Assembly of the Prothrombinase Complex

Mann, Kenneth G.; Nesheim, Michael E.; Tracy, PB; Hibbard, Lyndon S.; Bloom, James W.

doi:10.1016/s0006-3495(82)84624-9

Cited by 34 publications

(14 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Speculations on the molecular details of the nature of the protein-membrane interface over the past 20 years have resulted in conflicting models. Some groups have proposed that the vitamin K-dependent blood clotting proteins bind to acidic lipid membranes through calcium ions that bridge ␥-carboxyglutamic acid residues on the protein to the phosphate head groups on the lipid membrane (Lim et al, 1977;Mann et al, 1982;Schwalbe et al, 1989). This paradigm requires that the side chains of the ␥-carboxyglutamic acid residues extend into the solvent and are available on the surface to link to the membrane through calcium ion bridges.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX

Freedman

Blostein

Baleja

et al. 1996

Journal of Biological Chemistry

123

107

View full text Add to dashboard Cite

The blood coagulation and regulatory proteins that contain ␥-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to ␥-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca 2؉ , Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH 2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

show abstract

Section: Discussionmentioning

confidence: 99%

Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX

Freedman

Blostein

Baleja

et al. 1996

Journal of Biological Chemistry

123

107

View full text Add to dashboard Cite

show abstract

“…Partially purified carboxylase (2.0 ml) was applied to a 2-thiopyridyl activated thiol-Sepharose column (6 ml of gel; 1 x 10 cm) equilibrated in buffer B [20 mM sodium phosphate, pH 7.4/0.15 M NaCl/0.1% CHAPS/15% (vol/vol) glycerol/l mM EDTA/1 mg per ml phospholipid, composed of 91% (wt/wt) chicken egg L-a-phosphatidylcholine (type V-E, Sigma), 4.5% (wt/wt) Folch fraction III bovine brain extract, and 4.5% (wt/wt) bovine heart L-a-phosphatidylethanolamine (type VII, Sigma)] at 40C and the flow was stopped for 3 hr. The column was then washed with buffer B at 1 ml/hr.…”

Section: Methodsmentioning

confidence: 99%

“…Of the vitamin K-dependent proteins, prothrombin, factor IX, factor X, factor VII, protein C, and protein S are plasma proteins involved in blood coagulation or the regulation of coagulation (3,4). These proteins bind to calcium ions through -carboxyglutamic acid and undergo a calcium-induced conformational transition that is associated with the expression of a membrane binding site (5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

Hubbard

Ulrich

Jacobs

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form rcarboxygutam acid residues within the vitamin K-dependent proteins. This enzyme renizes the 'y-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood cogulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of igh concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25°C by reductive cleavage ofthe enzyme-propeptide complex in the presence ofdetergent and phospholipids. The eluted protein (Mr, 77,000)

show abstract

“…fXa generated on the platelet surface binds fVa to form prothrombinase, the enzyme complex that converts prothrombin to thrombin. 26 The rate of prothrombin activation by fXa assembled in the prothrombinase complex is approximately 10 6 -fold faster than that of fXa alone. 27 Consequently, each molecule of fXa generates approximately 1000 molecules of thrombin; this is a critical amplification step in coagulation that results in a burst of thrombin generation at sites of injury, thereby facilitating rapid hemostatic plug formation.…”

Section: Fxa: the Gatekeeper Of Coagulationmentioning

confidence: 99%

Oral Direct Factor Xa Inhibitors

Fredenburgh

Weitz

2012

Circulation Research

View full text Add to dashboard Cite

Vitamin K antagonists, such as warfarin, have been the mainstay of oral anticoagulation for many decades. Although effective, warfarin has numerous limitations, including a variable dose requirement from patient to patient because of differences in dietary vitamin K intake, common genetic polymorphisms, and multiple drug interactions that affect its pharmacodynamics and metabolism. Consequently, warfarin requires frequent monitoring to ensure that a therapeutic anticoagulant effect has been achieved because excessive anticoagulation can lead to bleeding, and because insufficient anticoagulation can result in thrombosis. Such monitoring is burdensome for patients and physicians and is costly for the health care system. These limitations have prompted the development of new oral anticoagulants that target either factor Xa or thrombin. Although the path to the development of these drugs has been long, the new drugs are at least as effective and safe as warfarin, but they streamline clinical care because they can be administered in fixed doses without routine coagulation monitoring. This article focuses on rivaroxaban, apixaban, and edoxaban, the oral factor Xa inhibitors in the most advanced stages of development. After 20 years of discovery research, these agents are already licensed for several indications. Thus, the long path to finding replacements for warfarin has finally reached fruition. Therefore, development of the oral factor Xa inhibitors represents a translational science success story.

show abstract

Assembly of the Prothrombinase Complex

Cited by 34 publications

References 4 publications

Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX

Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

Oral Direct Factor Xa Inhibitors

Contact Info

Product

Resources

About