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1.
Cell Mol Bioeng ; 14(2): 161-175, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33868498

ABSTRACT

INTRODUCTION: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. OBJECTIVE: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. METHODS: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. RESULTS: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. CONCLUSION: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

2.
J Thromb Haemost ; 16(10): 2044-2049, 2018 10.
Article in English | MEDLINE | ID: mdl-30007049

ABSTRACT

Essentials Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model. FIX-deficient mice displayed a hemostatic defect and FXI-deficient mice were similar to wild type mice. Infusion of FXI or over-expression of FXI in FIX-deficient mice improved hemostasis. FXI may affect the phenotype of FIX-deficiency (hemophilia B). SUMMARY: Background In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B). Methods Wild-type mice and mice lacking either FIX (F9- ) or FXI (F11-/- ) were tested in the SVB model. The plasma levels of FXI in F11-/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9- mice showed a significant defect in the SVB model, whereas F11-/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9- mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9- mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11-/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9- mice through FIX-independent pathways.


Subject(s)
Factor XI Deficiency/drug therapy , Factor XI/administration & dosage , Hemophilia B/drug therapy , Hemostasis/drug effects , Animals , Disease Models, Animal , Factor IX/genetics , Factor IX/metabolism , Factor XI/genetics , Factor XI/metabolism , Factor XI Deficiency/blood , Factor XI Deficiency/genetics , Genetic Predisposition to Disease , Hemophilia B/blood , Hemophilia B/genetics , Hemostasis/genetics , Infusions, Intravenous , Male , Mice, Inbred C57BL , Mice, Knockout , Phenotype
3.
Haemophilia ; 24(4): 519-521, 2018 07.
Article in English | MEDLINE | ID: mdl-29808936
4.
Thromb Res ; 156: 134-141, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28644959

ABSTRACT

BACKGROUND: The plasma protease factor XIa (FXIa) has become a target of interest for therapeutics designed to prevent or treat thrombotic disorders. METHODS: We used a solution-based, directed evolution approach called systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that target the FXIa catalytic domain. RESULTS: Two aptamers, designated 11.16 and 12.7, were identified that bound to previously identified anion binding and serpin bindings sites on the FXIa catalytic domain. The aptamers were non-competitive inhibitors of FXIa cleavage of a tripeptide chromogenic substrate and of FXIa activation of factor IX. In normal human plasma, aptamer 12.7 significantly prolonged the aPTT clotting time. CONCLUSIONS: The results show that novel inhibitors of FXIa can be prepared using SELEX techniques. RNA aptamers can bind to distinct sites on the FXIa catalytic domain and noncompetitively inhibit FXIa activity toward its primary macromolecular substrate factor IX with different levels of potency. Such compounds can be developed for use as therapeutic inhibitors.


Subject(s)
Anticoagulants/metabolism , Aptamers, Nucleotide/metabolism , Factor XIa/metabolism , Humans
5.
J Thromb Haemost ; 14(4): 828-38, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26748875

ABSTRACT

BACKGROUND: Allosteric inhibition is a promising approach for developing a new group of anticoagulants with potentially reduced bleeding consequences. Recently, we designed sulfated ß-O4 lignin (SbO4L) as an allosteric inhibitor that targets exosite 2 of thrombin to reduce fibrinogen cleavage through allostery and compete with glycoprotein Ibα to reduce platelet activation. OBJECTIVE: To assess: (i) the antithrombotic potential of a novel approach of simultaneous exosite 2-dependent allosteric inhibition of thrombin and competitive inhibition of platelet activation; and (ii) the promise of SbO4L as the first-in-class antithrombotic agent. METHODS: A combination of whole blood thromboelastography, hemostasis analysis, mouse arterial thrombosis models and mouse tail bleeding studies were used to assess antithrombotic potential. RESULTS AND CONCLUSIONS: SbO4L extended the clot initiation time, and reduced maximal clot strength, platelet contractile force, and the clot elastic modulus, suggesting dual anticoagulant and antiplatelet effects. These effects were comparable to those observed with enoxaparin. A dose of 1 mg of SbO4L per mouse prevented occlusion in 100% of arteries, and lower doses resulted in a proportionally reduced response. Likewise, the time to occlusion increased by ~ 70% with a 0.5-mg dose in the mouse Rose Bengal thrombosis model. Finally, tail bleeding studies demonstrated that SbO4L does not increase bleeding propensity. In comparison, a 0.3-mg dose of enoxaparin increased the bleeding time and blood volume loss. Overall, this study highlights the promise of the allosteric inhibition approach, and presents SbO4L as a novel anticoagulant with potentially reduced bleeding side effects.


Subject(s)
Anticoagulants/chemistry , Platelet Aggregation Inhibitors/chemistry , Platelet Glycoprotein GPIb-IX Complex/chemistry , Thrombin/chemistry , Allosteric Site , Animals , Binding Sites , Bleeding Time , Blood Coagulation/drug effects , Blood Coagulation Tests , Computer Simulation , Enoxaparin/pharmacology , Fibrinolytic Agents/chemistry , Hemorrhage , Hemostasis , Heparin/therapeutic use , Humans , Hydrolysis , Lignin/chemistry , Mice , Mice, Inbred C57BL , Platelet Activation , Platelet Aggregation/drug effects , Platelet-Rich Plasma , Protein Binding , Risk , Thrombelastography , Thrombin/immunology , Thrombosis
6.
Haemophilia ; 21(6): 761-5, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25930174

ABSTRACT

INTRODUCTION: Haemophilia A is an X-linked recessive bleeding disorder that primarily affects males. Emerging data support evidence for increased bleeding in female haemophilia A carriers despite factor VIII activity within the normal range. AIM: Data regarding the effect of increased bleeding on health-related quality of life (HR-QOL) in haemophilia A carriers is sparse. We tested the hypothesis that haemophilia A carriers have reduced HR-QOL related to bleeding symptoms. METHODS: We conducted a cross-sectional study at Vanderbilt University. Case subjects were obligate or genetically verified haemophilia A carriers age 18-60 years. Control subjects were mothers of children with cancer who receive care at the Vanderbilt paediatric haematology-oncology clinic. Trained interviewers administered the Rand 36-Item Health Survey 1.0, a validated questionnaire evaluating eight health concepts that may affect HR-QOL, to each study participant. Mann-Whitney U-tests were used to compare median scores for the eight health domains between the case and control groups. RESULT: Forty-two haemophilia A carriers and 36 control subjects were included in analyses. Haemophilia A carriers had significantly lower median scores for the domains of 'Pain' (73.75 vs. 90; P = 0.02) and 'General health' (75 vs. 85; P = 0.01) compared to control subjects. CONCLUSION: Haemophilia A carriers in our study demonstrated significantly lower median scores on the Rand 36-item Health Survey 1.0 in the domains of 'Pain' and 'General Health' compared to women in the control group. Our findings highlight the need for further investigation of the effect of bleeding on HR-QOL in this population.


Subject(s)
Health , Hemophilia A/genetics , Heterozygote , Quality of Life , Adolescent , Adult , Cross-Sectional Studies , Female , Hemophilia A/complications , Hemorrhage/complications , Humans , Male , Middle Aged , Young Adult
7.
J Thromb Haemost ; 13(8): 1383-95, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25976012

ABSTRACT

The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa (FXa) or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and FX. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on FXII, the zymogen of a protease (FXIIa) that initiates contact activation when blood is exposed to foreign surfaces, and FXI, the zymogen of the protease FXIa, which links contact activation to the thrombin generation mechanism. In the case of FXI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of FXI may be more effective than low molecular weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here, we review data on the role of FXI and FXII in thrombosis and results of preclinical and human trials for therapies targeting these proteins.


Subject(s)
Anticoagulants/therapeutic use , Blood Coagulation/drug effects , Factor XII/antagonists & inhibitors , Factor XI/antagonists & inhibitors , Fibrinolytic Agents/therapeutic use , Venous Thrombosis/drug therapy , Animals , Anticoagulants/adverse effects , Disease Models, Animal , Drug Design , Enzyme Activation , Factor XI/metabolism , Factor XII/metabolism , Fibrinolytic Agents/adverse effects , Hemorrhage/chemically induced , Humans , Molecular Targeted Therapy , Risk Factors , Treatment Outcome , Venous Thrombosis/blood , Venous Thrombosis/diagnosis
8.
Curr Mol Med ; 14(9): 1173-85, 2014.
Article in English | MEDLINE | ID: mdl-25324000

ABSTRACT

Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines and hormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg(9) bradykinin. It does so by cleaving a C-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identify PRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of the human PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acid sequence. Using structural and functional studies, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCP peptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning the opening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelial cells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro 637 (P(637)) and Ala 638 (A(638)). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codon is activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PK activation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is not identical to that of FXIIa.


Subject(s)
Carboxypeptidases/chemistry , Prekallikrein/chemistry , Amino Acid Sequence , Catalytic Domain , Cell Line , Enzyme Activation , Enzyme Assays , Humans , Kinetics , Molecular Sequence Data , Proteolysis , Substrate Specificity
12.
J Thromb Haemost ; 11(11): 2020-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24118982

ABSTRACT

BACKGROUND: Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. AIMS: To determine the importance of FXI ABSs to polyP enhancement of FXI activation. METHODS: Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model. RESULTS: In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation. CONCLUSIONS: The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes.


Subject(s)
Factor XI/chemistry , Polyphosphates/chemistry , Animals , Anions , Antithrombins/chemistry , Binding Sites , Blood Coagulation , Cattle , Factor IX/chemistry , Factor XIa/chemistry , Heparin/chemistry , Humans , Mice , Mice, Inbred C57BL , Polyelectrolytes , Polymers/chemistry , Recombinant Proteins/chemistry , Thrombin/chemistry , Thrombosis/metabolism
13.
J Thromb Haemost ; 11(12): 2118-27, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24152424

ABSTRACT

BACKGROUND: Factor XIa is traditionally assigned a role in FIX activation during coagulation. However, recent evidence suggests this protease may have additional plasma substrates. OBJECTIVE: To determine whether FXIa promotes thrombin generation and coagulation in plasma in the absence of FIX, and to determine whether FXI-deficiency produces an antithrombotic effect in mice independently of FIX. METHODS: FXIa, FXIa variants and anti-FXIa antibodies were tested for their effects on plasma coagulation and thrombin generation in the absence of FIX, and for their effects on the activation of purified coagulation factors. Mice with combined FIX and FXI deficiency were compared with mice lacking either FIX or FXI in an arterial thrombosis model. RESULTS: In FIX-deficient plasma, FXIa induced thrombin generation, and anti-FXIa antibodies prolonged clotting times. This process involved FXIa-mediated conversion of FX and FV to their active forms. Activation of FV by FXIa required the A3 domain on the FXIa heavy chain, whereas activation of FX did not. FX activation by FXIa, unlike FIX activation, was not a calcium-dependent process. Mice lacking both FIX and FXI were more resistant to ferric chloride-induced carotid artery occlusion than FXI-deficient or FIX-deficient mice. CONCLUSION: In addition to its predominant role as an activator of FIX, FXIa may contribute to coagulation by activating FX and FV. As the latter reactions do not require calcium, they may make important contributions to in vitro clotting triggered by contact activation. The reactions may be relevant to FXIa's roles in hemostasis and in promoting thrombosis.


Subject(s)
Blood Coagulation/physiology , Factor IX/physiology , Factor XIa/physiology , Animals , Electrophoresis, Polyacrylamide Gel , Factor IX/immunology , Factor XIa/immunology , Humans , Mice , Mice, Inbred C57BL , Proteolysis
14.
J Thromb Haemost ; 11(7): 1341-52, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23659638

ABSTRACT

BACKGROUND: Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI. OBJECTIVES: The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation. METHODS AND RESULTS: Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. CONCLUSIONS: We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.


Subject(s)
Blood Coagulation , Factor XII/metabolism , Factor XI/metabolism , Polyphosphates/blood , Animals , Antibodies, Neutralizing/pharmacology , Blood Coagulation/drug effects , Factor XI/antagonists & inhibitors , Factor XI Deficiency/blood , Factor XIIa/antagonists & inhibitors , Factor XIIa/metabolism , Factor XIa/metabolism , Humans , Plant Proteins/pharmacology , Prothrombin/metabolism , Thrombin/metabolism , Thrombosis/blood , Thrombosis/prevention & control , Time Factors
15.
J Thromb Haemost ; 11(7): 1374-84, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23617568

ABSTRACT

BACKGROUND: A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an exosite required for binding of FIX to activated FXI (FXIa). OBJECTIVE: To test the effects of the Arg184Gly substitution on FIX activation, and to characterize the FIX-binding site on FXIa. METHODS: Recombinant FXIa and FIX variants were used to identify residues involved in FIX activation by FXIa. Analysis of the FXI structure was used to identify potential FIX-binding sites. RESULTS: The Km for FIX activation by FXIa-Gly184 was approximately three-fold higher than for FXIa, suggesting that Arg184 is part of the exosite. Arg184 and the adjacent residues, Ile183 and Asp185, contribute to charged and hydrophobic areas that are not present in the FXI homolog prekallikrein (PK). Replacing residues 183-185 with alanine abolished exosite activity, similarly to replacement of the entire A3 domain with the A3 domain from PK (FXIa/PKA3). Reintroducing FXI residues 183-185 into FXIa/PKA3 partially restored the exosite, and replacing residues 183-185 and 260-264 completely restored exosite function. FIX in which the Ω-loop (residues 4-11) was replaced with the FVII Ω-loop was activated poorly by FXIa, suggesting that the FIX Ω-loop binds to FXIa. CONCLUSIONS: The results support a model in which the Ω-loop of FIX binds to an area on FXIa composed of residues from the N-terminus and C-terminus of the A3 domain. These residues are buried in zymogen FXI, and must be exposed upon conversion to FXIa to permit FIX binding.


Subject(s)
Blood Coagulation , Factor IX/metabolism , Factor XI Deficiency/blood , Factor XIa/metabolism , Arginine , Blood Coagulation Tests , Enzyme Activation , Factor IX/chemistry , Factor IX/genetics , Factor XI Deficiency/genetics , Factor XIa/chemistry , Factor XIa/genetics , Glycine , HEK293 Cells , Humans , Kinetics , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Structure-Activity Relationship , Transfection
16.
Curr Med Chem ; 19(24): 4194-206, 2012.
Article in English | MEDLINE | ID: mdl-22664251

ABSTRACT

Preclinical pharmacological characterization of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) was investigated. We synthesized and evaluated a library of proline-based analogs as prospective recombinant PRCP (rPRCP) inhibitors and inhibitors of PRCP-dependent prekallikrein (PK) activation on human pulmonary artery endothelial cells (HPAEC). Among the newly synthesized compounds, UM8190 was further characterized in vivo using methods that encompassed a mouse carotid artery thrombosis model and animal model of food consumption. (S)-N-dodecyl-1-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide [Compound 3 (UM8190)] was selected for further evaluation from the initial assessment of its PRCP inhibitory action (K(i)= 43 µM) coupled with its ability to block PRCP-dependent PK activation on HPAEC (K(i)= 34 µM). UM8190 demonstrated excellent selectivity against a panel of carboxypeptidases and serine proteases and blocked bradykinin (BK) generation and BK-induced permeability by 100%, suggesting that it may be useful in preventing the local production of large amounts of BK. Furthermore, UM8190 showed an anorexigenic effect when systemically administered to fasted mice, reducing food intake in a dose- and time-dependent manner. In a mouse carotid artery thrombosis model, it also demonstrated an antithrombotic effect. UM8190 is a selective PRCP inhibitor and it may represent a new anorexigenic, and antithrombotic drug, that works by inhibiting PRCP-mediated mechanisms.


Subject(s)
Appetite/drug effects , Carboxypeptidases/antagonists & inhibitors , Proline/analogs & derivatives , Protease Inhibitors/chemistry , Animals , Appetite Depressants/chemical synthesis , Appetite Depressants/chemistry , Appetite Depressants/pharmacology , Bradykinin/metabolism , Carboxypeptidases/metabolism , Cell Line , Disease Models, Animal , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/therapeutic use , Humans , Mice , Mice, Inbred C57BL , Nitric Oxide/metabolism , Prekallikrein/metabolism , Proline/chemistry , Proline/pharmacology , Proline/therapeutic use , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , Thrombosis/drug therapy , Thrombosis/pathology
17.
J Thromb Haemost ; 8(6): 1295-301, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20796202

ABSTRACT

BACKGROUND: Laminin is the most abundant non-collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis. OBJECTIVE: To investigate the ability of laminin to promote coagulation and support thrombus formation under shear. RESULTS AND METHODS: Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI- and FXII-dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin-coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX. CONCLUSION: This study identifies a role for laminin in the initiation of coagulation and the formation of platelet-rich thrombi under shear conditions in a FXII-dependent manner.


Subject(s)
Blood Coagulation/physiology , Factor XII/physiology , Laminin/physiology , Thrombosis , Humans
18.
J Thromb Haemost ; 7(11): 1840-2, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19694943
19.
J Thromb Haemost ; 7 Suppl 1: 75-8, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19630773

ABSTRACT

Factor XI (FXI) has structural and mechanistic features that distinguish it from other coagulation proteases. A relatively recent addition to vertebrate plasma coagulation, FXI is a homodimer, with each subunit containing four apple domains and a protease domain. The apple domains form a disk structure with binding sites for platelets, high molecular weight kininogen, and the substrate factor IX (FIX). FXI is converted to the active protease FXIa by cleavage of the Arg369-Ile370 bond on each subunit. This converts the catalytic domains to the active forms, and unmasks exosites on the apple domains required for FIX binding. FXI activation by factor XIIa or thrombin proceeds through an intermediate with only one activated submit (1/2-FXIa). 1/2-FXIa activates FIX in a similar manner to FXIa. While the importance of the homodimeric structure of FXI is not certain, it may represent a strategy for binding to FIX and a platelet surface simultaneously.


Subject(s)
Factor XI/chemistry , Factor XI/physiology , Catalytic Domain , Enzyme Activation , Humans , Protein Conformation , Protein Subunits
20.
J Thromb Haemost ; 6(11): 1876-83, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18761718

ABSTRACT

BACKGROUND: Previous reports have noted that factor (F) XI and FXII and prekallikrein (the contact phase proteases) are absent in fish. OBJECTIVES: A broad survey of recently completed genomes was undertaken to find where during the course of vertebrate evolution these coagulation factors appeared. METHODS: BLAST searches were conducted for the various factors on genomes of lamprey, puffer fish, zebra fish, frog, chicken, platypus, and opossum. RESULTS: It was confirmed that FXII is absent from fish; it is present in frog, platypus, and opossum, but is absent in chicken, an apparent example of gene loss. A single gene corresponding to the evolutionary predecessor of FXI and prekallikrein occurs in frog, chicken, and platypus. The opossum (a marsupial) has both prekallikrein and FXI, completing the full complement of these genes that occurs in eutherian mammals. CONCLUSIONS: The step-by-step accrual of genes for these factors by a series of timely gene duplications has been confirmed by phylogenetic analysis and other considerations.


Subject(s)
Biological Evolution , Blood Coagulation/genetics , Animals , Computational Biology , Factor XII/genetics , Phylogeny , Prekallikrein/genetics , Vertebrates
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