An orthosteric/allosteric bivalent peptide agonist comprising covalently linked protease-activated receptor-derived peptides mimics in vitro and in vivo activities of activated protein C.

BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.

Activated protein C and PAR1-derived and PAR3-derived peptides are anti-inflammatory by suppressing macrophage NLRP3 inflammasomes.

Essentials Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective effects. We tested whether APC or non-canonical PAR-derived peptides suppress inflammasome activity. APC or PAR1- and PAR3-derived peptides restrict inflammasome-dependent caspase-1 activity. Combined PAR1-derived and PAR3-derived peptides synergistically suppress caspase-1 activity. ABSTRACT: Background Activated protein C (APC) has been shown to restrict murine inflammasome activity. However, whether APC can exert anti-inflammatory activity in part through suppression of inflammasome activation in human systems is unknown. Objectives Studies were made to determine whether either APC or protease activated receptor (PAR)-derived peptides can reduce NLRP3 inflammasome activity in differentiated human THP-1 macrophage-like cells or in primary human monocytes stimulated to activate the inflammasome. Methods Human THP-1 cells or primary human monocytes were differentiated, treated with APC or PAR-derived peptides, and then stimulated with lipopolysaccharide and ATP to induce caspase-1 activity, a product of inflammasome activation. Results Activated protein C or noncanonical PAR1-derived or PAR3-derived peptides significantly reduced caspase-1 activity, detection of fluorescent NLRP3, and IL-1ß release from THP-1 cells. At low concentrations where no effect was observed for each individual peptide, combinations of the PAR1-derived peptide and the PAR3-derived peptide resulted in a significant synergistic decrease in caspase-1 and IL-1ß release. Caspase-1 activity was also reduced in primary human monocytes. Studies using blocking antibodies and small molecule PAR1 inhibitors suggest that EPCR, PAR1, and PAR3 each play roles in the observed anti-inflammatory effects. Several shortened versions of the PAR1- and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. Conclusions The results indicate that both APC and certain PAR1- and PAR3-derived peptides, which are biased agonists for PAR1 or PAR3, can reduce inflammasome activity in stimulated human monocytes as measured by caspase-1 activity and IL-1ß release and that PAR-derived biased peptide agonist combinations are synergistically anti-inflammatory.

Hepatic thrombopoietin gene silencing reduces platelet count and breast cancer progression in transgenic MMTV-PyMT mice.

In humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm3 combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Antithrombotic Strategies After Bioprosthetic Aortic Valve Replacement: A Systematic Review.

BACKGROUND: The optimal antithrombotic regimen after bioprosthetic aortic valve replacement (bAVR) is unclear. We conducted a systematic review of various anticoagulation strategies following surgical or transcatheter bAVR (TAVR). METHODS: We searched Medline, PubMed, Embase, Evidence-Based Medicine Reviews, and gray literature through June 2017 for controlled clinical trials and cohort studies that directly compared different antithrombotic strategies in nonpregnant adults who had undergone bAVR. We assessed risk of bias and graded the strength of the evidence using established methods. RESULTS: Of 4,554 titles reviewed, 6 clinical trials and 13 cohort studies met inclusion criteria. We found moderate-strength evidence that mortality, thromboembolic events, and bleeding rates are similar between aspirin and warfarin after surgical bAVR. Observational data suggest lower mortality and thromboembolic events with aspirin combined with warfarin compared with aspirin alone after surgical bAVR, but the effect size is small and the combination is associated with a substantial increase in bleeding risk. We found insufficient evidence for all other treatment comparisons in surgical bAVR. In TAVR patients, we found moderate-strength evidence that mortality, stroke, and major cardiac events are similar between dual antiplatelet therapy and aspirin alone, though a nonsignificantly lower rate of bleeding occurred with aspirin alone. CONCLUSIONS: Treatment with warfarin or aspirin leads to similar outcomes after surgical bAVR. Combining aspirin with warfarin may lead to a small decrease in thromboembolism and mortality, but is accompanied by increased bleeding. For TAVR patients, aspirin is equivalent to dual antiplatelet therapy for reducing thromboembolism and mortality, with a possible decrease in bleeding.

Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function.

The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

The BCR-ABL inhibitor ponatinib inhibits platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling, platelet activation and aggregate formation under shear.

BACKGROUND: Treatment of chronic myelogenous leukemia (CML) with the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib significantly improves patient outcomes. As some patients are unresponsive to imatinib, next generation BCR-ABL inhibitors such as nilotinib have been developed to treat patients with imatinib-resistant CML. The use of some BCR-ABL inhibitors has been associated with bleeding diathesis, and these inhibitors have been shown to inhibit platelet functions, which may explain the hemostasis impairment. Surprisingly, a new TKI, ponatinib, has been associated with a high incidence of severe acute ischemic cardiovascular events. The mechanism of this unexpected adverse effect remains undefined. OBJECTIVE AND METHODS: This study used biochemical and functional assays to evaluate whether ponatinib was different from the other BCR-ABL inhibitors with respect to platelet activation, spreading, and aggregation. RESULTS AND CONCLUSIONS: Our results show that ponatinib, similar to other TKIs, acts as a platelet antagonist. Ponatinib inhibited platelet activation, spreading, granule secretion, and aggregation, likely through broad spectrum inhibition of platelet tyrosine kinase signaling, and also inhibited platelet aggregate formation in whole blood under shear. As our results indicate that pobatinib inhibits platelet function, the adverse cardiovascular events observed in patients taking ponatinib may be the result of the effect of ponatinib on other organs or cell types, or disease-specific processes, such as BCR-ABL+cells undergoing apoptosis in response to chemotherapy, or drug-induced adverse effects on the integrity of the vascular endothelium in ponatinib-treated patients.

Thrombin mutant W215A/E217A treatment improves neurological outcome and attenuates central nervous system damage in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination and axonal damage of the central nervous system. The pathogenesis of MS has also been linked to vascular inflammation and local activation of the coagulation system, resulting in perivascular fibrin deposition. Treatment of experimental autoimmune encephalomyelitis (EAE), a model of human MS, with antithrombotic and antiinflammatory activated protein C (APC) reduces disease severity. Since recombinant APC (Drotecogin alfa), originally approved for the treatment of severe sepsis, is not available for human MS studies, we tested the hypothesis that pharmacologic activation of endogenous protein C could likewise improve the outcome of EAE. Mice were immunized with murine myelin oligodendrocyte glycoprotein (MOG) peptides and at the onset of EAE symptoms, were treated every other day with either WE thrombin (25 µg/kg; i.v.), a selective recombinant protein C activator thrombin analog, or saline control. Mice were monitored for changes in disease score until euthanized for ex vivo analysis of inflammation. Administration of WE thrombin significantly ameliorated clinical severity of EAE, reduced inflammatory cell infiltration and demyelination, suppressed the activation of macrophages comprising the CD11b + population and reduced accumulation of fibrin (ogen) in the spinal cord. These data suggest that symptomatic MS may respond to a treatment strategy that involves temporal pharmacological enhancement of endogenous APC generation.

Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation.

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.