A high affinity,antidote‐controllable prothrombin and thrombin‐binding RNA aptamer inhibits thrombin generation and thrombin activity

Background:  The conversion of prothrombin to thrombin is one of two non‐duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti‐thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D‐14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. Objectives: We sought to explore the structure of R9D‐14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA_R9D‐14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results:  RNA_R9D‐14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I‐mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA_R9D‐14T significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC‐183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA_R9D‐14T anticoagulation in vitro. Conclusions:  Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA_R9D‐14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.     

Prohibitins are involved in protease‐activated receptor 1‐mediated platelet aggregation

Summary. Background: Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs have been reported to be involved in typhoid fever, obesity, and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown.Objectives: To investigate the role of PHBs in platelet aggregation. Methods and results: PHB1 and PHB2 were detected on the surfaces of human platelets by flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease‐activated receptor 1 (PAR1), as determined by Bm‐TFF2 (a PAR1 agonist)‐affinity chromatography, coimmunoprecipitation, and confocal microscopy. The platelet aggregation, α_IIbβ₃ activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U mL^?1) or PAR1‐activating peptide (PAR1‐AP) (20 μm ) were reduced or abolished as a result of the blockade of PHBs by anti‐PHB antibodies or their Fab fragments; however, the same results were not obtained with induction by high concentrations of thrombin (0.6 U mL^?1) or protease‐activated receptor 4‐activating peptide (300 μm ). The calcium mobilization in MEG‐01 megakaryocytes stimulated by PAR1‐AP was significantly suppressed by PHB depletion with RNA interference against PHB1 and PHB2. Conclusions: PHBs are localized on the human platelet membrane and are involved in PAR1‐mediated platelet aggregation. Until recently, PHBs were unknown as regulators of PAR1 signaling, and they may be effective targets for antiplatelet therapy.     

Heparin‐induced thrombocytopenia – therapeutic concentrations of danaparoid,unlike fondaparinux and direct thrombin inhibitors,inhibit formation of platelet factor 4–heparin complexes

Summary. Background: Treatment of heparin‐induced thrombocytopenia (HIT), a disorder in which anti‐platelet factor 4 (PF4)–heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non‐heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin‐mimicking pentasaccharide). PF4–heparin complexes form at optimal stoichiometric ratios. Objectives: To compare the effects of these various non‐heparin anticoagulants in disrupting the formation of PF4–heparin complexes, and PF4‐containing immune complexes. Patients/methods: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4–heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Results: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4–heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4‐transfected HEK‐293 EBNA cell line expressing the PF4 receptor CXCR3‐B (P = 0.0408), reduced PF4–heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4–heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4–heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Conclusions: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4‐containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.     

Anticoagulant characteristics of HD1‐22, a bivalent aptamer that specifically inhibits thrombin and prothrombinase

Summary. Background: HD1‐22 is a bivalent aptamer that binds to thrombin with high affinity (K_d = 0.65 nm ) and occupies both anion binding exosites without blocking the active centre of the enzyme. HD1‐22 has been developed by connecting the exosite 1 binding aptamer HD1 and the exosite 2 binding aptamer HD22 through a poly‐dA linker. Objectives: To characterize the anticoagulant profile of HD1‐22 in comparison to the clinically established direct acting thrombin inhibitors bivalirudin and argatroban, and to test the efficacy of antidote‐oligodeoxynucleotides. Methods and Results: HD1‐22 prolongs clotting times of the thrombin time, activated partial thromboplastin time, ecarin clotting time, and lag‐time of the tissue factor triggered thrombin generation assay in a dose‐dependent manner. On a molar basis, its anticoagulant activity was nearly identical to bivalirudin and superior to argatroban. Thrombin‐induced platelet aggregation was more effectively inhibited by HD1‐22 than by bivalirudin. The HD1‐22 aptamer retains the ability of the HD1‐moiety to bind to (pro)exosite 1 of prothrombin and inhibits the prothrombinase activity nearly 2‐fold better than HD1. The anticoagulant activities of HD1‐22 are fully reversed by addition of antidote‐oligodeoxynucleotides. Conclusions: The strong thrombin‐inhibiting activity, together with the availability of a rapid acting antidote strategy, makes HD1‐22 an interesting anticoagulant candidate, especially for use in clinical situations where effective anticoagulation and rapid reversal of the anticoagulant effect are required. The data obtained warrant further clinical studies.     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

Diannexin,an annexin A5 homodimer,binds phosphatidylserine with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet‐mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS‐containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet‐fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser‐induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with K_D values of 0.6 and 5 nm , respectively, and both bind to the same subpopulation of PS‐exposing platelets. Diannexin inhibited thrombin generation and platelet‐fibrin clot formation in vitro at 10 nm (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 μg g^?1 (P < 0.05) and activation at 0.25 μg g^?1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g^?1 (P < 0.01). Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation.     

Release of sphingosine‐1‐phosphate from human platelets is dependent on thromboxane formation

Summary. Background: Platelets release the immune‐modulating lipid sphingosine‐1‐phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin‐layer chromatography in [³H]sphingosine‐labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease‐activated receptor‐1‐activating peptide (PAR‐1‐AP, 100 μm ). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500‐mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U‐46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC‐transport. Furthermore, thrombin‐induced release of S1P was attenuated in platelets from TP‐deficient mice. Supernatants from PAR‐1‐AP‐stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P‐dependent manner via S1P receptors‐1 and ‐3. These effects were inhibited by ASA‐pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti‐inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.     

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials

• RAS proteins are expressed in platelets but their functions are largely uncharacterized.
• TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo.
• TC21 regulates platelet aggregation by control of α_IIbβ₃ integrin activation, via crosstalk with Rap1b.
• This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Summary

Background

Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS‐induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored.

Objectives

To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis.

Results

We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI–FcRγ immunoreceptor tyrosine‐based activation motif (ITAM)‐containing collagen receptor, in an Src‐dependent manner. GPVI‐induced platelet aggregation, integrin α_IIbβ₃ activation, and α‐granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal‐regulated kinase, were inhibited in TC21‐deficient platelets ex vivo. In contrast, these responses were normal in TC21‐deficient platelets following stimulation with P2Y, protease‐activated receptor 4 and C‐type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI–FcRγ‐ITAM‐specific. TC21 was required for GPVI‐induced activation of Rap1b. TC21‐deficient mice did not show a significant delay in injury‐induced thrombosis as compared with wild‐type controls; however, thrombi were unstable. Hemostatic responses showed similar effects.

Conclusions

TC21 is essential for GPVI–FcRγ‐mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

     

Platelet collagen receptor Glycoprotein VI‐dimer recognizes fibrinogen and fibrin through their D‐domains,contributing to platelet adhesion and activation during thrombus formation

Essentials

• Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi.
• GPVI‐dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D‐domains.
• Collagen, D‐fragment and D‐dimer may share a common or proximate binding site(s) on GPVI‐dimer.
• GPVI‐dimer–fibrin interaction supports spreading, activation and adhesion involving αIIbβ3.

Summary

Background

Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin.

Objectives

To determine if GPVI‐dimer, GPVI‐monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site.

Methods

Recombinant GPVI monomeric extracellular domain (GPVI_ex) or dimeric Fc‐fusion protein (GPVI‐Fc₂) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti‐GPVI‐dimer or anti‐αIIbβ3.

Results

Under static conditions, GPVI_ex did not bind to any fibrinogen substrate. GPVI‐Fc₂ exhibited specific, saturable binding to both D‐fragment and D‐dimer, which was inhibited by mFab‐F (anti‐GPVI‐dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI‐Fc₂ binding to D‐fragment or D‐dimer was abrogated by collagen type III, Horm collagen or CRP‐XL (crosslinked collagen‐related peptide), suggesting proximity between the D‐domain and collagen binding sites on GPVI‐dimer. Under low shear, adhesion of normal platelets to D‐fragment, D‐dimer, fibrinogen and fibrin was inhibited by mFab‐F (inhibitor of GPVI‐dimer) and abolished by Eptifibatide (inhibitor of αIIbβ3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbβ3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab‐F, supporting some independent GPVI‐dimer involvement in this interaction.

Conclusion

Only dimeric GPVI interacts with fibrinogen D‐domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.     

Neuropharmacological effects of lipoic acid and ubiquinone on δ‐aminolevulinic dehydratase,Na+, K+‐ATPase,and Mg2+‐ATPase activities in rat hippocampus after pilocarpine‐induced seizures

In this study, we investigated the effects of lipoic acid (LA) in the hippocampus oxidative stress caused by pilocarpine‐induced seizures in adult rats. Wistar rats were treated with 0.9% saline (i.p., control group), LA (10 mg/kg, i.p., LA group), ubiquinone [20 mg/kg, i.p., ubiquinone (UQ) group], pilocarpine (400 mg/kg, i.p., P400 group), and the association of LA (10 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.) or UQ (20 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of P400 (LA plus P400 group and UQ plus P400 group, respectively). After the treatments, all groups were observed for 1 h. The enzyme activities (δ‐aminolevulinic dehydratase (δ‐ALA‐D), Mg²⁺‐ATPase, and Na⁺, K⁺‐ATPase) were measured using spectrophotometric methods, and the results compared to values obtained from saline and pilocarpine‐treated animals. Protective effects of LA and UQ were also evaluated on the same parameters. We reported here for the first time that Na⁺, K⁺‐ATPase and δ‐ALA‐D activities inhibition and Mg²⁺‐ATPase stimulation in the pilocarpine model are probably attributed to the oxidative stress caused by seizures in the rat hippocampus. The addition of the antioxidants LA and UQ may reverses the previously mentioned Na⁺, K⁺‐ATPase and δ‐ALA‐D inhibitions and Mg²⁺‐ATPase stimulation. Conclusions: The oxidative stress plays an important signaling role in pilocarpine‐induced seizures, and antioxidant drugs might be considered as therapeutical tools in this pathology.     

Human corneal fibroblast migration and extracellular matrix synthesis during stromal repair: Role played by platelet‐derived growth factor‐BB,basic fibroblast growth factor,and transforming growth factor‐β1

The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1‐integrin and syndecan‐4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet‐derived GF (PDGF‐BB) and transforming GF‐β1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF‐BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.     

Impact of polymorphisms of the major histocompatibility complex class II,interleukin‐10, tumor necrosis factor‐α and cytotoxic T‐lymphocyte antigen‐4 genes on inhibitor development in severe hemophilia A

Summary. Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case‐controlled cohort study, 260 severely affected, mutation‐type‐matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin‐10 (IL‐10), tumor necrosis factor‐α (TNF‐α) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P < 0.05]. In TNF‐α, the A allele of the ?308G>A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL‐10, the ?1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high‐TNF‐α/high‐IL‐10 producers, as compared with 3% of non‐inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA‐4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the ?308G>A polymorphism in TNF‐α and ?1082A>G in IL‐10 in inhibitor patients confirmed the earlier published data. The CT60 single‐nucleotide polymorphism in CTLA‐4 is of apparently less importance.     

Involvement of α2‐adrenoceptors,imidazoline, and endothelin‐A receptors in the effect of agmatine on morphine and oxycodone‐induced hypothermia in mice

Potentiation of opioid analgesia by endothelin‐A (ET_A) receptor antagonist, BMS182874, and imidazoline receptor/α₂‐adrenoceptor agonists such as clonidine and agmatine are well known. It is also known that agmatine blocks morphine hyperthermia in rats. However, the effect of agmatine on morphine or oxycodone hypothermia in mice is unknown. The present study was carried out to study the role of α₂‐adrenoceptors, imidazoline, and ET_A receptors in morphine and oxycodone hypothermia in mice. Body temperature was determined over 6 h in male Swiss Webster mice treated with morphine, oxycodone, agmatine, and combination of agmatine with morphine or oxycodone. Yohimbine, idazoxan, and BMS182874 were used to determine involvement of α₂‐adrenoceptors, imidazoline, and ET_A receptors, respectively. Morphine and oxycodone produced significant hypothermia that was not affected by α₂‐adrenoceptor antagonist yohimbine, imidazoline receptor/α₂ adrenoceptor antagonist idazoxan, or ET_A receptor antagonist, BMS182874. Agmatine did not produce hypothermia; however, it blocked oxycodone but not morphine‐induced hypothermia. Agmatine‐induced blockade of oxycodone hypothermia was inhibited by idazoxan and yohimbine. The blockade by idazoxan was more pronounced compared with yohimbine. Combined administration of BMS182874 and agmatine did not produce changes in body temperature in mice. However, when BMS182874 was administered along with agmatine and oxycodone, it blocked agmatine‐induced reversal of oxycodone hypothermia. This is the first report demonstrating that agmatine does not affect morphine hypothermia in mice, but reverses oxycodone hypothermia. Imidazoline receptors and α₂‐adrenoceptors are involved in agmatine‐induced reversal of oxycodone hypothermia. Our findings also suggest that ET_A receptors may be involved in blockade of oxycodone hypothermia by agmatine.