RESUMO
Resolution of the inflammatory response requires coordinated regulation of pro- and anti-inflammatory mediator production, together with clearance of recruited inflammatory cells. Many different receptors have been implicated in phagocytosis of apoptotic cells (efferocytosis), including Mer, a receptor tyrosine kinase that can mediate recognition and subsequent internalization of apoptotic cells. In this manuscript, we examine the expression and function of the Tyro3/Axl/Mer (TAM) family of receptors by human monocytes. We demonstrate that the Mer ligand, protein S, binds to the surface of viable monocytes via phosphatidylserine-dependent and -independent mechanisms. Importantly, we have identified a novel role for receptor tyrosine kinase signaling in the augmentation of monocyte cytokine release in response to LPS. We propose that low-level phosphatidylserine exposure on the plasma membrane of viable monocytes allows protein S binding that leads to TAM-dependent augmentation of proinflammatory cytokine production. Our findings identify a potentially important role for TAM-mediated signaling during the initiation phase of inflammation.
Assuntos
Inflamação/imunologia , Monócitos/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Monócitos/metabolismo , Proteína S/imunologia , Proteína S/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina Quinase/imunologia , c-Mer Tirosina Quinase/metabolismoRESUMO
Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.
Assuntos
Coagulação Sanguínea , Fator VIII/metabolismo , Fator VIIa/metabolismo , Fator Xa/metabolismo , Tromboplastina/metabolismo , Fator VIIIa/metabolismo , Humanos , Trombina/metabolismoRESUMO
The anticoagulant activity of heparin administered during medical interventions must be reversed to restore normal clotting, typically by titrating with protamine. Given the acute toxicity associated with protamine, we endeavored to generate safer heparin antagonists by engineering bacteriophage Qß virus-like particles (VLPs) to display motifs that bind heparin. A particle bearing a single amino acid change from wild-type (T18R) was identified as a promising candidate for heparin antagonism. Surface potential maps generated through molecular modeling reveal that the T18R mutation adds synergistically to adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region that is replicated 180 times over the capsid. Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle. Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin. Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP. Dynamic light scattering (DLS) experiments suggested that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent, and in vitro clotting assays confirmed the 1:1 binding stoichiometry as full antagonism of heparin is achieved. Biolayer interferometry and backscattering interferometry corroborated the strong interaction of T18R with heparin, yielding Kd â¼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists.
Assuntos
Allolevivirus/química , Antagonistas de Heparina/química , Heparina/química , Nanopartículas/química , Anticoagulantes/química , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Cátions/química , Fluorescência , Protaminas/química , Ligação ProteicaRESUMO
PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 µg/mL), (super)FVa did not (6.7 µg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 µg/mL) or saline (5.6 µg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.
Assuntos
Fator Va/farmacocinética , Hemofilia A/tratamento farmacológico , Hemostáticos/farmacocinética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/farmacocinética , Animais , Modelos Animais de Doenças , Estabilidade de Medicamentos , Fator VIII/genética , Fator VIII/metabolismo , Fator Va/administração & dosagem , Fator Va/genética , Fator Va/toxicidade , Feminino , Fibrina/metabolismo , Meia-Vida , Hemofilia A/sangue , Hemofilia A/genética , Hemostáticos/administração & dosagem , Hemostáticos/toxicidade , Humanos , Injeções Intravenosas , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Índice de Gravidade de Doença , Trombina/metabolismoRESUMO
Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 µM D4476, 100 µM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity â¼1.5-fold (158.7±4.8%, P<0.01) or â¼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.
Assuntos
Plaquetas/enzimologia , Proteína C/metabolismo , Proteínas Quinases/metabolismo , Proteína S/metabolismo , Sequência de Aminoácidos , Benzamidas/farmacologia , Sítios de Ligação/genética , Plaquetas/metabolismo , Caseína Quinase I/antagonistas & inibidores , Caseína Quinase I/sangue , Caseína Quinase I/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/sangue , Caseína Quinase II/metabolismo , Ativação Enzimática , Humanos , Imidazóis/farmacologia , Immunoblotting , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Tempo de Tromboplastina Parcial , Fosforilação , Proteínas Quinases/sangue , Proteína S/química , Proteína S/genética , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Treonina/química , Treonina/genética , Treonina/metabolismoRESUMO
Proteases are critical in many physiological processes and the human genome encodes for 566 predicted proteolytic enzymes. Therefore, there is great interest in identifying and characterizing physiologic protease-substrate relationships. The coagulation cascade is a well-described network of serine proteases. However, new interactions of the coagulation cascade with other biological pathways have been discovered only recently. Therefore, we hypothesized that a non-biased protease degradomics analysis of the physiologic coagulation reaction would identify new interactions between the coagulation cascade and other pathways. We used the recently described PROTOMAP technique to profile the complete coagulation degradome. This analysis detected virtually all of the proteins of the coagulation cascade and identified a majority of the expected proteolytic events, suggesting significant coverage of the coagulation degradome. Multiple potential new proteolytic cleavages were detected, including two of transmembrane proteins that may be shed from the surface of blood cells. In addition, this analysis was able to identify several new potentially secreted proteins. A significant majority of the newly identified events were of proteins involved in innate immunity (complement and inflammation). This highlights potential new areas of crosstalk between these linked systems. Future studies will elucidate the details and functional consequences of these proteolytic events during coagulation.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos/métodos , Plasma/enzimologia , Proteômica/métodos , Coagulação Sanguínea/genética , Western Blotting , Cromatografia Líquida , Proteínas do Sistema Complemento/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/genética , Plasma/química , Especificidade por Substrato , Tripsina/metabolismoRESUMO
Activated protein C (APC) binds to its substrates activated factor V (FVa) and activated factor VIII (FVIIIa) with a basic exosite that consists of loops 37, 60, 70 and the autolysis loop. These loops have a high density of basic residues, resulting in a positive charge on the surface of APC. Many of these residues are important in the interaction of APC with FVa and FVIIIa. The current study focused on the function of the autolysis loop in the interaction with FVIIIa. This loop was previously shown to interact with FVa, and it inhibits APC inactivation by plasma serpins. Charged residues of the autolysis loop were individually mutated to alanine and the activity of these mutants was assessed in functional FVIIIa inactivation assays. The autolysis loop was functionally important for FVIIIa inactivation. Mutation of R306, K311 and R314 each resulted in significantly reduced FVIIIa inactivation. The inactivating cleavages of FVIIIa at R336 and R562 were affected equally by the mutations. Protein S and FV stimulated cleavage at R562 more than cleavage at R336, independent of mutations in the autolysis loop. Together, these results confirmed that the autolysis loop plays a significant role as part of the basic exosite on APC in the interaction with FVIIIa.
Assuntos
Autólise , Fator VIIIa/metabolismo , Proteína C/fisiologia , Autólise/genética , Fator V/farmacologia , Humanos , Mutação , Proteína C/genética , Proteína S/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
The goal of this review is to briefly summarize the two primary pathways of hemostasis, primary hemostasis and secondary hemostasis, as well as to summarize anticoagulant mechanisms and fibrinolysis. In addition, this review will discuss pathologies of hemostasis and the mechanisms of the various drugs that are available to impact these pathways to prevent either thrombosis or bleeding. While many of the main drugs that are used to treat disorders of hemostasis have been used for decades, greater understanding of hemostasis has led to development of various new drugs that have come onto the market recently or are close to coming onto the market. Thus, improved understanding of hemostasis continues to lead to benefits for patients.
Assuntos
Transtornos da Coagulação Sanguínea/terapia , Educação Continuada , Hemostasia , Animais , Anticoagulantes/farmacologia , Transtornos da Coagulação Sanguínea/patologia , Plaquetas/patologia , Fibrinólise , Fibrinolíticos/farmacologia , Hemorragia/prevenção & controle , Humanos , Trombose/prevenção & controleRESUMO
Coagulation factor V (FV) promotes inactivation of activated factor VIII (FVIIIa) by activated protein C (APC) and protein S. Loss of this APC cofactor activity is proposed to be partially responsible for the APC resistance phenotype of FV(Leiden). However, FVIIIa loses activity rapidly due to dissociation of the A2 domain, and this may be the primary mechanism of FVIIIa inactivation. APC/protein S also readily inactivates activated FV (FVa). We therefore hypothesized that FV can function as an anticoagulant cofactor for APC/protein S in the inactivation of FVa. FV was titrated into FV-deficient plasma, and the APC sensitivity ratio (APCsr; a measure of APC activity) was measured in a clotting assay that was not sensitive to FVIII. Our results showed an increase in APCsr as the FV concentration increased, suggesting an anticoagulant function for FV in this assay. FV(Leiden) showed APC resistance with an APCsr of 1.0. Therefore, under our experimental conditions, FV acted as an anticoagulant cofactor for APC in the inactivation of FVa.
Assuntos
Coagulação Sanguínea/fisiologia , Fator V/metabolismo , Fator Va/metabolismo , Proteína C/metabolismo , Fator V/genética , Fator VIIIa/metabolismo , Humanos , Fenótipo , Proteína S/metabolismo , Tempo de ProtrombinaRESUMO
Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs.
Assuntos
Fator Va , Inibidores do Fator Xa , Animais , Anticoagulantes , Células Endoteliais , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Hemorragia/prevenção & controle , Humanos , CamundongosRESUMO
Particles to the rescue! The construction of cationic amino acid motifs on the surface of bacteriophage Qbeta by genetic engineering or chemical conjugation gives particles that are potent inhibitors of the anticoagulant action of heparin, which is a common anticlotting agent subject to clinical overdose.Polyvalent interactions allow biological structures to exploit low-affinity ligand-receptor binding events to affect physiological responses. We describe here the use of bacteriophage Qbeta as a multivalent platform for the display of polycationic motifs that act as heparin antagonists. Point mutations to the coat protein allowed us to generate capsids bearing the K16M, T18R, N10R, or D14R mutations; because 180 coat proteins form the capsid, the mutants provide a spectrum of particles differing in surface charge by as much as +540 units (K16M vs. D14R). Whereas larger poly-Arg insertions (for example, C-terminal Arg(8)) did not yield intact virions, it was possible to append chemically synthesized oligo-Arg peptides to stable wild-type (WT) and K16M platforms. Heparin antagonism by the particles was evaluated by using the activated partial thrombin time (aPTT) clotting assay; this revealed that T18R, D14R, and WT-(R(8)G(2))(95) were the most effective at disrupting heparin-mediated anticoagulation (>95 % inhibition). This activity agreed with measurements of zeta potential (ZP) and retention time on cation exchange chromatography for the genetic constructs, which distribute their added positive charge over the capsid surface (+180 and +360 for T18R and D14R relative to WT). The potent activity of WT-(R(8)G(2))(95), despite its relatively diminished overall surface charge is likely a consequence of the particle's presentation of locally concentrated regions with high positive charge density that interact with heparin's extensively sulfated domains. The engineered cationic capsids retained their ability to inhibit heparin at high concentrations and showed no anticlotting activity of the kind that limits the utility of antiheparin polycationic agents that are currently in clinical use.
Assuntos
Allolevivirus/química , Motivos de Aminoácidos , Cátions/química , Heparina/química , Allolevivirus/genética , Capsídeo/química , Cromatografia por Troca Iônica , Heparina/genética , Modelos Moleculares , Estrutura Molecular , Mutação Puntual , Conformação ProteicaRESUMO
The hypothesis that prothrombin (FII) protects coagulation factor Va (FVa) from proteolytic inactivation by activated protein C (APC) was tested using purified proteins. FII dose-dependently protected FVa from APC proteolysis under conditions where competition of proteins for binding to negatively-charged phospholipid surface was not relevant (i.e. either at high phospholipid vesicle concentrations or using soluble dicaproylphosphatidylserine at levels below its critical micellar concentration). Cleavages in FVa at both Arg(506) and Arg(306) by APC were inhibited by FII. FII did not alter the amidolytic activity of APC towards chromogenic oligopeptide substrates or inhibit FVIIIa inactivation by APC, implying that the FII-mediated protection of FVa from APC proteolysis was due to the ability of FII to inhibit protein-protein interactions between FVa and APC. FII also protected FVa from inactivation by Gla-domainless APC, ruling out a role for the APC Gla domain for these observations. To identify domains of FII responsible for the observed phenomenon, various forms or fragments of FII were employed. Biotin-Phe-ProArg-CMK-inhibited meizothrombin and fII-fragment 1*2 protected FVa from proteolysis by APC. In contrast, no significant protection of FVa from APC cleavage was observed for Gladomainless-FII, prethrombin-1, prethrombin-2, FII fragment 1 or active site inhibited-thrombin (DEGR-thrombin). Overall, these data demonstrate that the Gla domain of FII linked to kringle 1 and 2 is necessary for the ability of FII to protect FVa from APC cleavage and support the general concept that assembly of the FII activation complex (FXa*FVa*FII*lipid surface) protects FVa from APC inactivation so that the procoagulant, thrombin generating pathway can act unhindered by APC. Only following FII activation and dissociation of the FII Gla domain fragments from the FII-ase complex, can APC inactivate FVa and down-regulate thrombin generation.
Assuntos
Coagulação Sanguínea , Fator Va/metabolismo , Proteína C/metabolismo , Protrombina/metabolismo , Animais , Ligação Competitiva , Precursores Enzimáticos/metabolismo , Fator VIIIa/metabolismo , Fator Va/genética , Fator Xa/metabolismo , Humanos , Kringles , Mutação , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Trombina/metabolismo , Fatores de TempoRESUMO
Neutrophils and monocytes express cathepsin G and can also bind to activated platelets, thus they can be localized to the site of active coagulation. Previous studies have suggested that cathepsin G inactivated coagulation factor VIII (FVIII) and was thus anticoagulant. But other studies have indicated procoagulant functions for cathepsin G in activation of coagulation factor V or activation of platelets among other possible mechanisms. Therefore, it remains unclear if cathepsin G is anticoagulant or procoagulant. We investigated the effects of human neutrophil cathepsin G on FVIII/VIIIa. Cathepsin G activates FVIII to a partially active form while having only a minor inactivating effect on thrombin-activated FVIIIa. This inactivation is mostly due to decreased stability of FVIIIa since a disulfide bond that prevents A2 subunit dissociation from FVIIIa prevents any loss of activity due to cathepsin G proteolysis. FVIII that has been cleaved by cathepsin G can still be activated by thrombin if A2 subunit dissociation is prevented. Cathepsin G cleavages of FVIII are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. Therefore, it is possible that cathepsin G from neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII.
Assuntos
Coagulação Sanguínea , Catepsinas/metabolismo , Fator VIII/metabolismo , Fator VIIIa/metabolismo , Neutrófilos/enzimologia , Serina Endopeptidases/metabolismo , Catepsina G , Dissulfetos/metabolismo , Fator VIII/genética , Humanos , Técnicas In Vitro , Mutação , Tempo de Tromboplastina Parcial , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMO
With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Corantes Fluorescentes/química , Oligopeptídeos/química , Peptídeo Hidrolases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Humanos , Hidrólise , Cinética , Linfonodos/patologia , Metástase Linfática , Camundongos Endogâmicos BALB C , Proteólise , RatosRESUMO
Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/administração & dosagem , Fator VIIa/administração & dosagem , Fator Va/administração & dosagem , Hemofilia A/imunologia , Animais , Anticorpos/química , Relação Dose-Resposta a Droga , Fator VIII/antagonistas & inibidores , Feminino , Hemofilia A/sangue , Hemostasia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Trombina/metabolismoRESUMO
INTRODUCTION: We describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis. PATIENTS/METHODS: We enrolled the two affected patients, five unaffected family members and fifty-five normal controls. Clinical, laboratory, and radiological characteristics of patients were obtained. Exome sequencing was performed for the older affected child. PROC c.811 C>T was genotyped by PCR in patients, family members, and controls. Protein C amidolytic activity and antigen were measured using the STACHROM® protein C kit and ELISAs. To define functional abnormalities caused by the patients' mutation, recombinant wildtype protein C and its mutants R229W, R229Q and R229A were studied. RESULTS: For the two cousins, protein C amidolytic activity was 61% and 59% and antigen was 57% and 73% (nl 70-140%), respectively. Exome sequencing revealed a homozygous variant in exon 9 of the protein C (PROC) gene c.811 C>T (R229W). The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin interactions. Recombinant R229W-protein C mutant was strikingly defective in rate of activation by thrombin: thrombomodulin, suggesting an in vivo deficit in these children for generation of activated protein C. CONCLUSIONS: These cases emphasize that protein C and activated protein C are important in maintaining the integrity of the brain vascular endothelium in humans. Moreover, routine protein C assays utilizing snake venom protease fail to detect protein C mutants that are resistant to thrombin:thrombomodulin activation.
Assuntos
Hemorragias Intracranianas/genética , Mutação Puntual , Proteína C/genética , Trombose/genética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Homozigoto , Humanos , Recém-Nascido , Hemorragias Intracranianas/complicações , Hemorragias Intracranianas/metabolismo , Hemorragias Intracranianas/patologia , Linhagem , Proteína C/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo , Trombose/complicações , Trombose/metabolismo , Trombose/patologiaRESUMO
Procoagulant factor Va (FVa) is inactivated via limited proteolysis at three Arg residues in the A2 domain by the anticoagulant serine protease, activated protein C (APC). Cleavage by APC at Arg306 in FVa causes dissociation of the A2 domain from the heterotrimeric A1:A2:A3 structure and complete loss of procoagulant activity. To help distinguish inactivation mechanisms involving A2 domain dissociation from inactivation mechanisms involving unfavorable changes in factor Xa (FXa) affinity, we used our FVa homology model to engineer recombinant FVa mutants containing an interdomain disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (A2-SS-A3 mutants) in addition to cleavage site mutations, Arg506Gln and Arg679Gln. SDS-PAGE analysis showed that the disulfide bond in A2-SS-A3 mutants prevented dissociation of the A2 domain. In the absence of A2 domain dissociation from the A1:A2:A3 trimer, APC cleavage at Arg306 alone caused a sevenfold decrease in affinity for FXa, whereas APC cleavages at Arg306, Arg506, and Arg679 caused a 70-fold decrease in affinity for FXa and a 10-fold decrease in the k(cat) of the prothrombinase complex for prothrombin without any effect on the apparent K(m) for prothrombin. Therefore, for FVa inactivation by APC, dissociation of the A2 domain may provide only a modest final step, whereas the critical events are the cleavages at Arg506 and Arg306, which effectively inactivate FVa before A2 dissociation can take place. Nonetheless, for FVa Leiden (Gln506-FVa) inactivation by APC, A2 domain dissociation may become mechanistically important, depending on the ambient FXa concentration.
Assuntos
Anticoagulantes/metabolismo , Dissulfetos/metabolismo , Fator Va/metabolismo , Proteína C/metabolismo , Fator Va/genética , Fator Xa/metabolismo , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Protrombina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismoRESUMO
Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.
Assuntos
Fator Va/metabolismo , Proteína C/química , Proteína C/metabolismo , Trombomodulina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Proteína C/genética , Coelhos , Eletricidade Estática , Trombina/farmacologiaRESUMO
OBJECTIVE: An increased risk of bleeding is observed in patients receiving activated protein C (APC), which may be a limiting factor for the application of novel APC therapies. Since APC's therapeutic effects often require its cytoprotective activities on cells but not APC's anticoagulant activities, an agent that specifically antagonizes APC's anticoagulant effects but not its cytoprotective effects could provide an effective means to control concerns for risk of bleeding. We hypothesized that superFVa, an engineered activated FVa-variant that restores hemostasis in hemophilia could reduce APC-induced bleeding. APPROACH AND RESULTS: SuperFVa was engineered with mutations of the APC cleavage sites (Arg506/306/679Gln) and a disulfide bond (Cys609-Cys1691) between the A2 and A3 domains, which augment its biological activity and cause high resistance to APC. SuperFVa normalized APC-prolonged clotting times and restored APC-suppressed thrombin generation in human and murine plasma at concentrations where wild-type (wt) FVa did not show effects. Following intravenous injection of APC into BALB/c mice, addition to whole blood ex vivo of superFVa but not wt-FVa significantly normalized whole blood clotting. Blood loss following tail clip or liver laceration was significantly reduced when superFVa was administered intravenously to BALB/c mice prior to intravenous APC-treatment. Furthermore, superFVa abolished mortality (â¼50%) associated with excessive bleeding following liver laceration in mice treated with APC. CONCLUSIONS: Our results provide proof of concept that superFVa is effective in preventing APC-induced bleeding and may provide therapeutic benefits as a prohemostatic agent in various situations where bleeding is a serious risk.
Assuntos
Anticoagulantes/efeitos adversos , Fator Va/farmacologia , Hemorragia/etiologia , Hemorragia/prevenção & controle , Proteína C/efeitos adversos , Proteínas Recombinantes/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Fator Va/administração & dosagem , Feminino , Hemorragia/mortalidade , Hemostasia/efeitos dos fármacos , Camundongos , Modelos Animais , Tempo de Protrombina , Proteínas Recombinantes/administração & dosagem , Trombina/metabolismoRESUMO
Anticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the "thrombin-sensitive region", abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities ("PS-direct"). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PS-direct that was similar to or greater than that of Zn(2+)-containing pdPS and much greater than that of Zn(2+)-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticle-bound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.