ions show site specificity and three bound ions (Mn, Cd, Lanthanides) are sufficient to cause the conformational change. Calcium serves a second role in these protein-membrane interactions which appears to involve the acidic phospholipid residues. This second calcium-dependent process is also cooperative with respect to metal ion and shows a similar affinity for calcium. Approximately four calcium ions and eight acidic phospholipid residues are required for this second process. Metal ion specificity is observed in this step with calcium and strontium fully while magnesium and manganese fail to function at all. The precise structure of the protein-membrane interface is not known but studies with phospholipid bilayers and monolayers indicate involvement of the phospholipid headgroup only. The favored model is calcium bridging between ligands of the protein and the phospholipid. Rapid kinetic studies indicate that the metal ions responsible for maintaining protein conformation are in rapid exchange with solvent ions even in the membrane-bound state.