Structural insights into the activation of blood coagulation factor XI zymogen by thrombin: A computational molecular dynamics study

Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

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Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency

Blood ◽

10.1182/blood.v79.6.1435.1435 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1435-1440 ◽

Cited By ~ 67

Author(s):

JC Meijers ◽

EW Davie ◽

DW Chung

Keyword(s):

Blood Coagulation ◽

Messenger Rna ◽

Coagulation Factor ◽

Coagulation Cascade ◽

Mutant Protein ◽

Chain Molecule ◽

Single Chain ◽

Factor Xi ◽

Bhk Cells ◽

Type Iii

Abstract Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

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Platelets and Factor XI Bypass the Contact System of Blood Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615838 ◽

1999 ◽

Vol 82 (08) ◽

pp. 234-242 ◽

Cited By ~ 10

Author(s):

Peter Walsh

Keyword(s):

Blood Coagulation ◽

Coagulation Factor ◽

Bleeding Complications ◽

Ashkenazi Jews ◽

Factor Xi ◽

Contact Phase ◽

Platelet Factor ◽

Proteolytic Activation ◽

Factor Xi Deficiency ◽

Plasma Factor

IntroductionFactor XI is a plasma glycoprotein (concentration ∼30 nM) that was first identified by Rosenthal et al1 as a plasma coagulation factor deficiency in patients with abnormal hemostasis, particularly common among Ashkenazi Jews.2,3 In spite of recent advances in our understanding of the structure of factor XI and its gene, the structure-function relationships of the protein, and the molecular genetics of factor XI deficiency, considerable confusion about the physiologic role and clinical relevance of factor XI has arisen from both clinical and biochemical observations. One problem arises from the fact that, until recently, the only known pathway for activation of factor XI involved proteolytic activation by factor XIIa and interactions with coagulation proteins of the contact phase of blood coagulation.4-7 The problem arose from the clinical observation that patients with deficiencies of factor XI are subject to bleeding complications, whereas patients with deficiencies of the contact proteins are not.2,3,8-13 A related unanswered question concerns the lack of correlation in many reported patients between plasma levels of factor XI and the severity of clinical bleeding manifestations.2,3,8,9,13-15 In addition, some patients with severe factor XI deficiency experience significant bleeding complications, whereas others appear to be hemostatically normal. These clinical and biochemical observations have motivated investigations focused on alternative mechanisms for activation of factor XI independent of contact phase protein.19-22 Additional studies have focused on the identification and characterization of platelet factor XI, which is postulated to be an alternative splicing product of the factor XI gene. Platelet factor XI is present in platelet membranes and might substitute for plasma factor XI in hemostasis and account for the absence of bleeding complications in some patients with severe plasma factor XI deficiency.14,16-18,23-26 Studies addressing these two important and related problems are presented and discussed in this chapter.

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Role of hydrophobic mutations on the binding affinity and stability of blood coagulation factor VIIIa: A computational molecular dynamics and free-energy analysis

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2014.06.043 ◽

2014 ◽

Vol 450 (1) ◽

pp. 735-740 ◽

Cited By ~ 2

Author(s):

Divi Venkateswarlu

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Blood Coagulation ◽

Binding Affinity ◽

Energy Analysis ◽

Coagulation Factor ◽

Factor Viiia ◽

Free Energy Analysis

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ISOLATION AND FUNCTIONAL PROPERTIES OF MONOMERIC BLOOD COAGULATION FACTOR XI

10.1055/s-0038-1642803 ◽

1987 ◽

Author(s):

M Berrettini ◽

M J Heeb ◽

J H Griffin

Keyword(s):

Blood Coagulation ◽

Gel Filtration ◽

Coagulation Factor ◽

Fluid Phase ◽

Factor Ix ◽

Contact Activation ◽

Factor Xi ◽

Dimeric Structure ◽

Sds Page ◽

Mild Reduction

To evaluate the significance of the normal dimeric structure (160,000 MW) of blood coagulation Factor XI (F.XI), a monomeric form (80,000 MW) was produced by mild reduction and alkylation of native F.XI. Since initial efforts to reduce and alkylate F.XI in solution inactivated the molecule, F.XI was bound to high MW kininogen (HMWK) to stabilize the native structure. Purified F.XI was bound to HMWK-Sepharose, and the column was washed for 2 h with 40 μM dithiothreitol in 4mM acetate buffer, 2mM EDTA, 1mM benzamidine, pH 7.8, and then for 2 h with 50 μM iodoacetamide in the same buffer. Elution with 0.5 M NaCl gave a preparation containing ∼ 85% F.XI monomer and ∼ 15% dimer, as judged by nonreduced SDS-PAGE and by gel filtration of the radiolabeled preparation. The monomeric F.XI preparation had only 10% of the clotting activity of dimeric F.XI (per mole of enzymatic site) as measured in APTT clotting assays using F.XI deficient plasma. After activation with β-Factor XIIa in solution, the monomer F.XIa preparation exhibited 85% of the clotting activity of native F.XIa in unactivated PTT assays using F.XI deficient plasma. In addition, when compared to native F.XIa, monomeric F.XIa gave 65% amidolytic activity against the substrate, S-2366, and 75% activity against Factor IX in assays of the release of the activation peptide from 3H-Factor IX. Polystyrene tubes were coated with HMWK then blocked with 1% BSA to study the binding of 125I-F.XI to HWMK. When the binding of the 125I-labeled preparations of monomeric and dimeric forms of F.XI to HMWK was studied, two distinct components were identified in the association of dimeric F.XI, one with high affinity (Kd ∼ 2.5 X 10-9M) and one with less affinity (Kd ∼ 1.7 X 10-8M), while the binding of monomeric F.XI occurred with a single low affinity component (Kd ∼ 1.1 X 10-8M). These observations suggest that the dimeric structure of F.XI is required for efficient binding of the molecule to HMWK and for normal activation by the contact activation system in plasma, but that the dimeric structure of F.XIa does not play a role in the expression of the enzymatic activity against Factor IX in fluid phase.

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