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Nejman, Alicja, Irena Kamińska, Izabela Jasińska, Grzegorz Celichowski e Małgorzata Cieślak. "Influence of Low-Pressure RF Plasma Treatment on Aramid Yarns Properties". Molecules 25, n.º 15 (30 de julho de 2020): 3476. http://dx.doi.org/10.3390/molecules25153476.
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The aim of the study was to modify the surface free energy (SFE) of meta- (mAr) and para-aramid (pAr) yarns by their activation in low-pressure air radio frequency (RF) (40 kHz) plasma and assessment of its impact on the properties of the yarns. After 10 and 90 min of activation, the SFE value increased, respectively, by 14% and 37% for mAr, and by 10% and 37% for pAr. The value of the polar component increased, respectively by 22% and 57% for mAr and 20% and 62% for pAr. The value of the dispersion component for mAr and pAr increased respectively by 9% and 25%. The weight loss decreased from 49% to 46% for mAr and 62% to 50% for pAr after 90 min of activation. After 90 min, the specific strength for mAr did not change and for pAr it decreased by 40%. For both yarns, the 10 min activation in plasma is sufficient to prepare their surface for planned nanomodification.
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Antoniak, Silvio, Kohei Tatsumi, Michael Bode, Swetha Vanja, Julie C. Williams e Nigel Mackman. "Protease-Activated Receptor 1 Enhances Poly I:C Induction of the Antiviral Response in Macrophages and Mice". Journal of Innate Immunity 9, n.º 2 (8 de novembro de 2016): 181–92. http://dx.doi.org/10.1159/000450853.
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The coagulation cascade is activated during viral infections as part of the host defense system. Coagulation proteases activate cells by cleavage of protease-activated receptors (PARs). Recently, we reported that the activation of PAR-1 enhanced interferon (IFN)β and CXCL10 expression in cardiac fibroblasts and in the hearts of mice infected with Coxsackievirus B3. In this study, we used the double-stranded RNA mimetic polyinosinic:polycytidylic acid (poly I:C) to induce an antiviral response in macrophages and mice. Activation of PAR-1 enhanced poly I:C induction of IFNβ and CXCL10 expression in the murine macrophage cell line RAW264.7, bone-marrow derived mouse macrophages (BMM) and mouse splenocytes. Next, poly I:C was used to induce a type I IFN innate immune response in the spleen and plasma of wild-type (WT) and PAR-1-/- mice. We found that poly I:C treated PAR-1-/- mice and WT mice given the thrombin inhibitor dabigatran etexilate exhibited significantly less IFNβ and CXCL10 expression in the spleen and plasma than WT mice. These studies suggest that thrombin activation of PAR-1 contributes to the antiviral response in mice.
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Sparkenbaugh, Erica, John H. Griffin, Laurent O. Mosnier e Rafal Pawlinski. "Biased PAR-1 Signaling Regulates Thrombo-Inflammation in a Mouse Model of Sickle Cell Disease". Blood 136, Supplement 1 (5 de novembro de 2020): 16–17. http://dx.doi.org/10.1182/blood-2020-139862.
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Thrombin and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses. Protease activated receptor-1 (PAR-1) is a G-protein coupled receptor (GPCR) activated by proteolytic cleavage of the extracellular N-terminus. PAR-1 cleavage at R41 by thrombin or at R46 by APC creates unique tethered ligands that activate different signaling pathways. On endothelial cells, thrombin employs PAR-1 to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC employs PAR-1 to stabilize endothelial barrier and provides anti-inflammatory and anti-apoptotic activities. These opposing effects of PAR-1 activation are a classic example of "GPCR biased signaling". We have previously demonstrated that PAR-1 deficiency in all hematopoietic cells reduces heme-induced vascular stasis in sickle mice. In contrast it does not affect the elevated plasma levels of IL-6 or thrombin-antithrombin (TAT) complexes observed in sickle cell disease (SCD) mice compared to non-SCD controls during steady state. In this study, we investigated the effects of thrombin- and APC-induced PAR-1 activation on thromboinflammation in the well-characterized Townes mouse model of SCD. Procoagulant and proinflammatory responses were analyzed in control (AA) and sickle (SS) mice at steady state (saline injection) and 5 hours after TNFα (2 µg/kg, i.p.) to mimic the pro-inflammatory milieu that occurs during painful crisis in SCD patients. As previously described, SS mice had elevated plasma levels of TAT compared to AA mice (8.6± 2.4 vs 3.5±0.7ng/mL; p<0.05), that were further enhanced in SS mice by TNFα injection (21.5 ± 4.2 ng/mL, p<0.001). A similar pattern was observed for plasma levels of IL-6 (AA: 2.4±2 vs SS: 18±7.8 ng/mL, p<0.05; SS+TNFα: 231 ± 32 ng/mL, p<0.0001 vs SS). Using these experimental conditions, we first investigated the role of endogenous APC in SCD. We used a rat anti-mouse protein C mAb (SPC-54), which inhibits the active site of APC, thereby blocking all its enzymatic activities, including PAR-1-dependent signaling and anticoagulant actions. SS mice were injected with anti-APC SPC54 (10 mg/kg) or control IgG antibodies. Eighteen hours later both groups of mice were injected with either saline or TNFα and plasma samples were collected 5 hours later. We TNFαSSdata suggest that endogenous APC has anticoagulant and anti-inflammatory roles for both the steady state and after TNFα challenge. To address the individual roles of thrombin and APC signaling on vascular inflammation in SCD, we used mice with point mutations in PAR-1 at R41 or R46. Mutation of R41 to Glu (R41Q) renders PAR-1 insensitive to thrombin while preserving APC-mediated cleavage at R46. Mutation of R46 to Glu (R46Q) ablates APC's cytoprotective effects. Bone marrow (BM) from SS mice was transplanted into PAR-1WT, PAR-1R41Q, or PAR-1R46Q mice to generate SSBM mice with either normal PAR-1 (SSBM/PAR-1WT) or the mutated form of this receptor on all non-hematopoietic cells (SSBM/PAR-1R41Q and SSBM/PAR-1R46Q). Four months after BM transplantation, plasma levels of TAT, IL-6, and HMGB1 were analyzed in all 3 groups at steady state or 5 hours after TNFα challenge. Neither the PAR-1R41Q nor PAR-1R46Q mutation on non-hematopoietic cells affected plasma levels of TAT, IL-6, or HMGB1 in SS mice at steady state. Interestingly, plasma levels of IL-6 were significantly elevated in SSBM/PAR-1R46Q mice and reduced in SSBM/PAR-1R41Q mice compared to SSBM/PAR-1WT mice after TNFα challenge, with a statistically significant difference between the two PAR-1 mutant groups. Furthermore plasma levels of TAT and HMGB1 were significantly reduced in SSBM/PAR-1R41Q but not changed in SSBM/PAR-1R46Q mice after TNFα challenge (Figure 2A-C). These data suggest that PAR-1 activation at R41 regulates TAT, IL-6, and HMGB1, whereas activation of R46 negatively regulates IL-6 in sickle mice during TNFα challenge. Our data imply that pharmacological approaches aiming to eliminate thrombin-dependent, while preserving APC-dependent PAR-1 activation, may be effective in attenuating thrombo-inflamatory complications associated with SCD. Studies evaluating the effects of R41Q and R46Q mutations on microvascular stasis in SS mice are currently ongoing. Disclosures No relevant conflicts of interest to declare.
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Chantrathammachart, Pichika, Erica M. Sparkenbaugh, Nigel Mackman, Nigel S. Key e Rafal Pawlinski. "Protease Activated Receptor 2 (PAR-2) Promotes Vascular Inflammation in a Mouse Model of Sickle Cell Disease". Blood 120, n.º 21 (16 de novembro de 2012): 375. http://dx.doi.org/10.1182/blood.v120.21.375.375.
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Abstract Abstract 375 Sickle cell disease (SCD) is a hematologic disorder caused by a single nucleotide mutation of the beta-globin gene. It is associated with increased tissue factor (TF) expression, activation of coagulation and chronic vascular inflammation. Using two mouse models of SCD (BERK and Townes mice), we have recently demonstrated that inhibition of TF with a rat anti-mouse TF (1H1) antibody not only abolishes activation of coagulation (measured by plasma levels of thrombin anti-thrombin (TAT) complexes) but also reduces inflammation and endothelial cell (EC) injury, indicated by attenuation of plasma levels of IL-6 and sVCAM-1, respectively. Furthermore, we showed that EC-specific deletion of TF gene significantly reduced plasma levels of IL-6 but had no effect on activation of coagulation (TAT) or EC injury (sVCAM-1). These data suggest that EC-TF is primarily involved in signaling rather than activation of coagulation. Since TF:factor VIIa complex-dependent activation of protease activated receptor-2 (PAR-2) has been shown to promote inflammation, we have now investigated the role of PAR-2 expressed by non-hematopoietic cells in the pathology of SCD. PAR-2+/+ and PAR-2−/− mice were lethally irradiated and transplanted with bone marrow from BERK SS (sickle cell mice) or BERK AA (non-sickle control) mice(n=6–10). Four months after bone marrow transplantation, mice were sacrificed and the reconstitution of bone marrow was confirmed by electrophoretic analysis of the different forms of hemoglobin. PAR-2+/+ mice transplanted with bone marrow from BERK SS mice had reduced number of red blood cells and hematocrit compared to PAR-2+/+ mice transplanted with bone marrow from BERK AA mice. PAR-2 deficiency in all non-hematopoietic cells had no effect on these hematologic parameters. Furthermore, PAR-2+/+ mice transplanted with bone marrow from BERK SS mice had increased number of monocytes (3.1 fold, p<0.0001) and neutrophils (2.5 fold, p<0.05) in the blood. Interestingly, sickle cell mice lacking PAR-2 in non-hematopoietic cells had significantly reduced neutrophil counts compared to the sickle cell mice with normal levels of PAR-2 (1.9+/−0.2 vs. 5.4+/−1.4 X103/ul; p<0.05), whereas monocytes counts were not affected. Compared to non-sickle controls, sickle cell mice had increased plasma levels of TAT (1.9 fold, p<0.01), IL-6 (6.8 fold, P<0.0001), serum amyloid protein SAP (6.5 fold, p<0.01; mouse homolog of human C reactive protein) and sVCAM-1 (1.4 fold, p<0.01). Moreover, increased levels of myeloperoxidase (MPO) were observed in the livers of sickle cell mice (3 fold, p<0.0001). Importantly, sickle cell mice lacking PAR-2 expression in all non-hematopoietic cells demonstrated significant reduction of plasma levels of IL-6 (9.4+/−0.9 vs. 18.9+/−4.5 pg/ml; p<0.05) and SAP (60.5+/−12.9 vs. 182.8+/−62.5ug/ml; p<0.05) compared to sickle cell mice with normal levels of PAR2 expression. In addition, deletion of PAR-2 also significantly reduced MPO levels in the liver of sickle cell mice (53.7+/−3.5 vs. 117.4+/−16.9 ng/mg protein; p<0.0001). In contrast, PAR-2 deficiency in non-hematopoietic cells had no effect on activation of coagulation (TAT) or EC injury (sVCAM-1) in sickle mice. Our data demonstrate that vascular inflammation observed in a mouse model of sickle cell disease is mediated, in part, by PAR-2 expressed by non-hematopoietic cells. Activation of EC (sVCAM-1) was not affected by PAR-2 deficiency. Ongoing studies are investigating the possible contribution of the TF-thrombin-PAR1 pathway to the EC activation in SCD. Disclosures: No relevant conflicts of interest to declare.
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Tatsumi, Kohei, Silvio Antoniak e Nigel Mackman. "Role of the Thrombin-PAR-1 Pathway in Coxsackievirus Induced Hepatitis". Blood 124, n.º 21 (6 de dezembro de 2014): 1470. http://dx.doi.org/10.1182/blood.v124.21.1470.1470.
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Abstract Objective: Coxsackievirus B3 (CVB3) can infect different tissues including the heart and liver. Recently, we found that activation of the coagulation cascade and protease-activated receptor 1 (PAR-1) enhances toll-like receptor-3 (TLR3) mediated interferon-β (IFN-β) expression and protects mice from CVB3-induced myocarditis. Here, we investigated the role of PAR-1 in early anti-viral responses in mice and isolated hepatocytes. Methods: Wild-type (WT) and PAR-1 deficient (PAR-1-/-) mice were infected with CVB3 intraperitoneally. The innate immune response, viral load, liver enzyme plasma levels, and inflammation levels were analyzed. Bone-marrow transplantation experiments with the combination of WT mice PAR-1-/- mice were performed to identify the cellular source of PAR-1 contributing to the innate immune response to CVB3. We also analyzed the effect of the direct thrombin inhibition with dabigatran etexilate on CVB3 hepatitis. In addition, we analyzed the effect of PAR-1 activation on TLR3-dependent interferon (IFN)-β expression in primary mouse hepatocytes and the human hepatocyte cell line PH5CH8 in vitro. Results: PAR-1-/- mice exhibited a reduced early innate immune response in the liver at day 4 after infection, which was associated at later times (day 8) to higher viral titers in the liver, increased alanine transaminase plasma levels and more remarkable inflammation compared to control WT mice. Bone marrow transplantation experiments demonstrated that PAR-1 on non-hematopoietic played the major role in the innate immune response of CVB3 hepatitis. Stimulation of PAR-1 with either thrombin or agonist peptide on primary mouse hepatocytes and human PH5CH8 cells in vitro enhanced the antiviral response to dsRNA by increasing IFN-β and C-X-C motif chemokine 10 (CXCL10) expressions, supporting the results of in vivo experiments. Conclusion: Our results suggest that activation of PAR-1 on hepatocytes enhances the innate immune response to CVB3 in the liver. Disclosures No relevant conflicts of interest to declare.
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McRedmond, James P., Patrick Harriott, Brian Walker e Desmond J. Fitzgerald. "Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1". Blood 95, n.º 4 (15 de fevereiro de 2000): 1301–8. http://dx.doi.org/10.1182/blood.v95.4.1301.004k24_1301_1308.
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Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A2production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S741A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1.
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Tholanikunnel, Baby, Berhane Ghebrehiwet, Allen Kaplan e Kusumam Joseph. "Interaction of high molecular weight kininogen binding proteins on endothelial cells". Thrombosis and Haemostasis 91, n.º 01 (2004): 61–70. http://dx.doi.org/10.1160/th03-07-0471.
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SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.
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Azim, A. C., K. Barkalow, J. Chou e J. H. Hartwig. "Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor". Blood 95, n.º 3 (1 de fevereiro de 2000): 959–64. http://dx.doi.org/10.1182/blood.v95.3.959.003k22_959_964.
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Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1–mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.
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Tatour, Mifleh, Ma'anit Shapira, Elena Axelman, Shourouk Ghanem, Anat Keren-Politansky, Lilach Bonstein, Benjamin Brenner e Yona Nadir. "Thrombin is a selective inducer of heparanase release from platelets and granulocytes via protease-activated receptor-1". Thrombosis and Haemostasis 117, n.º 07 (2017): 1391–401. http://dx.doi.org/10.1160/th16-10-0766.
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SummaryHeparanase, known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of factor Xa. Platelets and granulocytes contain abundant amounts of heparanase that may enhance the coagulation system upon discharge. It was the aim of this study to identify the inducer and pathway of heparanase release from these cells. Platelets and granulocytes were purified from pooled normal plasma and were incubated with ATP, ADP, epinephrine, collagen, ristocetin, arachidonic acid, serotonin, LPS and thrombin. Heparanase levels were assessed by ELISA, heparanase procoagulant activity assay and western blot analysis. The effects of selective protease-activated receptor (PAR)-1 and 2 inhibitors and PAR-1 and 4 activators were studied. An in-house synthesised inhibitory peptide to heparanase was used to evaluate platelet heparanase involvement in activation of the coagulation system. Heparanase was released from platelets only by thrombin induction while other inducers exerted no such effect. The heparanase level in a platelet was found to be 40 % higher than in a granulocyte. Heparanase released from platelets or granulocytes increased factor Xa generation by three-fold. PAR-1 activation via ERK intracellular pathway was found to induce heparanase release. In conclusion, heparanase is selectively released from platelets and granulocytes by thrombin interacting with PAR-1. Heparanase derived from platelets and granulocytes is involved in activation of the extrinsic coagulation pathway. The present study implies on a potential anticoagulant effect, in addition to anti-platelet effect, of the new clinically studied PAR-1 inhibitors.
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Antoniak, Silvio, Kohei Tatsumi e Nigel Mackman. "The Tissue Factor/Thrombin/Protease-Activated Receptor 1 Pathway Enhances Double-Strand RNA Induced Immune Responses in Macrophages". Blood 124, n.º 21 (6 de dezembro de 2014): 4114. http://dx.doi.org/10.1182/blood.v124.21.4114.4114.
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Abstract Introduction: Co-regulation of the immune response and the coagulation cascade after infection is thought to be an ancient response to limit pathogen spread. Recently, we showed that activation of the thrombin receptor, protease-activated receptor 1 (PAR1), on fibroblasts enhanced the innate immune responses to RNA virus infection. Here, we investigated whether PAR1 activation by the extrinsic coagulation pathway contributes to dsRNA-induced innate immune responses in macrophages. Methods: Activation of the type-I interferon (IFN) pathway in the murine macrophage cell line RAW264.7 and bone-marrow derived macrophages (BMDM) from WT and PAR1-/- was analyzed after dsRNA (poly I:C) and/or PAR-1 stimulation. In addition, innate immune responses in the spleen were analyzed in vivo 4 hours after poly I:C (8mg/kg) injection in mice with reduced tissue factor expression (LowTF) or global PAR1 deletion (PAR1-/-) as well as in WT mice with a thrombin inhibitor (dabigatran etexilate, 10g/kg chow) or PAR-1 inhibitor (SCH79797, 25μg/kg). Lastly, we investigated the innate immune response in the spleen of WT and PAR1-/-mice after infection with the single-stranded RNA virus coxsackievirus B3. Results: RAW264.7 and BMDM exhibited a toll-like receptor 3 dependent induction of IFNβ and CXCL10 after poly I:C stimulation. Activation of PAR-1 with either thrombin or agonist peptide enhanced poly I:C induction of IFNβ and CXCL10. A deficiency of tissue factor levels, thrombin inhibition, PAR-1 inhibition or PAR1 deficiency resulted in reduced expression levels of type-I IFNs and IFN-response genes such as CXCL10 in the spleen and plasma in mice given poly I:C. Last, PAR1-/-mice exhibited impaired IFNβ immune response 4 days after coxsackievirus B3 infection compared to WT mice. Conclusion: Our study indicates that the coagulation dependent activation of PAR1 on macrophages is important for anti-viral responses to dsRNA. We speculate that PAR1 inhibition may interfere with anti-viral responses in humans. Disclosures No relevant conflicts of interest to declare.
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Didiasova, Miroslava, Sebastian Berscheid, Katarzyna Piskulak, Brigitte Taborski, Dariusz Zakrzewicz, Grazyna Kwapiszewska, Malgorzata Wygrecka, Klaus Preissner e Philipp Markart. "Protease-activated receptors (PAR)-1 and -3 drive epithelial-mesenchymal transition of alveolar epithelial cells – potential role in lung fibrosis". Thrombosis and Haemostasis 110, n.º 08 (2013): 295–307. http://dx.doi.org/10.1160/th12-11-0854.
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SummaryExtravascular activation of the coagulation cascade in the lung is commonly observed in pulmonary fibrosis. Coagulation proteases may exert profibrotic cellular effects via protease-activated receptors (PARs)-1 and -2. Here, we investigated the potential role of two other members of the PAR family, namely PAR-3 and PAR-4, in the pathobiology of lung fibrosis. Elevated expression of PAR-3, but not PAR-4, was detected in the lungs of idiopathic pulmonary fibrosis (IPF) patients and in bleomycin-induced lung fibrosis in mice. Increased PAR-3 expression in fibrotic lungs was mainly attributable to alveolar type II (ATII) cells. Stimulation of primary mouse ATII, MLE15 and A549 cells with thrombin (FIIa) – that may activate PAR-1, PAR-3 and PAR-4 – induced epithelial-mesenchymal transition (EMT), a process that has been suggested to be a possible mechanism underlying the expanded (myo)fibroblast pool in lung fibrosis. EMT was evidenced by morphological alterations, expression changes of epithelial and mesenchymal phenotype markers, and functional changes. Single knockdown of FIIa receptors, PAR-1, PAR-3, or PAR-4, had no major impact on FIIa-induced EMT. Simultaneous depletion of PAR-1 and PAR-3, however, almost completely inhibited this process, whereas only a partial effect on FIIa-mediated EMT was observed when PAR-1 and PAR-4, or PAR-3 and PAR-4 were knocked down. PAR-1 and PAR-3 co-localise within ATII cells with both being predominantely plasma membrane associated. In conclusion, our study indicates that PARs synergise to mediate FIIa-induced EMT and provides first evidence that PAR-3 via its ability to potentiate FIIa-triggered EMT could potentially contribute to the pathogenesis of pulmonary fibrosis.
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May, Carl J., Gavin I. Welsh, Musleeha Chesor, Phillipa J. Lait, Lauren P. Schewitz-Bowers, Richard W. J. Lee e Moin A. Saleem. "Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation". American Journal of Physiology-Renal Physiology 317, n.º 4 (1 de outubro de 2019): F913—F921. http://dx.doi.org/10.1152/ajprenal.00093.2019.
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The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.
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Mashimo, Masato, Akane Morozumi, Akari Nobeyama, Misato Kanzaki, Shigeru Negi, Jiro Kato, Joel Moss, Atsuo Nomura e Takeshi Fujii. "Poly(ADP-ribose) Polymerase 1 Mediates Rab5 Inactivation after DNA Damage". International Journal of Molecular Sciences 23, n.º 14 (15 de julho de 2022): 7827. http://dx.doi.org/10.3390/ijms23147827.
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Parthanatos is programmed cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1) after DNA damage. PARP1 acts by catalyzing the transfer of poly(ADP-ribose) (PAR) polymers to various nuclear proteins. PAR is subsequently cleaved, generating protein-free PAR polymers, which are translocated to the cytoplasm where they associate with cytoplasmic and mitochondrial proteins, altering their functions and leading to cell death. Proteomic studies revealed that several proteins involved in endocytosis bind PAR after PARP1 activation, suggesting endocytosis may be affected by the parthanatos process. Endocytosis is a mechanism for cellular uptake of membrane-impermeant nutrients. Rab5, a small G-protein, is associated with the plasma membrane and early endosomes. Once activated by binding GTP, Rab5 recruits its effectors to early endosomes and regulates their fusion. Here, we report that after DNA damage, PARP1-generated PAR binds to Rab5, suppressing its activity. As a result, Rab5 is dissociated from endosomal vesicles, inhibiting the uptake of membrane-impermeant nutrients. This PARP1-dependent inhibition of nutrient uptake leads to cell starvation and death. It thus appears that this mechanism may represent a novel parthanatos pathway.
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Sharma, Ruchika, Amanda P. Waller, Adam Guess, Shipra Agrawal, Berend Isermann, William E. Smoyer, Marvin T. Nieman e Bryce A. Kerlin. "Thrombin Induces Apoptosis in Human and Rat Podocytes in a Protease Activated Receptor (PAR)-Dependent Manner". Blood 124, n.º 21 (6 de dezembro de 2014): 2808. http://dx.doi.org/10.1182/blood.v124.21.2808.2808.
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Abstract INTRODUCTION Nephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes. There is evidence to indicate that thrombin generation is enhanced in nephrotic syndrome. The massive protein loss in nephrotic syndrome includes loss of key coagulation regulators, leading to a complex coagulopathy, enhanced thrombin activating capacity and, consequently, increased risk for devastating thromboembolic complications. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner. METHODS Experiments were performed with differentiated, conditionally immortalized human and rat podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. In human podocytes, specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. We have established that human thrombin is hemostatically active and regulated in rat plasma. Thus, rat podocytes were exposed to human thrombin, to determine toxicity, and specific PAR antibodies to determine if blockade of PARs could ameliorate thrombin mediated toxicity. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM). RESULTS Thrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p<0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p<0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p<0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p<0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. Figure 1 Figure 1. Figure 2 Figure 2. In rat podocytes human thrombin had a similar effect with increased apoptosis from 1% to 33.6% (p<0.05). We also demonstrated that this may be a PAR-mediated mechanism as blockade of PAR-1 and PAR-4 with specific antibodies ameliorated thrombin induced apoptosis [1.94% with PAR-1 ab and 3.5% with PAR-4ab] while blockade of PAR-2 and PAR-3 did not have a similar effect (p<0.05). Figure 3 Figure 3. CONCLUSIONS Thrombin-induced injury is mediated in a PAR-dependent fashion in both humans and rats. Specifically, in this in vitro model, thrombin induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner in human podocytes but in a PAR-1/-4 manner in rat podocytes. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on PAR heterodimerization. Our findings collectively suggest that interrupting thrombin-mediated podocyte injury may offer a novel future therapeutic approach to reduce podocyte injury in nephrotic syndrome. Disclosures No relevant conflicts of interest to declare.
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Bock, Ashley, Marguerite Kelher, Samina Khan, Kirk Hansen, Monika Dzieciatkowska e Christopher C. Silliman. "Thrombin Activation of Protease-Activated Receptor-2 on Neutrophils Primes the Respiratory Burst." Blood 116, n.º 21 (19 de novembro de 2010): 1119. http://dx.doi.org/10.1182/blood.v116.21.1119.1119.
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Abstract Abstract 1119 Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF. Disclosures: No relevant conflicts of interest to declare.
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Tarandovskiy, Ivan D., Paul W. Buehler e Elena Karnaukhova. "C1-inhibitor influence on platelet activation by thrombin receptors agonists". Clinical and Applied Thrombosis/Hemostasis 28 (janeiro de 2022): 107602962211204. http://dx.doi.org/10.1177/10760296221120422.
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Introduction Protease activated receptors 1 (PAR1) and 4 (PAR4) agonists are used to study platelet activation. Data on platelet activation are extrapolated across experimental settings. C1-inhibitor (C1INH) is a protease inhibitor present in plasma but not in isolated platelet suspensions. Here we show that C1INH affects platelet activation through PAR1 and PAR4 agonists. Methods Platelets were isolated from healthy donor whole blood and then labeled with anti-CD62P and PAC1 antibodies. The platelet suspensions were exposed to PAR1 agonists SFLLRN, TFLLR and TFLLRN; PAR4 agonists AYPGKF and GYPGQV; ADP and thrombin. Flow-cytometric measurements were performed in 5, 10 and 15 min after activation. Results 0.25 mg/ml C1INH addition made platelets to faster expose CD62P and glycoprotein IIb/IIIa complex after activation with PAR1 agonists. Conversely, C1INH addition led to inhibition of platelet activation with PAR4 agonists and thrombin. Activation with ADP was not affected by C1INH. Conclusions Our results suggest that C1INH can modify platelet activation in the presence of synthetic PAR agonists used in platelet research. These observations may be relevant to the development of new methods to assess platelet function.
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Ploug, M., T. Plesner, E. Ronne, V. Ellis, G. Hoyer-Hansen, NE Hansen e K. Dano. "The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria". Blood 79, n.º 6 (15 de março de 1992): 1447–55. http://dx.doi.org/10.1182/blood.v79.6.1447.1447.
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Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.
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Ploug, M., T. Plesner, E. Ronne, V. Ellis, G. Hoyer-Hansen, NE Hansen e K. Dano. "The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria". Blood 79, n.º 6 (15 de março de 1992): 1447–55. http://dx.doi.org/10.1182/blood.v79.6.1447.bloodjournal7961447.
Texto completo da fonteResumo:
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.
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De Simone, Ilaria, Constance C. F. M. J. Baaten, Martine Jandrot-Perrus, Jonathan M. Gibbins, Hugo ten Cate, Johan W. M. Heemskerk, Chris I. Jones e Paola E. J. van der Meijden. "Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1". International Journal of Molecular Sciences 23, n.º 18 (6 de setembro de 2022): 10203. http://dx.doi.org/10.3390/ijms231810203.
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Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbβ3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.
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Nieman, Marvin T. "Protease-activated receptors in hemostasis". Blood 128, n.º 2 (14 de julho de 2016): 169–77. http://dx.doi.org/10.1182/blood-2015-11-636472.
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Abstract Protease signaling in cells elicits multiple physiologically important responses via protease-activated receptors (PARs). There are 4 members of this family of G-protein–coupled receptors (PAR1-4). PARs are activated by proteolysis of the N terminus to reveal a tethered ligand. The rate-limiting step of PAR signaling is determined by the efficiency of proteolysis of the N terminus, which is regulated by allosteric binding sites, cofactors, membrane localization, and receptor dimerization. This ultimately controls the initiation of PAR signaling. In addition, these factors also control the cellular response by directing signaling toward G-protein or β-arrestin pathways. PAR1 signaling on endothelial cells is controlled by the activating protease and heterodimerization with PAR2 or PAR3. As a consequence, the genetic and epigenetic control of PARs and their cofactors in physiologic and pathophysiologic conditions have the potential to influence cellular behavior. Recent studies have uncovered polymorphisms that result in PAR4 sequence variants with altered reactivity that interact to influence platelet response. This further demonstrates how interactions within the plasma membrane can control the physiological output. Understanding the structural rearrangement following PAR activation and how PARs are allosterically controlled within the plasma membrane will determine how best to target this family of receptors therapeutically. The purpose of this article is to review how signaling from PARs is influenced by alternative cleavage sites and the physical interactions within the membrane. Going forward, it will be important to relate the altered signaling to the molecular arrangement of PARs in the cell membrane and to determine how these may be influenced genetically.
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Cheepala, Satish B., Kazumasa Takenaka, Tamara I. Pestina, Carl W. Jackson e John D. Schuetz. "The Role of ABC Transporter Abcc4 in Platelets Physiologic Function and Its Impact On Collagen Meditated Platelet Aggregation". Blood 120, n.º 21 (16 de novembro de 2012): 1063. http://dx.doi.org/10.1182/blood.v120.21.1063.1063.
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Abstract Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.
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Nemmar, Abderrahim, e Marc F. Hoylaerts. "Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization". International Journal of Molecular Sciences 23, n.º 2 (11 de janeiro de 2022): 744. http://dx.doi.org/10.3390/ijms23020744.
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Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05–0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.
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Petzold, Tobias, Manuela Thienel, Lisa Dannenberg, Philipp Mourikis, Carolin Helten, Aysel Ayhan, René M’Pembele et al. "Rivaroxaban Reduces Arterial Thrombosis by Inhibition of FXa-Driven Platelet Activation via Protease Activated Receptor-1". Circulation Research 126, n.º 4 (14 de fevereiro de 2020): 486–500. http://dx.doi.org/10.1161/circresaha.119.315099.
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Rationale: A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown. Objective: In this study, we hypothesized that rivaroxaban’s antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis. Methods and Results: In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban’s antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban’s anticoagulatory capacity. Conclusions: Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.
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Badolia, Rachit. "PAK and Akt Interactions Regulate PAR-Mediated Akt Translocation to Membrane in Platelets: A Novel PIP3-Independent Mechanism." Blood 120, n.º 21 (16 de novembro de 2012): 2166. http://dx.doi.org/10.1182/blood.v120.21.2166.2166.
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Abstract Abstract 2166 Akt has been shown to be an important signaling molecule regulating platelet aggregation. It was proposed that Akt translocates to the membrane for its activation where it is phosphorylated on Thr308 by PDK1 and Ser473 by mTORC2. However, the specific signaling events that regulate the Akt phosphorylation and its mechanism of translocation to the membrane are not clearly defined. It has been shown that Akt phosphorylation by many agonists requires signaling events downstream of Gi activation, involving PI3-kinaseb. In addition, it was proposed that Akt translocates to the plasma membrane by binding to PIP3 (Phosphatidylinositol-3,4,5-trisphosphate) generated by PI3-kinase. Our previous studies have revealed that Gq and G12/13 pathways enhance Akt phosphorylation in platelets, suggesting a role for these pathways independently of PIP3. Hence we investigated the mechanisms of Akt translocation in platelets. Stimulation of platelets with the PAR4 activation peptide (AYPGKF) caused translocation of Akt to the membrane rapidly (as early as 20 seconds) whereas, the phosphorylation of Akt occurred at the later time points (Figure 1). Pan-phoshatidylinositide 3-kinase (PI3-Kinase) inhibitors, LY-294002 (Figure 2) and Wortmannin inhibited Ser473 phosphorylation but failed to affect translocation of Akt to the membrane indicating that Akt translocation can occur independent of PI3-kinase activity. However, translocation of Akt was dramatically inhibited in the presence of a Gq-selective inhibitor, YM254890, indicating that Akt translocation is regulated downstream of Gqsignaling pathways (Figure 3). The p21-activated kinases (PAKs) are a family of serine/threonine kinases and are key regulators of actin polymerization and cell migration. PAK functions as a scaffolding protein and is reported to interact with numerous proteins including Akt. Here, we evaluated whether PAK activated by the Gqpathways plays a significant role in the translocation of Akt to the membrane. The co-immunoprecipitation studies revealed that Akt associates with PAK upon stimulation with AYPGKF (Figure 4). This association was abolished when platelets were pre-treated with YM254890 (Figure 5). These results suggest that PAK1/2 activation downstream of Gq pathway mediates Akt translocation to the membrane when stimulated by PAR agonist in platelets and that this translocation is PIP3- independent. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Jianying, Daibang Nie, Kelly Williamson, Jorge L. Rocha, MaCalus V. Hogan e James H.-C. Wang. "Selectively activated PRP exerts differential effects on tendon stem/progenitor cells and tendon healing". Journal of Tissue Engineering 10 (janeiro de 2019): 204173141882003. http://dx.doi.org/10.1177/2041731418820034.
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To understand the variable efficacy with platelet rich plasma (PRP) treatments for tendon injury, we determined the differential effects of proteinase-activated receptor (PAR)1- or PAR4-activated PRP (PAR1-PRP, PAR4-PRP) from humans on human patellar tendon stem/progenitor cells (TSCs) and tendon healing. We show that PAR1-PRP released VEGF, whereas PAR4-PRP released endostatin. Treatment of TSCs with PAR1-PRP increased collagen I expression and matrix metalloproteinase-1 (MMP-1), but cells treated with PAR4-PRP increased less collagen I and higher MMP-2 expression. The wound area treated with PAR4-PRP formed tendon-like tissues with well-organized collagen fibers and fewer blood vessels, while PAR1-PRP treatment resulted in the formation of blood vessels and unhealed tissues. These findings indicate that differential activation of PRP leads to different effects on TSCs and tendon healing. We suggest that based on acute or chronic type of tendon injury, selective activation of PRP should be applied in clinics in order to treat injured tendons successfully.
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Tucker, Nicole, Monika Dzieciatkowska, Kirk Hansen, Samina Khan, Marguerite Kelher, Anirban Banerjee, Ernest Moore e Christopher C. Silliman. "α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity". Blood 116, n.º 21 (19 de novembro de 2010): 3355. http://dx.doi.org/10.1182/blood.v116.21.3355.3355.
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Abstract Abstract 3355 A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods: Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results: HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.
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Hudák, Renáta, János Vincze, László Csernoch, Ildikó Beke Debreceni, Tamás Oláh, Ferenc Erdődi, Kenneth J. Clemetson e János Kappelmayer. "The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation". BioMed Research International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/9795271.
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The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.
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Ramström, Sofia, Maria Bjerke, Tomas Lindahl e Karin Vretenbrant. "Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development". Thrombosis and Haemostasis 97, n.º 03 (2007): 417–24. http://dx.doi.org/10.1160/th06-07-0397.
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SummaryThrombin is a pivotal enzyme formed in the coagulation cascade and an important and potent platelet activator. The two pro-tease-activated thrombin receptors on human platelets are denoted PARI and PAR4. The physiological relevance of PAR4 is still unclear, as both aggregation and secretion can be accomplished by PARI activation alone. In the present study we have investigated the role of PARs in platelet activation, blood coagulation, clot elasticity and fibrinolysis. Flow cytometry, free oscillation rheometry and thrombin generation measurements were used to analyze blood or platelet-rich plasma from healthy individuals. Maximum PARI activation with the peptide SFLLRN gave fewer fibrinogen-binding platelets with lower mean fluorescent intensity than maximum PAR4 activation with AYPGKF. Inhibition of any of the receptors prolonged clotting times. However, PARI is more important for fibrinolysis; inhibition of this receptor prolonged all the steps in the fibrinolytic process. Clot elasticity decreased significantly when the PAR4 receptor was inhibited. In the thrombin generation measurements, PAR4 inhibition delayed the thrombin generation start and peak, but did not affect the total amount of thrombin generated. PAR I inhibition had no significant impact on thrombin generation. We found that PAR4 is most likely activated by low concentrations of thrombin during the initial phase of thrombin generation and is of importance to the clotting time. Furthermore, we suggest that the PAR4 receptor may have a physiological role in the stabilisation of the coagulum.
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Preston, Roger JS, Jennifer A. Johnson, Fionnuala Ni Ainle, Shona Harmon, Owen P. Smith, Barry White e James S. O’Donnell. "Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement". Blood 112, n.º 11 (16 de novembro de 2008): 20. http://dx.doi.org/10.1182/blood.v112.11.20.20.
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Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.
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Haubold, Katja, Michael Rink, Brigitte Spath, Ali Amirkhosravi, John L. Francis, Barbara Eifrig, Carsten Bokemeyer e Florian Langer. "Microparticle-Associated Tissue Factor: A Molecular Link Between Coagulation Activation, Inflammation and Disease Progression in Early-Stage Prostate Cancer?" Blood 112, n.º 11 (16 de novembro de 2008): 3813. http://dx.doi.org/10.1182/blood.v112.11.3813.3813.
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Abstract Activation of coagulation and inflammation is a characteristic finding in patients with advanced malignancies, including prostate cancer. Tissue factor (TF), a molecule involved in hemostasis, thrombosis and pro-inflammatory signaling pathways, is over-expressed on tumor cells and cells of the tumor microenvironment (i.e. endothelial cells, fibroblasts and tissue macrophages). Moreover, the enhanced release of TF into plasma in association with sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently been associated with key events in molecular oncogenesis and cancer progression. In this study, we measured TF-specific procoagulant activity (PCA) of plasma MPs in 58 consecutive patients with clinically localized prostate cancer (mean age, 64±5 years) to explore its potential as a prognostic marker in this tumor entity. MPs were isolated from pre-operative plasma samples by sequential high-speed centrifugation for 1 h at 16,100 × g. TF-specific PCA of plasma MPs was quantified using a highly sensitive one-stage clotting assay in the presence and absence of inhibitory TF monoclonal antibody and calibration of clotting times against serial dilutions (1:10–1:105) of lipidated recombinant human full-length TF (rhTF1–263), showing a linear correlation in a log-log plot with R2>0.99. The lower detection limit of this assay for rhTF1–263 (33 kDa) was <5 pg/ml (<150 fM), and the intra- and inter-assay coefficients of variation were 7.3% and 5.4%, respectively. Total numbers of TF-positive MPs were measured by single-color flow cytometry using PE-conjugated TF monoclonal antibody (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization. TF antigen was quantified in plasma by ELISA. Calibrated automated thrombography (CAT) was used to monitor thrombin generation in platelet-free plasma samples over a 2-h period without the addition of exogenous TF or phospholipids. Intravascular coagulation activation was assessed by measuring plasma D-dimer. All assay systems were validated using MPs spontaneously shed from prostate cancer cell lines (PC-3, LNCaP and DU145) or from whole blood monocytes after challenge with endotoxin. Based on plasma fibrinogen and C-reactive protein levels, patients were stratified into those with (n=26) and those without (n=32) laboratory evidence of an acute-phase reaction (APR). Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer (0.46±0.19 vs. 0.21±0.05 mg/l) and TF-specific PCA of plasma MPs (563±301 vs. 292±74 U/ml) (P<0.001). Among patients, laboratory evidence of an APR was associated with a significant increase in MP-associated TF PCA (699±351 vs. 452±196 U/ml) (P=0.001). Overall, we found a significant correlation between MP-associated TF PCA and plasma D-Dimer (P=0.015), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. CAT also revealed significantly increased thrombin generation in patient compared to control plasmas, as indicated by a shortening in lag phase (25±4 vs. 29±5 min) and an increase in both peak thrombin generation (184±76 vs. 127±71 nM) and the endogenous thrombin potential, defined as the area under the thrombin generation curve (3576±509 vs. 2980±562 nM*min) (P<0.01). Importantly, TF-specific PCA of plasma MPs correlated neither with absolute numbers of TF-positive MPs nor with plasma TF antigen, suggesting that a substantial and variable fraction of the total plasma TF pool circulated as an inactive variant. Interestingly, systemic levels of IL-8, an inflammatory cytokine involved in TF/FVIIa-dependent, PAR-2-mediated pro-migratory signaling pathways in tumor cells and shown to be of biological relevance in advanced, hormone-refractory prostate cancer, were elevated in patients compared to controls (9±11 vs. 4±6 pg/ml) (P<0.01). In summary, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent an important molecular link between hypercoagulability, inflammation and disease progression. The above-described assay for the quantification of MP-associated TF PCA could thus be of prognostic value in the risk stratification of patients with localized prostate cancer with respect to thromboembolic complications and/or tumor recurrence.
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Schuliga, Michael, Jade Jaffar, Asres Berhan, Shenna Langenbach, Trudi Harris, David Waters, Peter V. S. Lee et al. "Annexin A2 contributes to lung injury and fibrosis by augmenting factor Xa fibrogenic activity". American Journal of Physiology-Lung Cellular and Molecular Physiology 312, n.º 5 (1 de maio de 2017): L772—L782. http://dx.doi.org/10.1152/ajplung.00553.2016.
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In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease-activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including those associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% vol/vol) or purified FXa (15–50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270 and 46%, respectively ( P < 0.05, n = 5–8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 μM, or antibodies raised against annexin A2 or PAR-1 (2 μg/ml). FXa-stimulated LFs from IPF patients ( n = 6) produced twice as much IL-6 as controls ( n = 10) ( P < 0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (* P < 0.05; n = 4–12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion, but lung levels of collagen were not. Our data suggest that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF.
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Akhavan, Sepideh, Raimondo De Cristofaro, Flora Peyvandi, Silvia Lavoretano, Raffaele Landolfi e Pier M. Mannucci. "Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype". Blood 100, n.º 4 (15 de agosto de 2002): 1347–53. http://dx.doi.org/10.1182/blood-2002-01-0243.
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In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene. Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1 × 107M−1s−1 and 9 × 105M−1s−1. Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM; Kd = 65.3 nM vs 2.1 nM, in FIIa-MT67 and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the absence of TM was normal. The Arg67His mutation had a dramatic effect on the cleavage of protease-activated G protein–coupled receptor 1 (PAR-1) 38-60 peptide (kcat/Km = 4 × 107M−1s−1 to 1.2 × 106M−1s−1). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second-order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulfate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the Arg67His mutation was associated with a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the Arg67His substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.
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Hall, Kellie J., Matthew L. Jones e Alastair W. Poole. "Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling". Biochemical Journal 406, n.º 3 (29 de agosto de 2007): 501–9. http://dx.doi.org/10.1042/bj20070244.
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PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.
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Riitano, Gloria, Antonella Capozzi, Serena Recalchi, Daniela Caissutti, Agostina Longo, Vincenzo Mattei, Fabrizio Conti et al. "Anti-β2-GPI Antibodies Induce Endothelial Cell Expression of Tissue Factor by LRP6 Signal Transduction Pathway Involving Lipid Rafts". Cells 11, n.º 8 (11 de abril de 2022): 1288. http://dx.doi.org/10.3390/cells11081288.
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In this study we analyzed whether anti-β2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-β2-GPI antibodies. Both LRP6 and β-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-β2-GPI antibodies induced LRP6 phosphorylation with consequent β-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-β-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-β2-GPI antibodies effect, indicating that the anti-β2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between β2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-β2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-β2-GPI binding triggers β-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).
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Hjortoe, Gertrud M., Lars C. Petersen, Tatjana Albrektsen, Brit B. Sorensen, Peder L. Norby, Samir K. Mandal, Usha R. Pendurthi e L. Vijaya Mohan Rao. "Tissue factor-factor VIIa–specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration". Blood 103, n.º 8 (15 de abril de 2004): 3029–37. http://dx.doi.org/10.1182/blood-2003-10-3417.
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Abstract Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we investigated the effect of FVIIa on IL-8 expression and cell migration in a breast carcinoma cell line, MDA-MB-231, a cell line that constitutively expresses abundant TF. Expression of IL-8 mRNA in MDA-MB-231 cells was markedly up-regulated by plasma concentrations of FVII or an equivalent concentration of FVIIa (10 nM). Neither thrombin nor other proteases involved in hemostasis were effective in stimulating IL-8 in these cells. Increased transcriptional activation of the IL-8 gene is responsible for increased expression of IL-8 in FVIIa-treated cells. PAR-2–specific antibodies fully attenuated TF-FVIIa–induced IL-8 expression. Additional in vitro experiments showed that TF-FVIIa promoted tumor cell migration and invasion, active site–inactivated FVIIa, and specific antibodies against TF, PAR-2, and IL-8 inhibited TF-FVIIa–induced cell migration. In summary, the studies described herein provide insight into how TF may contribute to tumor invasion. (Blood. 2004;103:3029-3037)
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Zarpellon, Alessandro, Antonella Zampolli, Patrizia Marchese, James R. Roberts, Grazia Loredana Mendolicchio e Zaverio M. Ruggeri. "GPIbα As a Selective Bidirectional Modulator Of α-Thrombin Prothrombotic Functions Influencing Fibrin Deposition and PAR-Dependent Platelet Activation". Blood 122, n.º 21 (15 de novembro de 2013): 32. http://dx.doi.org/10.1182/blood.v122.21.32.32.
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Abstract Background Generation of α-thrombin (FIIa) in response to vascular injury is a key host defense mechanism influencing thrombus formation and inflammation. Blood platelets express glycoprotein (GP) Ibα as the most abundant FIIa membrane binding site, as well as different protease activated receptors (PARs) with an effector role in platelet activation after proteolytic cleavage. The functional role of GPIbα, which is not a substrate for FIIa, relative to that of different PARs remains unclear. Aims Goal of these studies was to define with mechanistic understanding whether and how binding to GPIbα can modulate FIIa prothrombotic functions in vivo and ex vivo. Methods Endogenous mouse platelet GPIbα was replaced by the human (hu) counterpart with wild type (WT) sequence; or containing the single substitution of Asp277 (mutated to Asn), which interacts selectively with a site involving FIIa exosite 2; or with the combined substitution of post-translationally sulfated Tyr276, Tyr278 and Tyr279 (each mutated to Phe), which interact with FIIa residues in proximity of exosite 1 as well as exosite 2. These mice were evaluated in intravital models of arterial thrombosis. Moreover, their platelets were tested ex vivo for the response to FIIa-induced activation measuring changes in intracytoplasmic Ca2+ levels; and for effects on fibrinogen clotting and fibrin formation. Comparative ex vivo experiments were conducted with human and huGPIbα-WT mouse platelets in which FIIa binding was similarly blocked by the anti-human GPIbα monoclonal antibody, LJ-Ib10. Ex vivo FIIa effects on platelet activation/aggregation and fibrin clot formation were also evaluated concurrently in a model of thrombus formation in blood perfused over a thrombogenic surface under controlled flow conditions. Results Genetically modified mouse platelets expressed ≈9000 WT or mutant huGPIbα molecules; platelets with huGPIbα-WT bound ≈10,000 FIIa molecules with 1:1 stoichiometry and KD of ≈3 nM. FIIa binding to mutant huGPIbα was essentially abolished. Mice with defective FIIa binding to GPIbα exhibited a pronounced prothrombotic phenotype, with a shorter time to carotid artery occlusion following ferric chloride injury (median 550.5 seconds in 18 mutant huGPIbα, vs. 1980 seconds in 19 huGPIbα-WT mice; P<0.01). Accordingly, the platelet-rich plasma (PRP) of mutant huGPIbα mice exhibited a significantly shorter clotting time in the presence of 4 nM FIIa and significantly enhanced intracytoplasmic Ca2+ transients and platelet aggregation following stimulation by 0.5 nM FIIa. Human platelets, similar to mouse platelets, bound FIIa with a 1:1 stoichiometry relative to GPIbα and KD of ≈3 nM. Remarkably, blocking FIIa binding to GPIbα with antibody LJ-Ib10 essentially abolished activation by 1 nM FIIa in human platelets, in which FIIa effects are mediated predominantly by PAR1; this was in contrast to the enhanced activation seen under the same conditions in hu GPIbα-WT mouse platelets, in which FIIa acts through PAR3 and PAR4. Accordingly, the volume of platelet aggregates and fibrin formed in huGPIbα-WT mouse blood perfused over a thrombogenic surface was enhanced by blocking FIIa binding to platelets; in contrast, the volume of platelet aggregates, but not that of fibrin clots, was decreased under the same conditions in human blood. Antibody LJ-Ib10 shortened the clotting time of both huGPIbα-WT mouse and human PRP; however, in the absence of GPIbα-bound FIIa, fibrin associated with platelet aggregates had a less ordered fibrillar structure. Conclusions Our findings identify GPIbα as a relevant FIIa activity modulator. Through distinct mechanisms influenced by the expression of specific PAR subtypes, GPIbα can modulate FIIa function in hemostasis and thrombosis both enhancing and controlling prothrombotic responses and, thus, size and structure of platelet/fibrin thrombi. The effect of GPIbα on PAR4-mediated platelet activation, as well as fibrinogen clotting, can be explained by competition for FIIa exosites required for substrate binding, but the mechanism supporting the distinct GPIbα-PAR1 functional association remains to be elucidated. Disclosures: No relevant conflicts of interest to declare.
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Laurent, Marc, Remi Varin, Ulrich Joimel, Hong Li, He Lu, Emmanuel Blot, Shahsultan Mirshahi et al. "A Novel Mechanism of Action of Rivaroxaban: Inhibition of Monocyte and Macrophage Procoagulant Activity and Consequence On Inflammatory Process." Blood 114, n.º 22 (20 de novembro de 2009): 3124. http://dx.doi.org/10.1182/blood.v114.22.3124.3124.
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Abstract Abstract 3124 Poster Board III-61 Aim of the study Tissue factor is normally absent from monocytes in circulating blood. It can be induced by inflammatory mediators leading to the formation of monocyte-associated prothrombinase activity, which participates to thrombin generation. This activity appears to be important in both thrombosis (venous and arterial) and in chronic inflammation by inducing the release of inflammatory cytokines. This could be attributed to the activation of PAR-1 and PAR-2 (protease-activated receptors) by factor Xa or by thrombin. In this study, we compared the action of Rivaroxaban and Fondaparinux, two specific factor Xa inhibitors, on the activation of coagulation and on the secretion of inflammatory cytokines in both activated monocytes and activated human monocyte/macrophage cell line THP1. Methods 1-Monocytes were isolated from healthy volunteers and THP-1 cells were used as macrophages. 2- Activation of cells was performed by adding 1 μg/ml LPS for 2 hours at 37°C, in the presence of defibrinated human plasma which provides plasma coagulation factors, in the absence (control) or presence of Fondaparinux (500, 1000 and 1500 ng/ml) or Rivaroxaban (150, 250, 350 ng/ml, final concentrations). After incubation, the cells were isolated and their supernatants collected. 3- The procoagulant activity of cells was tested by measuring their effect on the clotting time of normal plasma in presence of calcium. 4- The release of cytokines was tested by antibody-cytokines array in the supernatants (RayBio®). 5-To test the neutralization of factor Xa bound to monocytes, activated monocytes were incubated with human purified factor Xa (100 ng/ml) for 10 min at room temperature. After isolation of the cells, 250 ng/ml Rivaroxaban or the appropriate solvent was added for 1, 10 and 30 min. The Xa activity associated to monocytes was measured by its amidolytic activity. Results 1- The procoagulant activity of monocytes and macrophages was reduced by Rivaroxaban and not by Fondaparinux. Results of prothrombinase activity in the presence of Rivaroxaban at 150, 250, 350 ng/ml, expressed as percentage of the control value was: 30 ± 3, 16 ± 4 and 12 ± 2 % for monocytes and: 43 ± 2, 24 ±3, 15 ± 1 % for THP-1 cells. In contrast, Fondaparinux did not modify the prothrombinase activity of monocytes (105 ± 12%). It was also shown that in the conditions used, Rivaroxaban (250 ng/ml) inhibited completely the amidolytic activity of factor Xa bound to activated monocytes. 2- Both Rivaroxaban and Fondaparinux modify the profile of chemokines secretion by activated monocytes and THP-1. In monocytes and THP-1 cells, LPS induced an important increase in Il-8 and angiogenin and a moderate increase in MIP-1d (Macrophage Inflammatory Protein-1) and RANTES, a member of the Il-8 superfamily. Both Rivaroxaban and Fondaparinux decreased the secretion of these chemokines to the basal level of secretion by non activated monocytes or THP-1. The secretion of leptin was only induced by LPS- treated THP-1 and was strongly decreased by both FXa inhibitors. In contrast, secretion of EGF (epithelial growth factor) was only induced by activated monocytes and strongly decreased by Rivaroxaban and moderately by Fondaparinux. Discussion and conclusion The results show that 1- Rivaroxaban induced a concentration-dependent inhibition of the procoagulant activity of activated monocytes and macrophages, whereas fondaparinux was devoid of this effect. This difference is attributed to a better access of Rivaroxaban to FXa bound to monocytes, as compared to the Fondaparinux-antithrombin complex. This inhibitory effect of Rivaroxaban could contribute to its antithrombotic activity. 2- Rivaroxaban inhibited the secretion of inflammatory chemokines by activated monocytes and THP-1. This decreased secretion was also observed with Fondaparinux, suggesting that it could be due to the decrease in thrombin generation in plasma, affecting the PAR-1 cell signaling system. As it was reported that that elevated IL-8 is associated with recurrent venous thrombosis and that these inflammatory cytokines are involved in plaque progression and rupture by recruitment of subpopulations of leukocytes and by potent angiogenic activity, this decrease in cytokines could also contribute to the antithrombotic efficacy of Rivaroxaban. This study received a support from Bayer Schering Pharma, France. Equal contribution of Marc Laurent and Rémi Varin Disclosures No relevant conflicts of interest to declare.
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Chatterjee, Madhumouli, Shannon L. Meeks, Valerie Anne Novakovic, Pete Lollar e Gary E. Gilbert. "Platelet Membranes and Activation Pathway Determine Inhibitory Activity of Anti-Factor VIII C2 Domain Antibodies". Blood 128, n.º 22 (2 de dezembro de 2016): 2567. http://dx.doi.org/10.1182/blood.v128.22.2567.2567.
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Abstract Inhibitory antibodies against factor VIII (fVIII) are the major complication of hemophilia A treatment, causing severe bleeding in spite of infused fVIII. Most antibodies are directed against the A2 or the C2 domain of fVIII and the degree of inhibition in clinical fVIII assays does not predict the severity of bleeding risk. We recently discovered that fVIII binds to a macromolecular complex including low molecular weight fibrin and the αIIbβ3integrin on membranes of thrombin-stimulated platelets rather than exposed phosphatidylserine (PS). Because clinical fVIII assays utilize PS-rich vesicles to support fVIII activity we asked whether the degree of inhibition of platelet-dependent activity by anti-C2 domain mAb's differs from inhibition on PS-rich phospholipid vesicles. Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides for PAR-1 and PAR-4, and either factor XIa (intrinsic pathway) or factor fXa (extrinsic pathway) without standard exogenous activators or vesicles. aPTT assays were performed on the same plasma-preparations using a commercial preparation of activator and phospholipid vesicles. Assays were performed using BBL fibrometers, electromechanical devices. A panel of 12 defined, epitope-mapped mAb's against the fVIII-C2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies. mAb's at 10 µg/ml were pre-incubated with fVIII for 60 min in the absence of VWF before mixing the fVIII-antibody mixture with plasma. Results: Platelets from nine donors supported a similar, near log-linear relationship between fVIII concentration and fibrin strand formation spanning at least 10,000-fold (0.0001 - 1 unit/ml). Time to fibrin strand formation was inversely related to platelet concentration over a range of at least 1 x 106 - 2 x 108/ml. Factor XIa and factor Xa concentrations of 20 pM and 0.01 pM, respectively were chosen emulating the intrinsic and extrinsic pathways. Four mAb's inhibited fVIII activity <10% in the aPTT assay, four others by 50-80%, with the final four in the 90-99% range. fVIII activity in the intrinsic APT had little correlation to inhibition in the aPTT assay with four mAb's causing less inhibition of the APT and the remaining 8 antibodies having 5-10 fold more inhibition. No mAb caused inhibition that was within 2-fold for the aPTT and APT assays. For the extrinsic APT, triggered by fXa, all antibodies produced greater than 99% inhibition of fVIII activity with 10 antibodies having 2-10 fold greater inhibition than with the intrinsic pathway. Conclusions: These results show that both platelet membranes and the pathway to fVIII activation determine the degree of inhibition by anti-C2 antibodies. The inhibition of platelet-dependent fVIII activity has little correlation to inhibition of the aPTT fVIII activity. Further, anti-C2 antibodies show greater inhibition of platelet-based activity when the fVIII activation pathway is via factor Xa vs. factor XIa. Additional studies will be needed to determine whether residual fVIII activity from the intrinsic APT or extrinsic APT better correlates to bleeding risk and whether the basis for activation pathway-dependent inhibition correlates to different proteolytic cleavage sites for factor VIII. Disclosures Meeks: Genentech: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Novakovic:Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity. Gilbert:Baxalta/Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity; Grifols: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Tran, F., A. Scharmacher, H. Grasshoff, S. Schinke, N. Kaeding, J. Bernades, J. Y. Humrich et al. "POS1203 INCREASED PROTEASE-ACTIVATED RECEPTOR 1 AUTOANTIBODIES ARE ASSOCIATED WITH SEVERE COVID-19". Annals of the Rheumatic Diseases 81, Suppl 1 (23 de maio de 2022): 930.1–930. http://dx.doi.org/10.1136/annrheumdis-2022-eular.947.
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BackgroundIn acute COVID-19 infection, growing evidence hints towards a broad activation of plasma cells and the presence of pathologic autoantibodies (abs). A systematic screening for abs confirmed induction of diverse functional abs by SARS-CoV-2 infection (1, 2). Immune-mediated thrombosis, involving platelet activation, has been identified as one of the key pathogenic mechanisms in COVID-19 and is linked to morbidity and mortality (3). As natural abs against G protein-coupled receptors, functional abs against the thrombin receptor type-1 (PAR-1) might predispose for increased activation of the coagulation system present in COVID-19 infection.ObjectivesThe aim of this study is to identify the diagnostic value of anti-PAR1 antibodies and their capacity to predict the outcome of COVID-19 infection.Methods82 serum samples from 55 individuals with COVID-19 derived from three different hospitals in Schleswig-Holstein, Germany, and 88 single time point samples from healthy controls were subjected to ELISA-based quantification of anti-PAR-1 abs (CellTrend GmbH Luckenwalde, Germany). The levels of anti-AT1R abs were compared with clinical and laboratory parameters.ResultsCOVID-19 patients revealed markedly increased levels of circulating anti-PAR1 abs in hospitalized patients particularly in those required intensive care treatment in comparison to controls (p < 0.0001, Figure 1a). Anti-PAR1 ab levels were highest in patients with fatal outcome (p = 0.006, Figure 1a). Receiver operating characteristic (ROC) analysis of PAR1 abs levels in COVID-19 patients revealed a sensitivity of 84.00% and a specificity 79.25% for patients requiring intensive care unit (ICU) treatment and a sensitivity of 87.50 % and a specificity 84.51 % to distinguish fatal vs. non-fatal disease outcome (Figure 1b). We found correlation of circulating anti-PAR1 abs with D dimers.Figure 1.Levels of anti-PAR-1 abs in healthy controls (HC) versus COVID-19 positive patients with different disease severity and in non survivors (left). ROC curves are shown to discriminate patients requiring intensive care unit (ICU) or survivors in COVID-19 infection.ConclusionThe increased anti-PAR1 abs, their prediction to identify patients requiring ICU and fatal outcome, and the correlation with markers for blood clotting suggest a role for antibodies against PAR1 in the disease development of blood clotting in COVID-19.References[1]Bastard P, Rosen LB, Zhang Q, et al. Autoantibodies against type I IFNs in patients with life-threatening COVID-19. Science 2020: 370(6515).[2]Wang EY, Mao T, Klein J, et al. Diverse functional autoantibodies in patients with COVID-19. Nature 2021.[3]Bonaventura A, Vecchie A, Dagna L, et al. Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19. Nat Rev Immunol 2021: 21(5): 319-329.Disclosure of InterestsNone declared
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Cheepala, Satish Babu, Kazumasa Takenaka, Tamara I. Pestina, Carl W. Jackson e Schuetz John. "The Abcc4 Knockout Reveals An Important Role for Abcc4 in Platelet Aggregation". Blood 118, n.º 21 (18 de novembro de 2011): 1141. http://dx.doi.org/10.1182/blood.v118.21.1141.1141.
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Abstract Abstract 1141 Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides. We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels. Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10. This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures: No relevant conflicts of interest to declare.
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Hamad, Osama A., Ioannis Mitroulis, Karin Fromell, Huda Kozarcanin, Triantafyllos Chavakis, Daniel Ricklin, John D. Lambris, Kristina N. Ekdahl e Bo Nilsson. "Contact activation of C3 enables tethering between activated platelets and polymorphonuclear leukocytes via CD11b/CD18". Thrombosis and Haemostasis 114, n.º 12 (2015): 1207–17. http://dx.doi.org/10.1160/th15-02-0162.
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SummaryComplement component C3 has a potential role in thrombotic pathologies. It is transformed, without proteolytic cleavage, into C3(H2O) upon binding to the surface of activated platelets. We hypothesise that C3(H2O) bound to activated platelets and to platelet-derived microparticles (PMPs) contributes to platelet-PMN complex (PPC) formation and to the binding of PMPs to PMNs. PAR-1 activation of platelets in human whole blood from normal individuals induced the formation of CD16+/CD42a+ PPC. The complement inhibitor compstatin and a C5a receptor antagonist inhibited PPC formation by 50 %, while monoclonal antibodies to C3(H2O) or anti-CD11b inhibited PPC formation by 75–100 %. Using plasma protein-depleted blood and blood from a C3-deficient patient, we corroborated the dependence on C3, obtaining similar results after reconstitution with purified C3. By analogy with platelets, PMPs isolated from human serum were found to expose C3(H2O) and bind to PMNs. This interaction was also blocked by the anti-C3(H2O) and anti-CD11b monoclonal antibodies, indicating that C3(H2O) and CD11b are involved in tethering PMPs to PMNs. We confirmed the direct interaction between C3(H2O) and CD11b by quartz crystal microbalance analysis using purified native C3 and recombinant CD11b/CD18 and by flow cytometry using PMP and recombinant CD11b. Transfectants expressing CD11b/CD18 were also shown to specifically adhere to surface-bound C3(H2O). We have identified contact-activated C3(H2O) as a novel ligand for CD11b/CD18 that mediates PPC formation and the binding of PMPs to PMNs. Given the various roles of C3 in thrombotic reactions, this finding is likely to have important pathophysiological implications.
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Honickel, Markus, Joanne van Ryn e Oliver Grottke. "Mechanistic Differences of Prothrombin Complex Concentrate and Idarucizumab in a Trauma Model Under Dabigatran Anticoagulation". Blood 126, n.º 23 (3 de dezembro de 2015): 1128. http://dx.doi.org/10.1182/blood.v126.23.1128.1128.
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Abstract Background: A proportion of trauma patients presenting with coagulopathy also receive anticoagulant therapy, which can exacerbate coagulopathy and increase bleeding. Emergency reversal of anticoagulation therapy may require therapy with specific reversal agents, as with idarucizumab for dabigatran or non-specific reversal agents, such as prothrombin complex concentrates (PCC). Methods: Using a lethal polytrauma model under dabigatran anticoagulation, we investigated the mechanism and procoagulant properties of a four-factor PCC or idarucizumab in dabigatran-anticoagulated pigs following multiple injuries. Blood markers of coagulation associated with procoagulant effects, platelet activation and hyperfibrinolysis were determined over time and related to blood loss. After ethical approval, dabigatran etexilate was given orally for 3 days and iv on day 4 to 18 pigs after anesthesia. Animals were randomized to receive idarucizumab (60 mg/kg), PCC (50 IU/kg) or saline (control), administered 15 minutes after bilateral femur fracture and blunt liver injury. Plasma dabigatran was determined using diluted thrombin time. Thrombin-antithrombin (TAT) complexes and fibrinopeptide A (FPA) were quantified using ELISA and D-Dimer with a clotting assay. Thrombin generation (ETP) in plasma was measured using Calibrated Automated Thrombogram. Platelet function was evaluated as aggregation using platelet-rich plasma in response to tissue factor, collagen, or adenosine diphosphate (ADP) agonism using light transmission aggregometry. Results: Mean dabigatran levels were 543±186 ng/ml prior to injury. Blood loss was 4056±537 mL after saline treatment (control), 1822±222 mL after PCC (P<0.0001 vs. control) and 1086±134 mL after idarucizumab (P<0.0001 vs. all). All controls developed severe coagulopathy and exsanguinated with a mean survival time of 93 min (range: 62-146 min). Post-injury and after PCC infusion, TAT, D-dimers and FPA were significantly increased vs idarucizumab and control and remained elevated over 300 minutes. Similarly thrombin generation (baseline ETP 267±84 nM*min) increased immediately after PCC administration and persisted elevated 848±114 nM*min at 120 min. In contrast, following therapy with idarucizumab ETP was restored to baseline values. Thrombin-dependent tissue factor-induced platelet aggregation was inhibited with dabigatran, partially restored with PCC and fully restored with idarucizumab. There was no effect with thrombin-independent collagen- and ADP-induced aggregation. Conclusion: Although four-factor PCC (50 IU/kg) is effective in reducing blood loss, its efficacy is less than therapy with idarucizumab (60 mg/kg). Most importantly the activation of coagulation is explained by different mechanisms. Supernormal activation of coagulation (e.g. TAT, FPA, ETP) was evident after PCC administration, whereas idarucizumab normalized ETP to baseline values. Since thrombin is a potent activator of platelets via PAR, restoration of active thrombin after PCC also partially restored aggregation. In contrast, idarucizumab binds dabigatran and forms an irreversible complex, inactivating dabigatran in plasma, thereby restoring hemostatic function. This difference may influence the choice of therapy when idarucizumab is clinically available. Disclosures van Ryn: Boehringer Ingelheim: Employment. Grottke:NovoNordisk: Research Funding; Portola Pharmaceuticals: Consultancy; CSL Behring: Honoraria, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Research Funding; Biotest: Research Funding.
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Pluthero, Fred G., Margaret L. Rand, Victor S. Blanchette e Walter H. Kahr. "Rapid Assessment of Platelet Function Using Thromboelastography and Small Volumes of Citrated Whole Blood." Blood 106, n.º 11 (16 de novembro de 2005): 3995. http://dx.doi.org/10.1182/blood.v106.11.3995.3995.
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Abstract Platelet function disorders are a key cause of abnormal bleeding, and diagnosis is challenging because: platelet abnormalities are diverse, affecting many aspects of function; variability in platelet function testing in clinical laboratories makes it difficult to compare results; large blood volumes required for platelet function analysis make it difficult to perform in neonatal patients; manipulation of platelet rich plasma used for platelet aggregation can lead to test variability; platelet aggregation curves are difficult to interpret in thrombocytopenic patients. We describe a method of testing platelet function using citrated whole blood and thromboelastography (TEG) that overcomes some of these limitations. Commercially-available platelet mapping kits allow the effects of the platelet agonists adenosine diphosphate (ADP) and arachidonic acid (AA) to be assessed via a TEG assay where reptilase and activated factor XIII produce fibrin clots independent of thrombin in heparinized whole blood. The activation and aggregation of platelets is quantified by measuring the difference in maximum amplitude (MA) between unstimulated samples, which form weak fibrin-only clots, and samples with agonists added, which form stronger clots containing fibrin and activated/aggregated platelets. Platelet mapping was used as the basis for a TEG assay which can be used to assess platelet responses to a wide range of stimuli - including ADP, AA, epinephrine, collagen, U46619 (thromboxane-A2 receptor agonist), SFLLRN (PAR-1 thrombin receptor activating peptide) and AYPGKF (PAR-4 activating peptide) - in small samples (330μL) of citrated native (CN) blood or plasma to which heparin is added to a concentration of 20U/mL. Samples were recalcified by adding calcium chloride to 10mM (necessary for the function of reptilase and FXIIIa), and other reagent volumes were the same as in platelet mapping assays, with fibrin activator prepared at 1/2 regular strength. The concentrations of platelet agonists were: collagen 51μg/ml, epinephrine 0.27μM, ADP 5.4μM, arachidonic acid 135μg/mL, U46619 2.6μM, SFLLRN 6.76μM and AYPGKF 34μM. These concentrations produced TEG MA values in heparinated fibrin-activated CN blood from a panel of normal individuals comparable to those obtained from recalcified CN blood in the absence of heparin (the fibrin/platelet response control). The platelet response was rapid with maximum amplitudes reached within 10 minutes for all agonists except collagen, which required >30 minutes to produce maximum amplitude. We have found this TEG platelet-response assay to be useful in detecting platelet function abnormalities, producing results which correlate with and extend those of other platelet function tests. For example in one patient a weak response to epinephrine corresponded to similar platelet aggregation results, and in another the TEG assay detected a weak PAR-1 response not specifically detected in other tests. The assay has also proven useful in assessing platelet function in blood and plasma having low platelet concentrations (<50 x 10E9/L) from experimental or pathological causes (e.g. thrombocytopenia), in titrating platelet responses to agonists and in assessing the effects of antiplatelet agents in vivo and in vitro. Thus this TEG platelet function assay has the advantages of speed, ease of use, flexibility, adaptability to low platelet concentrations and sample economy, requiring small volumes of citrated blood which can be used for other coagulation assays and platelet response tests.
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Azorsa, David, Sylvie Moog, Catherine Ravanat, Simone Schuhler, Gilles Folléa, Jean-Pierre Cazenave e François Lanza. "Measurement of GPV Released by Activated Platelets Using a Sensitive Immunocapture ELISA – Its Use to Follow Platelet Storage in Transfusion". Thrombosis and Haemostasis 81, n.º 01 (1999): 131–38. http://dx.doi.org/10.1055/s-0037-1614430.
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SummaryThrombin, the most potent platelet agonist, plays a central role in haemostasis and in the occurrence of thrombotic events. This agonist activates platelets by cleaving the PAR G-protein coupled receptors and by binding to glycoprotein (GP) Ib and also cleaves GPV at the platelet surface to liberate the soluble 69 kDa fragment GPVf1. Monoclonal antibodies (MoAbs) to GPV were developed as tools to study the mechanism of platelet GPV cleavage and measure release of GPV in pathological situations. Specificity of the MoAbs for GPV was confirmed by flow cytometry and immunoprecipitation of proteins from human platelets and Dami megakaryocytic cells. A sensitive immunocapture sandwich ELISA for soluble GPV was developed using two MoAbs recognizing different epitopes of GPV and purified platelet or recombinant GPV as reference protein. This ELISA was employed to determine the mean plasma concentration of GPV in 100 normal individuals (17.3 ng/ml), to demonstrate the dose-dependent release of GPVf1 from washed platelets stimulated with thrombin and to follow the progressive release of GPVf1 during storage of therapeutic platelet concentrates. The present report describes a sensitive GPV ELISA of direct application to survey the processing and storage of platelet concentrates for transfusion and of potential value to monitor platelet activation in thrombotic states.
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Tsiailanis, Antonios D., Constantinos C. Tellis, Paraskevi Papakyriakopoulou, Androniki D. Kostagianni, Vasileios Gkalpinos, Christos M. Chatzigiannis, Nikolaos Kostomitsopoulos, Georgia Valsami, Alexandros D. Tselepis e Andreas G. Tzakos. "Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen". Molecules 28, n.º 5 (2 de março de 2023): 2311. http://dx.doi.org/10.3390/molecules28052311.
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The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin’s potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4′-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4′-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4′-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4′-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4′-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4′-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.
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Butta, Nora, María Isabel Rivas Pollmar, María Teresa Álvarez Román, Monica Martín Salces, Ihosvany Fernandez Bello, Miguel Canales e Victor Jiménez Yuste. "Platelet Features from ITP Patients Responders to Different Therapeutic Treatments". Blood 126, n.º 23 (3 de dezembro de 2015): 4648. http://dx.doi.org/10.1182/blood.v126.23.4648.4648.
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Abstract Background: Patients with ITP have a wide variation in the presentation of the disease, platelet count and their clinical course. The decision to begin treatment is based on the hemorrhagic symptoms and platelet count. Intravenous immunoglobulin (IVIG) is usually associated with glucocorticoid administration in patients with severe bleeding or platelet counts <20x109/L and a quick response is required. Agonists of thrombopoietin receptor (TPO-AR) and splenectomy are other therapeutic tools for these patients. Materials and Methods: We recruited patients with ITP before and after responding to treatment with IVIG (n = 11) and AR-TPO (4 patients with romiplostim and 10 with eltrombopag), 5 splenectomized patients and 82 healthy controls. The percentage of reticulated platelets, platelet activation and binding of annexin-V were evaluated by flow cytometry. Plasma levels of TPO and "a proliferation-inducing ligand" (APRIL) were determined by ELISA. Procoagulant activity associated microparticles (MP) and the ability of plasma to generate thrombin were determined, respectively, with Zymuphen kit and calibrated automated thrombinography (CAT) triggered by 1 pM tissue factor and 4 micromolar phospholipid (PPP-low reagent, Diagnostica Stago, Spain). Results: Patients with ITP that respond to IGIV and AR-TPO treatments recovered platelet counts without reaching the levels of the control group, whereas the platelet count in splenectomized patients did not differ from it. Plasma levels of TPO and the number of immature platelets in the first two groups were higher than in controls before responding to treatment. Despite recovering platelet count, platelet capacity of being activated by agonists such as TRAP (thrombin receptor agonist for PAR-1) was less than that of the controls in all groups. This decrease was not due to a reduction in the expression of the fibrinogen receptor on platelets from ITP patients. Platelets from ITP patients before and after responding to all treatments studied, showed more phosphatidylserine exposure and greater microparticles-associated and plasma-associated procoagulant activity. Plasma levels of APRIL, a factor that stimulates B cells and antibody production, decreased in ITP patients who responded to the AR-TPO, reaching the levels observed in the control group. In the group of splenectomized patients a decrease of APRIL was also observed, but still remained higher than in healthy controls. Conclusions: ITP patients who respond to treatment with IVIG and AR-TPO and undergoing splenectomy recovered platelet count but not its function. The treatments did not modify the microparticles- and plasma-associated thrombogenic capacity. Among all the treatments studied, AR-TPO and splenectomy had an addittional benefical effect reducing APRIL plasma levels Disclosures No relevant conflicts of interest to declare.
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Preston, Roger JS, Shona Harmon, Fionnuala B. Ni Ainle, Jennifer A. Johnson, Moya Cunningham, O. Smith, Barry White e James S. O’Donnell. "Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement". Blood 112, n.º 11 (16 de novembro de 2008): 21. http://dx.doi.org/10.1182/blood.v112.11.21.21.
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Abstract Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (<2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.
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Patel, Nishi H., R. Alan Mitteer, Jamunabai M. Prakash, Oresta V. Borodevyc e Gerald Soslau. "New Clues To The Mechanism Of Cardioprotection By Estrogen". Blood 122, n.º 21 (15 de novembro de 2013): 4736. http://dx.doi.org/10.1182/blood.v122.21.4736.4736.
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Platelets are circulating anucleated cell fragments that play a major role in thrombosis and hemostasis in response to a number of stimuli. Platelet activation is a multi-step processes that can be modulated via several known mechanisms. It has long been suspected that sex hormones, like estrogen, have an effect on platelet aggregation and thrombosis; the exact role which estrogen plays in these processes remains unclear. Pre-menopausal women have a lower risk for coronary thrombosis than men– presumably due to higher estrogen levels. However, in postmenopausal women, hormone replacement paradoxically increases the risk for thrombotic events such as coronary and venous thrombosis. Current literature on the subject of estrogen effect on platelets varies considerably. Previous and current research indicates potential estrogen mechanisms for thrombosis; yet the complete biochemical pathway has not been elucidated. Current literature describes the aggregatory effects of estrogen, but little is said about the fact that premenopausal women still have a lower risk for coronary thrombosis in relation to their male counterparts despite having comparatively higher estrogen levels. The goal of our study was to better delineate estrogen mediated biochemical pathways involved in platelet aggregation. Blood was collected from healthy male donors and fractionated into platelet rich plasma (PRP). PRP was centrifuged to prepare platelet poor plasma (PPP) to be used as a control during platelet aggregation assays. PRP samples were incubated with varying physiologic concentrations of 17-b-estradiol for 1-10 minutes and then activated with agonist, and compared to activated control samples in the absence of estrogen. Agonists tested include TRAP-1 (activates PAR-1), gamma thrombin (activates PAR-4), collagen, and ADP. Our results show that estrogen at physiologic levels (60-80 nM) has a synergistic effect on platelet aggregation when combined with sub-threshold doses of agonist. We also showed that this synergy is at least in part mediated by lipid rafts, a fact demonstrated in other studies. A role for lipid rafts in the estrogen effect was demonstrated by incubating PRP with the lipid raft disruptor, methyl β cyclodextrin (MβCD), which resulted in the inhibition of estrogen-induced synergy. Incubation with MβCD alone had no effect on platelet aggregation. We demonstrated that estrogen-induced synergy was absent when platelets, derived from male donors, were washed free of plasma components. Upon the addition of PPP (10-25 uL), synergy was restored. We postulated the necessity of some plasma protein in presenting estrogen to its receptor (ERα and ERβ) for there to be any synergistic effect. Using immunoflourescent microscopy we were able to identify that platelets contain both ERα and ERβ and that the expression of each is altered upon binding of estrogen and TRAP-1. Using filtration techniques we were able to determine that the molecular weight of the necessary protein to restore synergy is greater than 90-100,000 kDa. The most likely candidate was the dimerized plasma sex hormone binding globulin (SHBG), due to its high affinity for estrogens. Support for this comes from the ability of anti-SHBG antibodies to block the estrogen-induced synergy of agonist-induced platelet aggregations. Platelets derived from premenopausal females showed no estrogen-induced synergy of platelet aggregations under the identical conditions used with platelets derived from males. However, platelets derived from postmenopausal women did show synergistic effects of estrogen mirroring effects observed with male platelets samples. In all cases where estrogen-induced synergy of aggregation was observed, incubation of the same platelet samples with estrogen for 2-24hrs totally reversed the synergistic effect. This would be comparable to the premenopausal woman whose platelets in vivo are constantly exposed to high levels of estrogen. Our studies demonstrate that the SHBG must present estrogen to the platelet membrane estrogen receptors and that these receptors are down-regulated upon prolonged exposure to estrogen resulting in reduced levels of platelet aggregation in the presence of sub-optimal concentrations of agonists. Results of our studies would account for the varied effects of estrogen on platelet activation reported in the literature. Studies are in progress with testosterone and second messengers. Disclosures: No relevant conflicts of interest to declare.
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Isermann, Berend, Madhusudhan Thati, Ilya Vinnikov, Stefanie Herzog, Sina Huntscha, Robert Bünder, Hartmut Weiler, Angelika Bierhaus e Peter P. Nawroth. "Loss of TM-Dependent PC-Activation Predisposes to Diabetic Nephropathy: Potential Role of Endothelial Apoptosis." Blood 106, n.º 11 (16 de novembro de 2005): 1029. http://dx.doi.org/10.1182/blood.v106.11.1029.1029.
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Abstract Increased levels of soluble thrombomodulin (TM) in patients with diabetes mellitus are considered a marker of endothelial damage. It is unknown whether the loss of endothelial TM-function contributes to the progression of vascular complications in diabetes mellitus. To address whether the loss of TM-dependent protein C (PC) activation contributes to diabetic complications such as diabetic nephropathy two animal models were employed: (1) TMPro mice, which have been previously described and carry a point mutation in the TM-gene (E404P), resulting in a loss of TM-dependent PC-activation, and (2) hPC mice, which carry a transgene resulting in the expression of a mutant “hyperactivatable” PC, which can be activated by thrombin in the absence of TM. The mutant PC could be captured from plasma samples of hPC mice and activated ex vivo by thrombin in the absence of TM. hPC mice had a prolonged bleeding time. Following induction of diabetes by streptozotocin TAT levels were increased in diabetic control (wild type) mice and to a larger extent in TMPro mice, but not in diabetic hPC mice. In comparison to diabetic control mice the kidney weight was increased in diabetic TMPro mice, but not in diabetic hPC mice. Albuminuria was increased in diabetic TMPro mice and reduced in diabetic hPC mice in comparison to diabetic control mice, indicating increased glomerular damage in TMPro mice and partial protection from glomerular damage in hPC mice. Consistently, using a histological score glomerular damage was more severe in diabetic TMPro mice in comparison to diabetic control mice, while diabetic hPC mice were protected. Preliminary data suggest that the observed changes are associated with increased apoptosis in glomeruli of diabetic TMPro mice. Using HUVECs we were able to establish that high glucose concentrations (30 mM) reduce TM-dependent PC activation. The reduced TM-dependent PC activation is associated with increased apoptosis. Glucose induced apoptosis in HUVECs is associated with an increased Bax/Bcl-2 ratio, increased translocation of Bax into mitochondria, and increased caspase-3 activation. Activated PC normalizes the Bax/Bcl-2 ratio, prevents translocation of Bax, and reduces caspase-3 activity. Further studies using TRAPs and inhibitory antibodies established that the antiapoptotic effect of aPC in glucose stressed endothelial cells is mediated through a Par-1 and EPCR-dependent mechanism. The current data strongly suggest that the loss of the endothelial TM-PC system is not just a marker of endothelial damage in diabetic patients, but rather contributes to the progression of diabetic vascular complications.
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Liao, Wei-Ju, Meng-Ying Wu, Chen-Chung Peng, Yi-Chung Tung e Ruey-Bing Yang. "Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation". Cardiovascular Research 116, n.º 1 (1 de março de 2019): 193–201. http://dx.doi.org/10.1093/cvr/cvz036.
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Abstract Aims SCUBE1 [signal peptide-CUB-epidermal growth factor (EGF) domain-containing protein 1], expressed in endothelial cells (ECs) and platelets, exists in soluble or membrane forms. We previously showed that soluble SCUBE1 is a biomarker for platelet activation and also an active participant of thrombosis. However, whether the adhesive module of its EGF-like repeats is essential and the specific contribution of SCUBE1 synthesized in ECs or platelets to thrombosis in vivo remain unclear. Methods and results We generated new mutant (Δ2) mice lacking the entire EGF-like repeats to evaluate the module’s functional importance during thrombogenesis in vivo. The Δ2 platelet-rich plasma showed markedly impaired platelet aggregation induced by agonists including adenosine diphosphate, collagen, the thrombin agonist PAR-4 peptide and the thromboxane A2 analogue U46619. Consistently, genetic ablation of the EGF-like repeats diminished arterial thrombosis and protected Δ2 mice against lethal thromboembolism. On flow chamber assay, whole blood isolated from Δ2 or wild-type (WT) mice pre-treated with blocking antibodies against the EGF-like repeats showed a significant decrease in platelet deposition and thrombus formation on collagen-coated surfaces under arterial shear rates. Moreover, we created animals expressing SCUBE1 only in ECs (S1-EC) or platelets (S1-PLT) by reciprocal bone-marrow transplantation between WT and Δ2 mice. The time of carotid arterial thrombosis induced by ferric chloride was normal in S1-PLT chimeric mice but much prolonged in S1-EC animals. Conclusions We demonstrate that platelet-derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF-like repeats in vivo and suggest targeting these adhesive motifs of SCUBE1 for potential anti-thrombotic strategy.