A Brief Historical Review of the Waterfall/Cascade of Blood Coagulation

Davie, Earl W.

doi:10.1074/jbc.x300009200

Cited by 135 publications

(87 citation statements)

References 131 publications

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“…7 Vitamin K antagonists, such as warfarin, exert their therapeutic effects by inhibiting VKOR, resulting in impaired recycling of vitamin K and a concomitant decrease in the generation of appropriately posttranslationally modified clotting factors II (prothrombin), VII, IX, and X. 8 Despite over 60 years of clinical use of warfarin and the identification of the genes encoding GGCX 9 and VKOR, 10,11 much remains to be learned about the function of the vitamin K cycle, the identities and roles of ancillary cellular factors that support its function, the detailed molecular mechanism whereby warfarin exerts its anticoagulant effects, and the nature of complex interactions with xenobiotics that impact warfarin therapy. Both VKOR and GGCX are integral transmembrane proteins, a feature that has necessitated their functional characterization in detergent-solubilized or microsomal preparations.…”

Section: Introductionmentioning

confidence: 99%

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Haque

McDonald

Kulman

et al. 2014

Blood

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• Factor IX glutamyl carboxylation in engineered HEK 293 cells recapitulates in vivo anticoagulant inhibition of vitamin K cycle activity.• Warfarin metabolite structureactivity analysis on vitamin K cycle antagonism determines their contributions to in vivo anticoagulation.Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K-dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of g-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293-derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal g-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular g-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R ) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of g-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo. (Blood. 2014;123(4):582-589)

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Section: Introductionmentioning

confidence: 99%

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Haque

McDonald

Kulman

et al. 2014

Blood

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“…Serine proteases are ubiquitous in living organisms and are involved in many physiologic processes, including digestion and respiration (1)(2)(3), blood coagulation and fibrinolysis (4,5), kinin formation and tumorigenesis (6), complement activation and phagocytosis (7), osteoarthritis and bone remodeling (8,9), as well as in ovogenesis and fertilization (10). Biologic activity of serine proteases is tightly regulated by their cognate inhibitors.…”

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confidence: 99%

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

Bajaj¹,

Ogueli

Kumar

et al. 2011

Journal of Biological Chemistry

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Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Fulllength TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2 position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2 residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ϳ6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ϳ8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

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“…As an anticoagulant protease, thrombin activates protein C in the presence of the cofactor thrombomodulin. Activated protein C (APC) inactivates factor Va and factor VIIIa (FVIIIa), down-regulating the generation of thrombin (1)(2)(3)(4)(5). Thromboembolic disorders are major causes of mortality and morbidity (6).…”

mentioning

confidence: 99%

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Koh

Kazimírová

Trimnell

et al. 2007

Journal of Biological Chemistry

102

101

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Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K i ϳ 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8 -14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.Blood coagulation is part of the physiological response to vascular injury, in which circulating zymogens of serine proteases are sequentially activated by limited proteolysis leading to the formation of a fibrin clot. Within this network of reactions, thrombin plays a central role in maintaining the integrity of hemostasis. Thrombin interacts with most of the zymogens and their cofactors, playing multiple procoagulant and anticoagulant roles in blood coagulation (1, 2). As a procoagulant protease, the first traces of thrombin generated in the initiation phase activate factor V (FV) 2 and factor VIII (FVIII) to provide positive feedback leading to the thrombin burst. Thrombin can also activate factor XI, triggering the intrinsic pathway. Thrombin cleaves fibrinogen to fibrin, forming insoluble clots. Fibrin polymers are further strengthened and stabilized through covalent cross-linking driven by thrombin-activated factor XIII. Thrombin also contributes to the generation of a platelet plug, possibly through two mechanisms: (a) it activates platelets by interacting with protease-activated receptors and glycoprotein V, and (b) it prevents destabilization of the platelet plug, by inactivating ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, that cleaves von Willebrand factor. As an anticoagulant protease, thrombin activates protein C in the presence of the cofactor thrombomodulin.

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A Brief Historical Review of the Waterfall/Cascade of Blood Coagulation

Cited by 135 publications

References 131 publications

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

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