P2Y12 Inhibitors Restore the Ability of GPIb-IX to Inhibit Outside-in Signaling Across alphaIIbbeta3.

Abstract Abstract 3006 Poster Board II-982 P2Y12 inhibitors decrease the reactivity of platelets. Since the P2Y12 receptor acts through Gαai-induced signals, which inhibit adenyl cyclase, P2Y12 inhibitors are thought to act through phosphorylation of protein kinase A (PKA) substrates. One PKA substrate is serine166 (S166) of GPIbβ. Using a phospho-specific antibody, Western blots, and FACS of whole blood, we found that ADP and epinephrine, which activate P2Y12 receptors caused a dose-dependent dephosphorylation of S166 while apyrase and a P2Y12 inhibitor prevented this dephosphorylation. Since S166 functions with S609 of GPIbαa to bind 14-3-3, we determined whether i) dephosphorylation was associated with release of 14-3-3 from GPIb, and ii) 14-3-3 regulated αaIIbβ3 function. GPIb bound ∼91 ± 5% of the 14-3-3, as determined by its insolubility in streptolysin O-permeabilized platelets and its solubilization by phospho-S609 and -S166 peptides. Quantitation of 14-3-3 in GPIb immunoprecipitates showed that graded Gαai-induced dephosphorylation of S166 was accompanied by graded release of 14-3-3 from GPIb. Evidence that 14-3-3 participates in αaIIbβ3 signaling came from its co-immunoprecipitation with αaIIbβ3 from sheared platelets. Moreover, when CHO/αaIIbβ3 cell 14-3-3 was sequestered by expression of GPIb, αaIIbβ3-induced activation of RhoGTPases and cell spreading were inhibited; overexpression of 14-3-3 restored these functions. These studies: i) show that 14-3-3 is critical for αaIIbβ3-induced spreading; ii) describe a novel function of GPIb in dampening the transition to firm adhesion through the regulated sequestration of 14-3-3; and iii) suggest that the availability of 14-3-3 for αaIIbβ3-mediated adhesion is determined by the Gai-modulated level of GPIb phosphorylation. Disclosures: No relevant conflicts of interest to declare.

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P2Y12 inhibitors in neuroendovascular surgery: An opportunity for precision medicine

Interventional Neuroradiology ◽

10.1177/1591019921991394 ◽

2021 ◽

pp. 159101992199139

Author(s):

Axel Rosengart ◽

Malie K Collins ◽

Philipp Hendrix ◽

Ryley Uber ◽

Melissa Sartori ◽

...

Keyword(s):

Adverse Events ◽

Precision Medicine ◽

Point Of Care ◽

Indirect Evidence ◽

Cost Benefit ◽

Interventional Cardiology ◽

Platelet Inhibition ◽

Response Monitoring ◽

P2y12 Inhibitor ◽

P2y12 Inhibitors

Introduction Dual antiplatelet therapy (DAPT), primarily the combination of aspirin with a P2Y12 inhibitor, in patients undergoing intravascular stent or flow diverter placement remains the primary strategy to reduce device-related thromboembolic complications. However, selection, timing, and dosing of DAPT is critical and can be challenging given the existing significant inter- and intraindividual response variations to P2Y12 inhibitors. Methods Assessment of indexed, peer-reviewed literature from 2000 to 2020 in interventional cardiology and neuroendovascular therapeutics with critical, peer-reviewed appraisal and extraction of evidence and strategies to utilize DAPT in cardio- and neurovascular patients with endoluminal devices. Results Both geno- and phenotyping for DAPT are rapidly and conveniently available as point-of-care testing at a favorable cost-benefit ratio. Furthermore, systematic inclusion of a quantifying clinical risk score combined with an operator-linked, technical risk assessment for potential adverse events allows a more precise and individualized approach to new P2Y12 inhibitor therapy. Conclusions The latest evidence, primarily obtained from cardiovascular intervention trials, supports that combining patient pharmacogenetics with drug response monitoring, as part of an individually tailored, precision medicine approach, is both predictive and cost-effective in achieving and maintaining individual target platelet inhibition levels. Indirect evidence supports that this gain in optimizing drug responses translates to reducing main adverse events and overall treatment costs in patients undergoing DAPT after intracranial stent or flow diverting treatment.

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Bile Acids Induce Platelet Activation Leading to Degranulation and a Prothrombotic Phenotype

Blood ◽

10.1182/blood-2019-126095 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4887-4887

Author(s):

Joachim Zobel ◽

Tanja Strini ◽

Martin Tischitz ◽

Sina Pohl ◽

Theresa Greimel ◽

...

Keyword(s):

Liver Fibrosis ◽

Bile Acids ◽

Conflicts Of Interest ◽

Annexin V ◽

Farnesoid X Receptor ◽

Fibrinogen Binding ◽

Model Study ◽

Serine Protease Activity ◽

Adult Volunteers ◽

Dose Dependent

Background: Previous articles have identified the farnesoid X receptor (FXR) as an integral part in the formation of coated platelets. Coated platelets are preactivated platelets featuring degranulation, increased fibrinogen binding, and increased serine protease activity leading to fibrin generation. Furthermore, phosphatidylserine exposure is increased and integrin α2bβIII is inhibited - leading to a prothrombotic phenotype despite decreased platelet aggregation. We hypothesize that bile acids, as natural ligands of FXR, lead to a change of platelet phenotype and therefore play a pivotal role in the formation of coated platelets, especially in presence of cholestasis. Methods: Based on previous findings, we incubated human washed platelets of healthy adult volunteers with the synthetic FXR ligand GW4064 in various concentrations (0, 10, 20, 50, 100µM) and used flow cytometry to detect a shift in p-selectin expression, PAC-1 binding and annexin-V-binding. Moreover, we used different concentrations (0, 100, 200, 400, 600µM) of three bile acids (ursodeoxycholic acid, UDCA; chenodeoxycholic acid, CDCA; glycochenodeoxycholic acid, GCDCA) to see if natural FXR ligands induce an effect on the platelet phenotype. Results: We observed a dose dependent shift in annexin-V-binding when treating washed platelets with GW4064 as well as CDCA and GCDCA. Similarly, GW4064 led to increased p-selectin expression while increased PAC-1-binding was only detected at the highest concentration. In contrast, CDCA and GCDCA showed merely slight changes in p-selectin expression whereas PAC-1-binding seemed to be unaffected. However, none of these effects were seen when using UDCA. Conclusion: We conclude that pretreatment of washed platelets with CDCA and GCDCA initiate a dose-dependent shift towards a prothrombotic platelet phenotype. Therefore, we assume that increased levels of certain bile acids drive thrombosis in patients with cholestatic liver injury. Furthermore, a recent mouse model study suggested that platelet derived growth factor β (PDGFβ), a component of α-granula, drives liver fibrosis. Hence, in addition to their prothrombotic effects, coated platelets might exacerbate liver fibrosis. Disclosures No relevant conflicts of interest to declare.

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The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz037 ◽

2019 ◽

Vol 25 (10) ◽

pp. 587-600 ◽

Cited By ~ 2

Author(s):

Héctor Zapata-Carmona ◽

Lina Barón ◽

Lidia M Zuñiga ◽

Emilce Silvina Díaz ◽

Milene Kong ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Current Knowledge ◽

Human Sperm ◽

Adenyl Cyclase ◽

Sperm Capacitation ◽

Time Treatment ◽

Proteasome Subunits ◽

Pka Pathway ◽

Kinase A

Abstract One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

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Mechanisms of PTH-induced rise in cytosolic calcium in adult rat hepatocytes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.267.5.g754 ◽

1994 ◽

Vol 267 (5) ◽

pp. G754-G763 ◽

Cited By ~ 1

Author(s):

M. Klin ◽

M. Smogorzewski ◽

H. Khilnani ◽

M. Michnowska ◽

S. G. Massry

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

G Protein ◽

Cytosolic Calcium ◽

Amino Terminal ◽

Kinase C ◽

Dose Dependent ◽

Kinase A ◽

Stimulation Of

Available data indicate that the liver is a target organ for parathyroid hormone (PTH) and that this effect is most likely mediated by PTH-induced calcium entry into hepatocytes. The present study examined the effects of both PTH-(1-84) and its amino-terminal fragment [PTH-(1-34)] on cytosolic calcium concentration ([Ca2+]i) of hepatocytes and explored the cellular pathways that mediate this potential action of PTH. Both moieties of PTH produced a dose-dependent rise in [Ca2+]i, but the effect of PTH-(1-84) was greater (P < 0.01) than an equimolar amount of PTH-(1-34). This effect required calcium in the medium and was totally [PTH-(1-34)] or partially [PTH-(1-84)] blocked by PTH antagonist ([Nle8,18,Tyr34]bPTH-(7-34)-NH2] and by verapamil or nifedipine. Sodium or chloride channel blockers did not modify this effect. 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and G protein activator also produced a dose-dependent rise in [Ca2+]i. Staurosporine abolished the effect of TPA, and both staurosporine and calphostin C partially inhibited the effect of PTH. Staurosporine and verapamil together produced greater inhibition of PTH action than each alone. Rp-cAMP, a competitive inhibitor of cAMP binding to the R subunit of protein kinase A, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, blocked the effect of both DBcAMP and PTH, but the effect of these agents was greater (P < 0.01) on DBcAMP action. G protein inhibitor and pertussis toxin partially blocked the action of PTH. The data indicate that 1) PTH increases [Ca2+]i of hepatocytes; 2) this action of the hormone is receptor mediated; 3) the predominant pathway for this PTH action is the stimulation of a G protein-adenylate cyclase-cAMP system, which then leads to stimulation of a calcium transport system inhibitable by verapamil or nifedipine or activation of L-type calcium channels; 4) activation of protein kinase C is also involved; and 5) the PTH-induced rise in [Ca2+]i is due, in major parts, to movement of extracellular calcium into the cell.

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AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.2866.2866 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2866-2866

Author(s):

Hisayuki Yao ◽

Eishi Ashihara ◽

Rina Nagao ◽

Shinya Kimura ◽

Hideyo Hirai ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Signaling Pathway ◽

Small Molecule ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Cancer Cell Line ◽

Dependent Manner ◽

Dose Dependent

Abstract Abstract 2866 Poster Board II-842 Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells. The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells. We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

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Oral Supplementation with Omega3 Fatty Acids Inhibits NFkB Activation In Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽

10.1182/blood.v116.21.4607.4607 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4607-4607

Author(s):

Oscar F. F Ballester ◽

Johannes Fahrmann ◽

Theodore Witte ◽

Gabriela Ballester ◽

W. Elaine Hardman

Keyword(s):

Conflicts Of Interest ◽

Early Stage ◽

Gradient Centrifugation ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Dependent Manner ◽

Red Cell ◽

Omega 3 ◽

Dose Dependent

Abstract Abstract 4607 Introduction: Nuclear factor kappa B (NFkB) is a critical transcription factor involved in the growth and survival of CLL cells. NFkB is recognized as an important target for the development of novel therapies for the treatment of various malignancies. In vitro and in experimental animal models, OMEGA-3 fatty acid (O3FA) supplementation has been shown to inhibit NFkB activity. Patients and Methods: Patients with early stage CLL (Rai stages 0-II) who required no therapy, where accrued to this phase I-II trial. O3FA supplements were given for a total of 12 months at doses ranging from 2250 mg (EPA plus DHA), escalated to 4500 mg and 6750 mg per day as tolerated. NFkB activity was measured in peripheral blood samples after separation of mononuclear cell by gradient centrifugation and expressed as luminescence units/μ g of protein. Baseline and multiple serial samples were obtained during the study period. In-vitro cytotoxicity assays to doxorubicin were conducted using standard LD50 methods. Compliance was monitored by analysis of red cell and lymphocyte membrane lipid composition by gas chromatography. Results: Fifteen patients have been accrued to the trial, 8 of them have currently completed the planned 12 months of the study period. No significant clinical changes in disease activity were noted. O3FA was well tolerated. Supplementation resulted in a dose-dependent increase of O3FA composition of red cell and lymphocyte membranes in a dose dependent manner. At baseline, CLL patients had NFkB above the range observed in normal controls (2.05 × 104 to 2.32 × 105 NFkB lum units/μ g). The median value in CLL patients at baseline was 11.60 × 106 NFkB lum units/μ g (range 0.9 × 105 to 23.12 × 106). Among 5 patients with the highest baseline levels of NFkB, a decrease in NFkB activity ranging from 0.02 to 0.19 of the baseline value, was noted at the 2 higher doses of O3FA supplementation. Similar results were seen in patients with relatively lower levels of baseline NFkB activity (0.9 × 105 to 2.96 × 106 lum units/μ g). In vitro, significant doxorubicin cytotoxicity (>50%) was noted in samples obtained during supplementation, at μ gM concentrations which produced no detectable cell kill in baseline samples. Conclusions: O3FA supplementation resulted in significant inhibition of NFkB activity in leukemic cells from patients with CLL. In-vitro, after O3FA supplementation CLL cells became more sensitive to doxorubicin. Preliminary analysis of whole genome micro arrays revealed significant down-regulation of multiple genes associated with O3FA supplementation. Disclosures: No relevant conflicts of interest to declare.

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Anti-CD45 Radioimmunotherapy Using the Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model

Blood ◽

10.1182/blood.v120.21.4096.4096 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4096-4096

Author(s):

Johnnie J. Orozco ◽

Aimee Kenoyer ◽

Tom Bäck ◽

Ethan R. Balkin ◽

Donald K. Hamlin ◽

...

Keyword(s):

Bone Marrow ◽

Renal Function ◽

Path Length ◽

Conflicts Of Interest ◽

Energy Levels ◽

Radiation Doses ◽

Cell Transplant ◽

Normal Ranges ◽

Dose Dependent ◽

Murine Leukemia

Abstract Abstract 4096 Background: Despite aggressive hematopoietic cell transplant (HCT) strategies, many patients with acute myeloid leukemia (AML) relapse. Our group has explored radioimmunotherapy (RIT) using anti-CD45 antibodies (Ab) labeled with β-emitting radionuclides such as 131I and 90Y as a means to augment cytotoxicity and reduce relapse. This approach has been limited by their low energy levels (0.66–2.3 MeV) and potential non-specific toxicities due to their relatively long path lengths (0.3–2.3 mm). Conversely, α-emitting agents display higher energy levels (8 MeV) delivered over a short path-length (∼60–80 μm) that can lead to superior therapeutic ratios of absorbed radiation doses that may reduce AML relapse. For this purpose 211At is an α-emitter with an attractive half-life (7.2 hours), energy profile (6.8 MeV averages of two alpha decays, 5.9 and 7.5 MeV) and path length (average range 55–70 μm). Therefore, we evaluated the efficacy and toxicity of anti-CD45 RIT using 211At in a clinically relevant CD45+disseminated murine leukemia model. Methods: SJL/J mice were given 105 syngeneic SJL leukemic peripheral blood mononuclear cells (PBMCs) via tail vein, followed two days later by211At-labeled anti-murine CD45 Ab-decaborate(2-) conjugate (30F11-B10) or 211At-labeled negative control Ab-decaborate(2-) conjugate (rat IgG-B10) in tissue biodistribution studies. Groups of 5 mice were euthanized 6, 24 and 48 hours later and organs were harvested and analyzed in a gamma counter to yield percent of the injected dose per gram (% ID/g). To assess toxicities associated with this approach, SJL/J mice were treated with 12–24 μCi of 211At-30F11-B10 and then evaluated weekly thereafter for impact on blood counts, as well as changes in hepatic and renal function. In HCT therapeutic studies, groups of 10 leukemic mice per dose were injected with 12–24 μCi of 211At-30F11-B10 or 211At-rat Ab-B10, followed two days later by rescue with 15 × 106 syngeneic bone marrow (BM) cells. Results: Delivery of 211At-30F1-B10 demonstrated excellent localization to the BM and spleen at 24 hours (79 and 18% ID/g, respectively) post injection with lower kidney and lung uptake (8.4 and 8.3% ID/g, respectively) at the same time point. Anti-CD45 RIT using 211At-30F11-B10 followed by syngeneic HCT led to a dose-dependent survival benefit in leukemic mice with a median survival (OS) of 120, 98 days, and 62 days for animals treated with 24, 20, and 12 μCi 211At-30F11-B10, respectively, compared with untreated control mice (median OS of 36 days) and mice treated with non-specific 211At-labeled rat Ab-B10 (median OS of 46 days) (Figure). Moreover, anti-CD45 RIT with 211At-30F11-B10 led to minimal toxicity with mild dose dependent leukopenia as the most pronounced lab abnormality. White blood cell count nadir was between 2.5 and 4.2 k/μL two weeks after HCT for mice treated with 24 and 12 μCi 211At-30F11-B10, respectively. Counts recovered to normal levels (6–8 K/μL) 4 weeks after HCT. Mild increases in transaminase levels were seen in mice that received 211At-30F11-B10, yet these values remained within the normal ranges (ALT 68–75 IU/L; AST 155–170 IU/L). Renal function after 211At-30F11-B10 did not significantly deviate from baseline (BUN 15–17 mg/dL; Cr 0.3–0.4 mg/dL). Conclusion: Taken together, these data suggest that anti-CD45 RIT using the α-emitting radionuclide 211At in conjunction with HCT is a promising therapeutic option for AML. Excellent targeting of radiation doses to BM and spleen was demonstrated with a favorable toxicity profile. Further investigation of anti-CD45 RIT for AML using 211At in clinical trials appears to be warranted. Disclosures: No relevant conflicts of interest to declare.

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The W–x–x–W Motif in the TSR1 of ADAMTS13 Is Important for Its Secretion

Blood ◽

10.1182/blood.v120.21.4389.4389 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4389-4389

Author(s):

Jing Ling ◽

Jian Su ◽

Zhenni Ma ◽

Changgeng Ruan

Keyword(s):

Shear Stress ◽

Binding Affinity ◽

Culture Medium ◽

Stress Condition ◽

Conflicts Of Interest ◽

Enzyme Linked Immunosorbent Assay ◽

High Shear ◽

High Shear Stress ◽

Wild Type ◽

Western Blots

Abstract Abstract 4389 Introduction: The W-x-x-W motif is commonly found in the thrombospondin type 1 repeat (TSR) of various extra cellular proteins called TSR super family proteins, including thrombospondins, ADAMTSs, F-spondin and properdin. The W–x–x–W motif is known to bind with heparin, heparan sulfate proteoglycans, collagen and transforming growth factor-β, suggesting functional significance in cell–cell interaction and/or cellular signaling. However, the function of W-x-x-W in ADAMTS13 is unclear. In this study, we investigated the role of the W-x-x-W motif of ADAMTS13. Materials and Methods: We generated a W-x-x-W mutant (W387A) construct of ADAMTS13, and expressed the mutant and the wild-type constructs in HELA cells. Percentage of the protein secretion was defined as the concentration in the culture medium divided by the concentrations in the culture medium and cell lysates, multiplied by 100%. The binding affinity of the mutant or wild-type ADAMTS13 was investigated by enzyme-linked immunosorbent assay. Measurement of ADAMTS13 proteolytic activity toward von Willebrand factor (VWF) multimers was based on the generation of a dimeric 176-kDa fragment resulting from cleavage of VWF at the Y1605-M1606 bond, under denaturing condition and high shear stress condition, analyzed by Western blots. Results: SDS-PAGE gel analysis showed that the W387A mutant was secreted less efficiently relative to the wild-type construct. As for the binding affinity for the VWF multimer, there was no difference between the wild-type and mutant ADAMTS13. The W387A mutant was less active under denaturing condition; the same result was reproduced when FRETS-VWF73 was used as the substrate. However, under high shear stress condition, the mutant was as efficient as the wild-type ADAMTS13. Conclusions: The W–x–x–W motif is necessary for efficient secretion of ADATMS13. Further studies are needed to determine the contribution of the motif to the VWF cleave activity of ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

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Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

Blood ◽

10.1182/blood.v122.21.1105.1105 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1105-1105

Author(s):

Erica A. Peterson ◽

Jonathan H Foley ◽

Michael J Krisinger ◽

Edward Conway

Keyword(s):

Flow Cytometry ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Complement Receptors ◽

Red Cell Membrane ◽

Western Blots ◽

Complement Pathways

Abstract Introduction The plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways. The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses. Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and Results Plasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces. We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%. We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. Conclusions Plasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

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The Parallel Signaling Pathways Of Phosphatidylserine (PS) Exposure Downstream Of Platelet FcγRIIa

Blood ◽

10.1182/blood.v122.21.3514.3514 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3514-3514 ◽

Cited By ~ 2

Author(s):

Pierrette Andre ◽

Steven E. McKenzie ◽

Wolfgang Bergmeier

Keyword(s):

Signaling Pathways ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Annexin V ◽

Therapeutic Benefit ◽

Human Platelets ◽

Molecular Signaling ◽

P2y12 Inhibitor ◽

Immune Mediated ◽

Static Conditions

Abstract Platelet FcγRIIa is an important component of heparin-induced thrombocytopenia and other immune-mediated thrombocytopenia and thrombosis syndromes. Platelet FcγRIIa, an ITAM receptor, signals not only to cause aggregation and secretion, but also to PS exposure in the platelet procoagulant response when platelets are co-stimulated via the GCPR PARs. The molecular mechanisms downstream of FcγRIIa that lead to PS exposure are incompletely understood. Recently, we (WB; Ahmad et al., JTH 2011; 9:2077) demonstrated that downstream of PAR and GPVI/FcRγ, another ITAM receptor, there are CalDAG-GEF I (CDGI)-dependent and CDGI-independent, ADP/P2Y12-dependent parallel signaling pathways to PS exposure. In this study, we investigated the molecular signaling requirements for PS exposure downstream of FcγRIIa + PAR dual stimulation. We studied the exposure of PS in FcγRIIa transgenic (tg) mouse platelets following dual stimulation through FcγRIIa via anti-mouse CD9 and through PAR4 via PAR4 activating peptide (PAR4AP; AYPGKF). Washed platelets from FcγRIIa-tg mice and FcγRIIa-tg/CDG1-/- mice were stimulated with varying concentrations of anti-mouse CD9 and PAR4-AP (200uM) under static conditions and immediately measured for PS exposure by labeled Annexin-V in flow cytometry. We observed that at 0.5ug/ml of anti-mouse CD9 that the PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 70% of the FcγRIIa-tg platelets. However, when the platelets are pre-incubated with P2Y12 inhibitor MesAMP at 100uM, the PS exposure of the FcγRIIa-tg platelets is decreased by approximately 50% (n=3). For the FcγRIIa-tg/CDG1-/- platelets in the presence of MesAMP, PS exposure is completely abolished (n=3). This indicates that CDG1 contributes part of the signal that leads to PS exposure, while ADP/P2Y12 contributes the other CDGI-independent part of PS exposure downstream of FcγRIIa and GPCR dual stimulation. At a lower concentration of anti-mouse CD9 (0.25ug/ml; near threshold), the level of PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 80% of the FcgRIIa-tg platelets. In addition, at 0.25 ug/ml of anti-CD9, FcγRIIa-tg/CDG1-/- platelets pre-incubated with the P2Y12 inhibitor revealed no stimulated PS exposure. This observation indicates that ADP/P2Y12 plays a more significant role in PS exposure as the concentration of FcγRIIa stimulant nears threshold. Eradication of procoagulant PS exposure may require targeting of both the CDGI-dependent and CDGI-independent pathways for optimum therapeutic benefit in HIT and other immune-mediated thrombocytopenia and thrombosis disorders. CDGI inhibitors useful in human platelets will allow translation of these findings. Disclosures: No relevant conflicts of interest to declare.

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