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1.
Blood ; 135(8): 558-567, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31800958

RESUMO

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.


Assuntos
Coagulação Sanguínea , Bradicinina/metabolismo , Pré-Calicreína/metabolismo , Substituição de Aminoácidos , Animais , Fator XII/metabolismo , Células HEK293 , Humanos , Cininogênio de Alto Peso Molecular/metabolismo , Camundongos Endogâmicos C57BL , Pré-Calicreína/química , Pré-Calicreína/genética , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Blood ; 133(10): 1152-1163, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30591525

RESUMO

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.


Assuntos
Fator XII/química , Fator XIa/química , Angioedema Hereditário Tipo III/sangue , Angioedema Hereditário Tipo III/genética , Angioedemas Hereditários , Animais , Arginina/química , Coagulação Sanguínea , Bradicinina/sangue , Catálise , Proteína Inibidora do Complemento C1/química , Fator XIIa/química , Células HEK293 , Humanos , Cininogênios/sangue , Lisina/química , Camundongos , Camundongos Endogâmicos C57BL , Calicreína Plasmática/química , Pré-Calicreína/química , Ligação Proteica , Proteínas Recombinantes/química , Propriedades de Superfície , Trombina/genética
3.
Blood ; 129(11): 1527-1537, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28069606

RESUMO

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.


Assuntos
Coagulação Sanguínea , Fator XII/metabolismo , Proteólise , Domínio Catalítico/genética , Fator XIIa/metabolismo , Humanos , Calicreínas/metabolismo , Propriedades de Superfície
4.
Curr Opin Hematol ; 24(5): 411-418, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28604413

RESUMO

PURPOSE OF REVIEW: Exposure of blood to foreign surfaces induces reciprocal conversion of the plasma proteins factor XII (fXII) and plasma prekallikrein (PPK) to the proteases α-fXIIa and α-kallikrein. This process, called contact activation, has a range of effects on host defence mechanisms, including promoting coagulation. The nature of the triggering mechanism for contact activation is debated. One hypothesis predicts that fXII has protease activity, either intrinsically or upon surface-binding, that initiates contact activation. We tested this by assessing the proteolytic activity of a recombinant fXII variant that cannot be converted to α-fXIIa. RECENT FINDINGS: The proteolytic activity of fXII-T (for 'triple' mutant), a variant with alanine substitutions for arginine at activation cleavage sites (Arg334, Arg344, and Arg353) was tested with known α-fXIIa substrates. FXII-T activates PPK in solution, and the reaction is enhanced by polyphosphate, an inducer of contact activation released from platelets. In the presence of polyphosphate, fXII-T converts fXII to α-fXIIa, and also converts the coagulation protein factor XI to its active form. SUMMARY: The findings support the hypothesis that contact activation is initiated through activity intrinsic to single-chain fXII, and indicate that preexisting α-fXIIa is not required for induction of contact activation.


Assuntos
Fator XIIa/metabolismo , Calicreína Plasmática/metabolismo , Pré-Calicreína/metabolismo , Fator XIIa/genética , Humanos , Calicreína Plasmática/genética , Pré-Calicreína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Am J Physiol Heart Circ Physiol ; 312(5): H907-H918, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28235789

RESUMO

Immune activation in chronic systolic heart failure (HF) correlates with disease severity and prognosis. Recombinant neuregulin-1 (rNRG-1) is being developed as a possible therapy for HF, based on the activation of ERBB receptors in cardiac cells. Work in animal models of HF led us to hypothesize that there may be direct effects of NRG-1 on immune system activation and inflammation. We investigated the expression of ERBB receptors and the effect of rNRG-1 isoform glial growth factor 2 (GGF2) in subpopulations of peripheral blood mononuclear cells (PB MNCs) in subjects with HF. We found that human monocytes express both ERBB2 and ERBB3 receptors, with high interindividual variability among subjects. Monocyte surface ERBB3 and TNF-α mRNA expression were inversely correlated in subjects with HF but not in human subjects without HF. GGF2 activation of ERBB signaling ex vivo inhibited LPS-induced TNF-α production, specifically in the CD14lowCD16+ population of monocytes in a phosphoinositide 3-kinase-dependent manner. GGF2 suppression of TNF-α correlated directly with the expression of ERBB3. In vivo, a single dose of intravenous GGF2 reduced TNF-α expression in PB MNCs of HF subjects participating in a phase I safety study of GGF2. These results support a role for ERBB3 signaling in the regulation of TNF-α production from CD14lowCD16+ monocytes and a need for further investigation into the clinical significance of NRG-1/ERBB signaling as a modulator of immune system function.NEW & NOTEWORTHY This study identified a novel role of neuregulin-1 (NRG-1)/ERBB signaling in the control of proinflammatory activation of monocytes. These results further improve our fundamental understanding of cardioprotective effects of NRG-1 in patients with heart failure.


Assuntos
Receptores ErbB/biossíntese , Inflamação/fisiopatologia , Monócitos , Transdução de Sinais , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Humanos , Técnicas In Vitro , Ativação de Macrófagos , Masculino , Pessoa de Meia-Idade , Neuregulina-1/metabolismo , Neuregulina-1/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-3/biossíntese , Receptor ErbB-3/genética , Proteínas Recombinantes/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
6.
Blood ; 125(9): 1488-96, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25587039

RESUMO

Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI.


Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Fator IX/metabolismo , Fator XIa/metabolismo , Fator Xa/metabolismo , Fibrina/metabolismo , Lipoproteínas/metabolismo , Plaquetas/citologia , Western Blotting , Células Cultivadas , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipoproteínas/genética , Mutação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Blood ; 123(11): 1739-46, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24408325

RESUMO

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.


Assuntos
Modelos Animais de Doenças , Deficiência do Fator XII/complicações , Fator XII/antagonistas & inibidores , Fator XII/fisiologia , Trombina/metabolismo , Trombose/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Coagulação Sanguínea , Fator XI/metabolismo , Fator XIIa/metabolismo , Fibrina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Papio , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Tromboplastina/metabolismo , Trombose/etiologia , Trombose/metabolismo
8.
Biochemistry ; 54(36): 5578-88, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26295742

RESUMO

With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.


Assuntos
Plaquetas/enzimologia , Fosfolipases A2 do Grupo IV/sangue , Lipídeos/sangue , Benzoatos/farmacologia , Plaquetas/química , Plaquetas/efeitos dos fármacos , Glicerofosfolipídeos/sangue , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Humanos , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/sangue , Espectrometria de Massas por Ionização por Electrospray , Estresse Mecânico , Sulfonamidas/farmacologia , Trombina/farmacologia
9.
Blood ; 121(19): 3962-9, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23515926

RESUMO

Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit.


Assuntos
Fator XI/química , Fator XI/metabolismo , Fator XIa/metabolismo , Animais , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/metabolismo , Fator XI/genética , Deficiência do Fator XI/genética , Deficiência do Fator XI/metabolismo , Fator XIIa/química , Fator XIIa/metabolismo , Fator XIa/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína
10.
Arterioscler Thromb Vasc Biol ; 33(7): 1670-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23559626

RESUMO

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fator XI/metabolismo , Fibrinolíticos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Trombose/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Derivação Arteriovenosa Cirúrgica , Tempo de Sangramento , Colágeno , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Fator XI/antagonistas & inibidores , Fator XI/genética , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Macaca fascicularis , Oligonucleotídeos Antissenso/toxicidade , Papio , Trombina/metabolismo , Trombose/sangue , Trombose/etiologia , Trombose/genética , Fatores de Tempo
11.
Blood ; 118(2): 437-45, 2011 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-21527525

RESUMO

The prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to ß-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by ß-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, ß-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.


Assuntos
Fator XI/metabolismo , Protrombina/metabolismo , Protrombina/fisiologia , Sequência de Aminoácidos , Coagulação Sanguínea/fisiologia , Testes de Coagulação Sanguínea , Domínio Catalítico , Células Cultivadas , Fator XI/química , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Protrombina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
12.
J Thromb Haemost ; 21(5): 1200-1213, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36696212

RESUMO

BACKGROUND: Titanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered compatible with blood, they appear to support plasma contact activation and may be thrombogenic. OBJECTIVES: The objective of this study was to compare Ti and titanium nitride (TiN) with known activators of contact activation (kaolin and silica) in plasma-clotting assays and to assess binding and activation of factor XII, (FXII), factor XI (FXI), prekallikrein, and high-molecular-weight kininogen (HK) with Ti/TiN. METHODS: Ti-based nanospheres and foils were compared with kaolin, silica, and aluminum in plasma-clotting assays. Binding and activation of FXII, prekallikrein, HK, and FXI to surfaces was assessed with western blots and chromogenic assays. RESULTS: Using equivalent surface amounts, Ti and TiN were comparable with kaolin and superior to silica, for inducing coagulation and FXII autoactivation. Similar to many inducers of contact activation, Ti and TiN are negatively charged; however, their effects on FXII are not neutralized by the polycation polybrene. Antibodies to FXII, prekallikrein, or FXI or coating Ti with poly-L-arginine blocked Ti-induced coagulation. An antibody to FXII reduced FXII and PK binding to Ti, kallikrein generation, and HK cleavage. CONCLUSION: Titanium compounds induce contact activation with a potency comparable with that of kaolin. Binding of FXII with Ti shares some features with FXII binding to soluble polyanions but may have unique features. Inhibitors targeting FXII or FXI may be useful in mitigating Ti-induced contact activation in patients with titanium-based implants that are exposed to blood.


Assuntos
Caulim , Pré-Calicreína , Humanos , Fator XI/metabolismo , Fator XII/metabolismo , Pré-Calicreína/metabolismo , Titânio
13.
Blood ; 116(19): 3981-9, 2010 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-20634381

RESUMO

Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.


Assuntos
Fator XIIa/fisiologia , Fator XI/fisiologia , Trombose/sangue , Trombose/etiologia , Animais , Anticorpos Monoclonais/farmacologia , Anticoagulantes/farmacologia , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/etiologia , Gatos , Modelos Animais de Doenças , Cães , Fator XI/antagonistas & inibidores , Deficiência do Fator XI/sangue , Deficiência do Fator XI/genética , Deficiência do Fator XI/fisiopatologia , Deficiência do Fator XII/sangue , Deficiência do Fator XII/genética , Deficiência do Fator XII/fisiopatologia , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Papio anubis , Tempo de Tromboplastina Parcial , Embolia Pulmonar/sangue , Embolia Pulmonar/etiologia , Coelhos , Especificidade da Espécie
14.
J Pathol ; 224(2): 203-11, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21557221

RESUMO

Goblet cell hyperplasia is a common feature of chronic obstructive pulmonary disease (COPD) airways, but the mechanisms that underlie this epithelial remodelling in COPD are not understood. Based on our previous finding of hypoxia-inducible factor-1α (HIF-1α) nuclear localization in large airways from patients with COPD, we investigated whether hypoxia-inducible signalling could influence the development of goblet cell hyperplasia. We evaluated large airway samples obtained from 18 lifelong non-smokers and 13 former smokers without COPD, and 45 former smokers with COPD. In these specimens, HIF-1α nuclear staining occurred almost exclusively in COPD patients in areas of airway remodelling. In COPD patients, 93.2 ± 3.9% (range 65-100%) of goblet cells were HIF-1α positive in areas of goblet cell hyperplasia, whereas nuclear HIF-1α was not detected in individuals without COPD or in normal-appearing pseudostratified epithelium from COPD patients. To determine the direct effects of hypoxia-inducible signalling on epithelial cell differentiation in vitro, human bronchial epithelial cells (HBECs) were grown in air-liquid interface cultures under hypoxia (1% O(2)) or following treatment with a selective HIF-1α stabilizer, (2R)-[(4-biphenylylsulphonyl)amino]-N-hydroxy-3-phenyl-propionamide (BiPS). HBECs grown in hypoxia or with BiPS treatment were characterized by HIF-1α activation, carbonic anhydrase IX expression, mucus-producing cell hyperplasia and increased expression of MUC5AC. Analysis of signal transduction pathways in cells with HIF-1α activation showed increased ERK1/2 phosphorylation without activation of epidermal growth factor receptor, Ras, PI3K-Akt or STAT6. These data indicate an important effect of hypoxia-inducible signalling on airway epithelial cell differentiation and identify a new potential target to limit mucus production in COPD.


Assuntos
Brônquios/patologia , Células Caliciformes/patologia , Fator 1 Induzível por Hipóxia/fisiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Remodelação das Vias Aéreas/fisiologia , Brônquios/metabolismo , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Células Cultivadas , Ativação Enzimática/fisiologia , Feminino , Células Caliciformes/metabolismo , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucina-5AC/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/fisiologia , Adulto Jovem
15.
J Biol Chem ; 285(11): 8278-89, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20053992

RESUMO

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH(2)-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K(D)) of 10 +/- 4 microm and 400 +/- 300 microm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k(obs)). SOS bound HCII with K(D) 1.45 +/- 0.30 mm, and this binding was tightened in the T.SOS.HCII complex, characterized by K(complex) of approximately 0.20 microm. Inactivation data were incompatible with a model solely depending on HCII.SOS but fit an equilibrium linkage model employing T.SOS binding in the pathway to higher order complex formation. Hirudin-(54-65)(SO(3)(-)) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54-65)(SO(3)(-)) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K(D) = 1600 +/- 300 microm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.


Assuntos
Anticoagulantes/metabolismo , Coagulação Sanguínea/fisiologia , Cofator II da Heparina/metabolismo , Sacarose/análogos & derivados , Trombina/metabolismo , Regulação Alostérica , Anticoagulantes/química , Anticoagulantes/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Coagulação Sanguínea/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/farmacologia , Cofator II da Heparina/química , Hirudinas/metabolismo , Hirudinas/farmacologia , Humanos , Cinética , Modelos Químicos , Plasma , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Protrombina/metabolismo , Espectrometria de Fluorescência , Sacarose/química , Sacarose/metabolismo , Sacarose/farmacologia , Trombina/química , Trombina/farmacologia
16.
Blood ; 114(2): 452-8, 2009 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-19351955

RESUMO

During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or alpha-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.


Assuntos
Fator XI/metabolismo , Trombina/metabolismo , Linhagem Celular , Fator XI/genética , Fator XII/metabolismo , Humanos , Especificidade por Substrato , Tromboplastina/metabolismo
17.
JRSM Cardiovasc Dis ; 9: 2048004020906994, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32110389

RESUMO

PURPOSE: Chronic thromboembolic pulmonary hypertension is characterized by incomplete thrombus resolution following acute pulmonary embolism, leading to pulmonary hypertension and right ventricular dysfunction. Conditions such as thrombophilias, dysfibrinogenemias, and inflammatory states have been associated with chronic thromboembolic pulmonary hypertension, but molecular mechanisms underlying this disease are poorly understood. We sought to characterize the molecular and functional features associated with chronic thromboembolic pulmonary hypertension using a multifaceted approach. METHODS: We utilized functional assays to compare clot lysis times between chronic thromboembolic pulmonary hypertension patients and multiple controls. We then performed immunohistochemical characterization of tissue from chronic thromboembolic pulmonary hypertension, pulmonary arterial hypertension, and healthy controls, and examined RNA expression patterns of cultured lymphocytes and pulmonary arterial specimens. We then confirmed RNA expression changes using immunohistochemistry, immunofluorescence, and Western blotting in pulmonary arterial tissue. RESULTS: Clot lysis times in chronic thromboembolic pulmonary hypertension patients are similar to multiple controls. Chronic thromboembolic pulmonary hypertension endarterectomized tissue has reduced expression of both smooth muscle and endothelial cell markers. RNA expression profiles in pulmonary arteries and peripheral blood lymphocytes identified differences in RNA transcript levels related to inflammation and growth factor signaling, which we confirmed using immunohistochemistry. Gene expression data also suggested significant alterations in metabolic pathways, and immunofluorescence and Western blot experiments confirmed that unglycosylated CD36 and adiponectin expression were increased in chronic thromboembolic pulmonary hypertension versus controls. CONCLUSIONS: Our data do not support impaired clot lysis underlying chronic thromboembolic pulmonary hypertension, but did demonstrate distinct molecular patterns present both in peripheral blood and in pathologic specimens of chronic thromboembolic pulmonary hypertension patients suggesting that altered metabolism may play a role in chronic thromboembolic pulmonary hypertension pathogenesis.

18.
Oncotarget ; 10(66): 7031-7042, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31903163

RESUMO

Growth arrest-specific 6 (Gas6) has been implicated in carcinogenesis through activation of its receptors, particularly MerTK. To investigate whether Gas6 plays a role in resistance to NF-κB inhibitors, which have not proven to be effective agents for lung cancer therapy, we studied lung cancer models induced by urethane injection or expression of mutant Kras (KrasG12D). We found that Gas6 is primarily produced by macrophages during tumorigenesis and that Gas6 is negatively regulated by NF-κB. Since Gas6 is a vitamin K dependent protein, we used low-dose warfarin to block Gas6 production and showed that this treatment inhibited tumorigenesis in both the urethane and KrasG12D models, most prominently in mice with targeted deletion of IKKß in myeloid cells (IKKßΔMye mice). In addition, MerTK deficient mice had reduced urethane-induced tumorigenesis. Inhibition of the Gas6-MerTK pathway in all these models reduced macrophages and neutrophils in the lungs of tumor-bearing mice. Analysis of mouse lung tumors revealed MerTK staining on tumor cells and in vitro studies showed that Gas6 increased proliferation of human lung cancer cell lines. To assess the therapeutic potential for combination treatment targeting NF-κB and Gas6-MerTK, we injected Lewis Lung Carcinoma cells subcutaneously and treated mice with Bay 11-70852 (NF-κB inhibitor) and/or Foretinib (MerTK inhibitor). While individual treatments were ineffective, combination therapy markedly reduced tumor growth, blocked tumor cell proliferation, reduced tumor-associated macrophages, and increased CD4+ T cells. Together, our studies unmask a role for Gas6-MerTK signaling in lung carcinogenesis and indicate that up-regulation of Gas6 production in macrophages could be a major mechanism of resistance to NF-κB inhibitors.

19.
J Thromb Haemost ; 17(9): 1449-1460, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31125187

RESUMO

BACKGROUND: The homologous plasma proteins prekallikrein and factor XI (FXI) circulate as complexes with high molecular weight kininogen. Although evidence supports an interaction between the prekallikrein-kininogen complexes and vascular endothelium, there is conflicting information regarding FXI binding to endothelium. OBJECTIVE: To study the interaction between FXI and blood vessels in mice. METHODS: C57Bl/6 wild-type or F11-/- mice in which variants of FXI were expressed by hydrodynamic tail vein injection, received intravenous infusions of saline, heparin, polyphosphates, protamine, or enzymes that digest glycosaminoglycans (GAGs). Blood was collected after infusion and plasma was analyzed by western blot for FXI. RESULTS AND CONCLUSIONS: Plasma FXI increased 5- to 10-fold in wild-type mice after infusion of heparin, polyphosphates, protamine, or GAG-digesting enzymes, but not saline. Similar treatments resulted in a much smaller change in plasma FXI levels in rats, and infusions of large boluses of heparin did not change FXI levels appreciably in baboons or humans. The releasable FXI fraction was reconstituted in F11-/- mice by expressing murine FXI, but not human FXI. We identified a cluster of basic residues on the apple 4 domain of mouse FXI that is not present in other species. Replacing the basic residues with alanine prevented the interaction of mouse FXI with blood vessels, whereas introducing the basic residues into human FXI allowed it to bind to blood vessels. Most FXI in mice is noncovalently associated with GAGs on blood vessel endothelium and does not circulate in plasma.


Assuntos
Endotélio Vascular/metabolismo , Fator XI/metabolismo , Glicosaminoglicanos/sangue , Animais , Sítios de Ligação , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Cloretos/toxicidade , Fator XI/química , Deficiência do Fator XI/sangue , Compostos Férricos/toxicidade , Heparina/farmacologia , Humanos , Cininogênios/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Papio , Pré-Calicreína/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Eletricidade Estática
20.
Res Pract Thromb Haemost ; 2(1): 168-173, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29354798

RESUMO

BACKGROUND: Factor XII (FXII) Locarno is a natural variant with proline replacing Arg353 at the activation cleavage site, preventing conversion to the fully active protease factor XIIa (FXIIa). Recently, we showed that FXII restricted to a single chain form (sc-FXII) by replacing Arg353 with alanine expresses proteolytic activity that is enhanced by cofactors such as polyphosphate. AIM: To determine if the Pro353 substitution affects the activity of sc-FXII. METHODS: Wild type FXII (FXII-WT), FXII-R353A, and FXII Locarno (FXII-R353P) were tested for their abilities to activate prekallikrein, and to induce thrombin generation and coagulation in plasma in a factor XI-dependent manner. RESULTS: FXII-WT is converted to FXIIa by autoactivation in the presence of polyphosphate, and by incubation with kallikrein. FXII-R353P and FXII-R353A were not converted to FXIIa by these methods. Despite this, FXII-R353A converts prekallikrein to kallikrein, and the reaction is enhanced by polyphosphate. FXII-R353P also converts prekallikrein to kallikrein, but at a slower rate than FXII-R353A. In FXII-deficient plasma induced to clot with silica, FXII-R353A is a better promoter of factor XI-dependent thrombin generation and coagulation than FXII-R353P. CONCLUSION: The activity of sc-FXII is sensitive to perturbations in the activation loop, which contains residue 353. Homology modeling based on the crystal structure of the FXII homolog tissue plasminogen activator suggests that Pro353 introduces changes in the shape and flexibility of the activation loop that disrupt key interactions that support an active conformation in sc-FXII.

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