RESUMO
Inorganic polyphosphate is widespread in biology and exhibits striking prohemostatic, prothrombotic, and proinflammatory effects in vivo. Long-chain polyphosphate (of the size present in infectious microorganisms) is a potent, natural pathophysiologic activator of the contact pathway of blood clotting. Medium-chain polyphosphate (of the size secreted from activated human platelets) accelerates factor V activation, completely abrogates the anticoagulant function of tissue factor pathway inhibitor, enhances fibrin clot structure, and greatly accelerates factor XI activation by thrombin. Polyphosphate may have utility as a hemostatic agent, whereas antagonists of polyphosphate may function as novel antithrombotic/anti-inflammatory agents. The detailed molecular mechanisms by which polyphosphate modulates blood clotting reactions remain to be elucidated.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Inflamação/etiologia , Polifosfatos/farmacologia , Animais , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Fibrina/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Inflamação/sangue , Inflamação/metabolismo , Modelos Biológicos , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Polifosfatos/química , Polifosfatos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trombina/metabolismoRESUMO
Inorganic polyphosphates are linear polymers of orthophosphate that modulate blood clotting and inflammation. Polyphosphate accumulates in infectious microorganisms and is secreted by activated platelets; long-chain polyphosphate in particular is an extremely potent initiator of the contact pathway, a limb of the clotting cascade important for thrombosis but dispensable for hemostasis. Polyphosphate inhibitors therefore might act as novel antithrombotic/anti-inflammatory agents with reduced bleeding side effects. Antipolyphosphate antibodies are unlikely because of polyphosphate's ubiquity and simple structure; and although phosphatases such as alkaline phosphatase can digest polyphosphate, they take time and may degrade other biologically active molecules. We now identify a panel of polyphosphate inhibitors, including cationic proteins, polymers, and small molecules, and report their effectiveness in vitro and in vivo. We also compare their effectiveness against the procoagulant activity of RNA. Polyphosphate inhibitors were antithrombotic in mouse models of venous and arterial thrombosis and blocked the inflammatory effect of polyphosphate injected intradermally in mice. This study provides proof of principle for polyphosphate inhibitors as antithrombotic/anti-inflammatory agents in vitro and in vivo, with a novel mode of action compared with conventional anticoagulants.
Assuntos
Anti-Inflamatórios/farmacologia , Fibrinolíticos/farmacologia , Inflamação/tratamento farmacológico , Polifosfatos/antagonistas & inibidores , Trombose/tratamento farmacológico , Animais , Anti-Inflamatórios/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Fibrinolíticos/isolamento & purificação , Hemostasia/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Inflamação/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Polifosfatos/sangue , Trombose/sangueRESUMO
Wnt-signaling pathway (WSP) alterations have been identified in patients with prostate cancer (PCa) and are implicated in disease progression and hormonal resistance. We utilized a multi-institutional dataset to characterize molecular alterations in the canonical and non-canonical WSP in PCa. Patients with PCa who underwent tissue-based genomic sequencing were investigated. Tumors with somatic activating mutations in CTNNB1 or RSPO2, or inactivating mutations in either APC or RNF43 were characterized as having aberrant canonical Wnt signaling (WSP-activated). Overall survival (OS) analyses were restricted to microsatellite stable (MSS) tumors lacking RNF43 G659fs* mutations. We also investigated non-canonical WSP by evaluation of ROR1, ROR2, and WNT5 in WSP-activated versus WSP wild-type (WSP-WT) tumors. Of 4,138 PCa samples, 3,684 were MSS. Among MSS tumors, 42.4% were from metastatic sites, of which 19.1% were WSP-activated, and 57.6% from the prostate, of which 10.1% were WSP-activated. WSP-activated tumors were more prevalent in metastatic sites than in primary PCa. WSP-activated PCa exhibited more SPOP mutations and higher expression of canonical WSP activators than WSP-WT tumors. ROR1 gene expression was elevated in WSP-activated tumors from both primary and metastatic sites. M2 macrophages predominated the tumor microenvironment in WSP-activated tumors. There was no significant difference in OS between WSP-activated and WSP-WT PCa patients. WSP-activated PCa demonstrated a more immunosuppressed tumor microenvironment and a pronounced upregulation of ROR1 gene expression, underscoring its potential involvement in the crosstalk between canonical and non-canonical Wnt signaling pathways. Implications: Our findings may provide rationale for developing novel therapeutic strategies targeting Wnt-activated PCa.
RESUMO
Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, ß-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis.
Assuntos
Fator XI/metabolismo , Fator XIa/metabolismo , Polifosfatos/farmacologia , Trombina/farmacologia , Ligação Competitiva , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Coagulantes/metabolismo , Coagulantes/farmacologia , Sinergismo Farmacológico , Hemostasia/efeitos dos fármacos , Humanos , Ativação Plaquetária , Polifosfatos/metabolismo , Ligação Proteica , Ressonância de Plasmônio de Superfície , Trombina/metabolismoRESUMO
Polyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (≥ 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (â¼ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required ≥ 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Tamanho da Partícula , Polímeros/química , Polímeros/farmacologia , Polifosfatos/química , Polifosfatos/farmacologia , Anticoagulantes/farmacologia , Fracionamento Químico , Fator Xa/metabolismo , Fibrina/metabolismo , Humanos , Lipoproteínas/farmacologia , Espectroscopia de Ressonância Magnética , Nefelometria e TurbidimetriaRESUMO
Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using (31)P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate result in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores, and immobilized onto solid supports, that immobilized polyphosphate can be readily used to quantify protein binding affinities, that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting, and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.
Assuntos
Amidas/química , Coagulação Sanguínea , Ácidos Fosfóricos/química , Polifosfatos/química , Polifosfatos/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Biotina/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Inibidores Enzimáticos/química , Fator VIIa/metabolismo , Fator XIa/metabolismo , Humanos , Calicreínas/metabolismo , Modelos Moleculares , Polifosfatos/farmacologia , Espermidina/metabolismo , Ressonância de Plasmônio de Superfície , Trombina/metabolismoRESUMO
PURPOSE: Unfractionated heparin reduces metastasis in many murine models. Multiple mechanisms are proposed, particularly anticoagulation and/or inhibition of P-selectin and L-selectin. However, the doses used are not clinically tolerable and other heparins are now commonly used. We studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. EXPERIMENTAL DESIGN: Five clinically approved heparins were evaluated for inhibition of P-selectin and L-selectin binding to carcinoma cells. Pharmacokinetic studies determined optimal dosing for clinically relevant anticoagulant levels in mice. Experimental metastasis assays using carcinoma and melanoma cells investigated effects of a single injection of various heparins. Heparins were compared for structural relationships to selectin inhibition. RESULTS: One (Tinzaparin) of three low molecular weight heparins showed increased selectin inhibitory activity, and the synthetic pentasaccharide, Fondaparinux, showed none when normalized to anticoagulant activity. Experimental metastasis models showed attenuation with unfractionated heparin and Tinzaparin, but not Fondaparinux, at clinically relevant anticoagulation levels. Tinzaparin has a small population of high molecular weight fragments not present in other low molecular weight heparins, enriched for selectin inhibitory activity. CONCLUSIONS: Heparin can attenuate metastasis at clinically relevant doses, likely by inhibiting selectins. Equivalent anticoagulation alone with Fondaparinux is ineffective. Clinically approved heparins have differing abilities to inhibit selectins, likely explained by size distribution. It should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. Caution is also raised about the current preference for smaller heparins. Despite equivalent anticoagulation, hitherto unsuspected benefits of selectin inhibition in various clinical circumstances may be unwittingly discarded.
Assuntos
Fibrinolíticos/farmacologia , Heparina/farmacologia , Trombose/tratamento farmacológico , Animais , Anticoagulantes/química , Linhagem Celular Tumoral , Dissacarídeos/química , Fator Xa/química , Fondaparinux , Heparina/química , Heparina de Baixo Peso Molecular/farmacologia , Cinética , Selectina L/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Oligossacarídeos/química , Selectina-P/metabolismo , Polissacarídeos/farmacologia , Ligação Proteica , Selectinas/metabolismo , Antígeno Sialil Lewis X , Temperatura , Fatores de Tempo , TinzaparinaRESUMO
Factor Va enhances the rate of prothrombin activation by factor Xa by four to five orders of magnitude. Production of initiating levels of factor Va from its precursor, factor V, is a critical event early in haemostasis, as factor V exhibits negligible cofactor activity. While thrombin is the most potent physiological back-activator of factor V, the first prothrombinase complexes require a source of factor Va prior to thrombin generation. A recent study by Whelihan et al. (J Thromb Haemost 2010; 8:1532-1539) identified factor XIa as a candidate for the initial thrombin-independent activation of factor V, although this reaction was slow and required relatively high concentrations of factors V and XIa. Activated platelets secrete polyphosphate, which we previously showed to be potently procoagulant. We now report that polyphosphate greatly accelerates factor V activation by factor XIa, and that this is supported by polyphosphate polymers of the size secreted by activated human platelets. This finding provides additional evidence that factor XIa-mediated generation of factor Va may contribute to the initiation of haemostasis.
Assuntos
Plaquetas/efeitos dos fármacos , Fator V/metabolismo , Fator XIa/metabolismo , Polifosfatos/farmacologia , Plaquetas/enzimologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Hemostasia/efeitos dos fármacos , Humanos , Cinética , Peso Molecular , Polifosfatos/sangue , Polifosfatos/química , Ligação ProteicaRESUMO
Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.