Inhibition Of Thrombus Formation Research Articles

LX0702, a novel snake venom peptide derivative, inhibits thrombus formation via affecting the binding of fibrinogen with GPIIb/IIIa

Based on the structure of AAP, a novel anti-thrombotic peptide from snake venom which we discovered in our previous study, more than 60 compounds were designed and synthesized. One of these termed as LX0702 exhibited stronger anti-platelet aggregation activity than AAP. This study aims to investigate the effects of LX0702 on anti-thrombotic formation and its underlying mechanism. We found that LX0702 inhibited platelet aggregation induced by ADP, thrombin and collagen in a dose dependent manner, with IC50 values of 49.90 ± 2.03, 50.65 ± 0.34 and 83.90 ± 2.06 μM, respectively. It also inhibited thrombus formation in the rat arterio-venous shunt model. In addition, the effect of LX0702 on hemostasis system was tested. Compared to control saline, bleeding time was not prolonged. Furthermore, the ELISA revealed that LX0702 inhibited fibrinogen binding with GPIIb/IIIa in a dose dependent manner with an IC50 value of 1.26 ± 0.13 μM. These findings clearly demonstrate that LX0702 has anti-platelet/anti-thrombotic effects without increased bleeding risk. Therefore it might be developed into an effective drug for the prevention or treatment of thrombotic diseases.

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar

AbstractProtease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand–binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein–coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways.

LVAD inflow cannula covered with a titanium mesh induces neointimal tissue with neovessels.

Thrombus formation at the interfaces of inflow cannulas and left ventricular apexes is considered to be one of the predominant sources of thromboembolic complication. Use of a fine titanium mesh-covered inflow cannula is expected to prevent such thrombus formation by inducing neointimal tissue around the cannula. Titanium pins (20 mm long, 3 mm diameter) covered with a fine titanium mesh (wire dia. 85 μm; volumetric porosity 40-70%) were developed to mimic the inflow cannulas of left ventricular assist devices (LVADs). Smooth-surface pins of the same size were also designed. The pins were implanted into the left ventricular apexes of rabbits. The rabbits were bled without anticoagulation for between 1 week and 1 year. The tissues around the pins were evaluated histologically. 28 rabbits (mesh group, 15; smooth group, 13) were evaluated. The mesh-covered pins inhibited thrombus formation to a remarkable degree throughout the entire observation period. The tissues around the mesh-covered titanium pins appeared to be in the process of conversion from thrombus formation to granulation, resulting in the replacement of fibrotic tissue containing myofibroblasts with endothelialized and angiogenic tissue. On the surface of the mesh-covered pins, endothelial cells were arranged without platelet adhesion. The tissue formed around the smooth-surface pins was partially organized into a thrombus without angiogenesis. The titanium-mesh surface modification prevented thrombus formation with formation of neointimal tissue with endothelialization and angiogenesis. This surface modification could prevent wedge thrombus formation among patients supported by LVADs.

Thrombolytic and Antiplatelet Effects of a Novel Plasminogen Activator from the Venom of Gloydius Brevicaudus Viper.

To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of the Gloydius brevicaudus viper (GBV-PA) in vitro and in vivo. Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis and in dog model of acute cerebral infarction. Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis. In vitro, we assayed the antithrombotic effect of GBV-PA on rabbit blood clots, euglobulin lysis time (ELT) of rabbit plasma, and ADP-induced platelet aggregation. GBV-PA intravenous administration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis. The arterial recanalization rates were dose- and time-dependently improved after the administration of GBV-PA in canine acute cerebral infarction model. Thrombus length and weight were significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models. Thrombus formation in the blood of rabbits that were administered of GBV-PA was also inhibited. GBV-PA radically reduced plasma ELT of the rabbit's blood clots. ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9 μg/mL. This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent. It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis. The mechanisms that underline its antithrombotic effects were related to GBV-PA's capabilities of increasing fibrinolytic activity and inhibition of platelet aggregation.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3β and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Exogenous Bradykinin Inhibits Tissue Factor Induction and Deep Vein Thrombosis via Activating the eNOS/Phosphoinositide 3-Kinase/Akt Signaling Pathway

Background/Aims: Bradykinin has been shown to exert a variety of protective effects against vascular injury, and to reduce the levels of several factors involved in the coagulation cascade. A key determinant of thrombin generation is tissue factor (TF). However, whether bradykinin can regulate TF expression remains to be investigated. Methods: To study the effect of bradykinin on TF expression, we used Lipopolysaccharides (LPS) to induce TF expression in human umbilical vein endothelial cells and monocytes. Transcript levels were determined by RT-PCR, protein abundance by Western blotting. In the in vivo study, bradykinin and equal saline were intraperitoneally injected into mice for three days ahead of inferior cava vein ligation that we took to induce thrombus formation, after which bradykinin and saline were injected for another two days. Eventually, the mice were sacrificed and tissues were harvested for tests. Results: Exogenous bradykinin markedly inhibited TF expression in mRNA and protein level induced by LPS in a dose-dependent manner. Moreover, the NO synthase antagonist L-NAME and PI3K inhibitor LY294002 dramatically abolished the inhibitory effects of bradykinin on tissue factor expression. PI3K/Akt signaling pathway activation induced by bradykinin administration reduced the activity of GSK-3ß and MAPK, and reduced <smlcap>NF-</smlcap>κ<smlcap>B </smlcap>level in the nucleus, thereby inhibiting TF expression. Consistent with this, intraperitoneal injection of C57/BL6 mice with bradykinin also inhibited the thrombus formation induced by ligation of inferior vena cava. Conclusion: Bradykinin suppressed TF protein expression in human umbilical vein endothelial cells and monocytes in vitro; in line with this, it inhibits thrombus formation induced by ligation of inferior vena cava in vivo.

Targeted antithrombotic protein micelles.

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.

Discovery of a Novel, Potent, Selective and Injectable Small Molecule Inhibitor of Blood Coagulation Factor XIa, ONO-8610539: in Vitro and in Vivo Pharmacological Profiles

Introduction: Congenital human blood coagulation factor XI (FXI) deficiency is associated with bleeding that is less severe than that observed in patients with other coagulation factor deficiencies, whereas severe FXI deficiency confers the decreased risk of deep vein thrombosis. FXI knockout mice show a reduction in venous thrombus formation with no bleeding. Therefore, FXI is considered to be a promising drug target for treatment and prevention of venous thromboembolism without increasing bleeding risk. Recently, we discovered a novel, potent, selective and injectable small molecule inhibitor of activated FXI (FXIa), ONO-IG-012, and evaluated the in vitro and in vivo pharmacological profiles of the compound.Methods: In anin vitro study, inhibitory effects of ONO-IG-012 on enzyme activities of human FXIa, other blood coagulation factors, and fibrinolytic factors were evaluated. Anticoagulant effects of ONO-IG-012 were also evaluated in human and rabbit plasma. In in vivo studies, the antithrombotic and hemorrhagic effects of ONO-IG-012 were compared to those of enoxaparin in rabbit models of deep venous thrombosis and femur hemorrhage. In the thrombosis model, under ketamine and xylazine anesthesia, inferior vena cava was isolated and partially ligated to reduce blood flow. The vein at the distal site was wrapped with filter paper saturated with ferric chloride (FeCl3) solution for 15 minutes to induce endothelial injury and subsequent thrombus formation. Sixty minutes after the application of FeCl3, the thrombus wet weight was measured. In the bleeding model, a puncture wound was made into the medullary canal at the epiphysis of femur using a drill under isoflurane anesthesia and mechanical ventilation. Blood was continuously collected with absorbent cotton for one hour, and the blood loss volume was calculated from its specific gravity. Intravenous administration of ONO-IG-012 or enoxaparin was initiated as a loading dose an hour before applying FeCl3 or producing a puncture wound, followed by their maintenance dose infusion. Blood was collected to measure APTT and PT just before the administration of the compounds and applying FeCl3 or producing a puncture wound.Results: ONO-IG-012 competitively inhibited human FXIa with a Ki value of 0.0019 μmol/L. Although ONO-IG-012 moderately inhibited human plasma kallikrein with a Ki value of 0.15 μmol/L, it had little effect on other human blood coagulation factors, and fibrinolytic factors [thrombin, FVIIa, FIXa, FXa, FXIIa, tPA, urokinase, and plasmin (Ki value >100 μmol/L)]. ONO-IG-012 prolonged APTT, and the concentration required to double the APTT was 0.098 μmol/L in human plasma and 0.30 μmol/L in rabbit plasma. However, prolongation of PT was not observed even at 33 μmol/L. ONO-IG-012 inhibited thrombus formation even at a dose as low as 0.1 mg/kg/h and achieved maximum antithrombotic effect at greater than or equal to 0.3 mg/kg/h. The ex vivo APTT was increased from baseline by 1.7 ± 0.0-fold at 0.1 mg/kg/h, by 2.8 ± 0.3-fold at 0.3 mg/kg/h, and by 5.4 ± 0.4-fold at 1 mg/kg/h, while PT showed no changes at any of the concentrations tested. Enoxaparin also inhibited thrombus formation at a dose of 10 IU/kg/h, and the antithrombotic effect at 30 IU/kg/h was comparable to that of ONO-IG-012 at 0.3 mg/kg/h (−87% vs. −80%). ONO-IG-012 did not affect the blood loss volume at all even at 10 mg/kg/h, which is 33-fold higher than the dose showing maximum antithrombotic effect (0.3 mg/kg/h). At 10 mg/kg/h, the blood loss volume was 0.7 ± 0.1 mL which is not statistically significant as compared to 1.2 ± 0.3 mL in the vehicle group, and the APTT ratio was 9.0 ± 1.1-fold. In contrast, enoxaparin increased the blood loss volume dose-dependently with the values of 4.5 ± 1.8 mL (not statistically significant) at 10 IU/kg/h, 8.4 ± 2.2 mL (P <0.01) at 30 IU/kg/h, and 23.1 ± 5.0 mL (P <0.001) at 100 IU/kg/h.Conclusions: ONO-IG-012 demonstrated a competitive, highly selective and potent inhibitory effect on FXIa among proteases involved in blood coagulation or fibrinolysis and a potent anticoagulant effect on APTT. ONO-IG-012 did not affect the blood loss volume at all even at 33-fold higher dose than the dose showing the maximum antithrombotic effect comparable to enoxaparin. ONO-IG-012 is expected to be a novel potent anticoagulant without an increased risk of bleeding for the treatment and prevention of venous thromboembolism. DisclosuresNo relevant conflicts of interest to declare.

ML359, a Small Molecule Inhibitor of Protein Disulfide Isomerase That Prevents Thrombus Formation and Inhibits Oxidoreductase but Not Transnitrosylase Activity

Vascular thiol isomerases comprise a family of enzymes including protein disulfide isomerase (PDI), ERp5, and ERp57 that are important in the process of thrombus formation. PDI is secreted at sites of vascular injury, and antibody-mediated PDI inhibition prevents thrombus formation in a mouse laser injury model. Our group has previously reported on the discovery of the small molecule PDI inhibitors quercetin-3-rutinoside and ML359. Identified as part of a high-throughput screen, ML359 is a second-generation PDI inhibitor that selectively blocks PDI oxidoreductase activity with approximately ten-fold the potency of quercetin-3-rutinoside. To better understand the mechanism of allosteric modulation of PDI by small molecules, we evaluated the association of ML359 with isolated domains of PDI, determined the effects of ML359 on a variety of PDI functions, and compared the activity of ML359 to that of quercetin-3-rutinoside. PDI is composed of four thioredoxin-like domains and an x-linker region in the sequence a-b-b’-x-a’. Major substrate binding is thought to occur in the b-b’ region while the a and a’ domains contain catalytically active cysteine motifs (CGHC) that mediate the oxidoreducase, nitrosylase, and thiol isomerase functions of PDI. In order to identify potential binding sites of ML359 on PDI, we constructed and expressed the domain fragments a, ab, abb’, b’xa’, and a’. These fragments were tested in the presence of 10 µM ML359 using an insulin turbidometric assay that measures the oxidoreductase activity of PDI. ML359 demonstrated full inhibition of oxidoreductase activity when full-length PDI and the b’xa’ fragment were used whereas no inhibition was observed with the other fragments assayed. These results are consistent with docking studies showing that ML359 likely binds in a pocket formed at the b’x interface. In contrast, when the same experiment was performed in the presence of 30 µM of quercetin-3-rutinoside, inhibition was only noted with full-length PDI and the abb’ and b’xa’ fragments, suggesting that binding was dependent on the b’ and not the x-linker region. To determine if ML359 has differential effects on the oxidoreductase and nitrosylase functions of PDI, we utilized a platelet-based assay in which fluorescence intensity stemming from the NO-sensitive intracellular dye DAF-FM was measured as an indicator of PDI-mediated translocation of NO from the extracellular surface into the cytosol (transnitrosylation). While quercetin-3-rutinoside potently inhibited PDI-mediated transnitrosylation activity, ML359 had no effect. These results are consistent with the idea that the transnitrosylase and oxidoreducase functions of PDI are separable and inhibition of either is specific to the small molecule used. We evaluated the ability of ML359 to inhibit thrombosis in a mouse laser injury model. Intravital microscopy was used to follow thrombus formation in mouse cremaster arterioles after laser-induced vascular injury. Infusion of ML359 resulted in inhibition of thrombus formation, in contrast to thrombosis seen after infusion of vehicle alone. In summary, ML359 is a second generation small molecule inhibitor of PDI that likely binds at the b’x interface of the enzyme. Furthermore, ML359 is able to selectively inhibit PDI oxidoreductase activity without affecting transnitrosylase activity. ML359 may prove a useful molecular probe to better understand the different functions of PDI relative to thrombus formation in vivo. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Protein Disulfide Isomerase By S-Nitrosylation Controls Its Function during Thrombus Formation

Protein disulfide isomerase (PDI) is an oxidoreductase that is essential for thrombus formation following vascular injury. Clinical trials testing the efficacy and safety of PDI inhibition in the setting of thrombotic disease are currently underway. Yet while preclinical and clinical trials of PDI in thrombosis have progressed rapidly, the mechanisms by which PDI is regulated in the vasculature and how it mediates thrombosis remain unknown. PDI has an a-b-b’-x-a’ domain structure, where the a and a’ domains contain a CGHC motif responsible for cleaving and forming disulfide bonds. The active site cysteines within the catalytic CGHC motif that perform oxidoreductive reactions can also undergo S-nitrosylation. We have evaluated the hypothesis that nitric oxide (NO) converts PDI into a nitrosylase and regulates PDI oxidoreductase activity in the vasculature during thrombus formation. Initial studies demonstrated that incubation of recombinant PDI with the NO donor, SNAP, resulted in an 83±1.4% decrease in its reductase activity. A transnitrosylase assay using the NO indicator DAF-FM showed that S-nitrosylated PDI (SNO-PDI) transferred NO into platelets and inhibited platelet aggregation. To define the molecular determinants of PDI nitrosylation activity, we evaluated mutant PDIs containing Cys -> Ala mutations of the CGHC (a domain)/CGHC (a’ domain) motifs in the platelet-based transnitrosylase assay. Wild-type PDI (CGHC/CGHC) demonstrated full reductase and nitrosylase activity and the enzymatically dead mutant (AGHA/AGHA) showed neither activity. In contrast, the CGHA/CGHA mutant maintained nitrosylase activity (41±0.23%), but had no reductase activity. This observation suggested that reductase and nitrosylase activities were separable. To further evaluate this supposition, we screened a series of PDI mutants in which intervening sequences of the CGHC domain had been modified. The screen identified CGPC/CGPC as a nitrosylase-biased mutant that showed a 59±2.31% decrease in reductase activity, but a 72±1.83% increase in nitrosylase activity compared to wild-type PDI. Another nitrosylase-biased mutant, CGRC/CGRC, showed a similar activity pattern. Since PDI is prothrombotic and SNO-PDI is antithrombotic, we compared the activity of nitrosylase-biased mutants with wild-type PDI in platelet aggregation studies in the presence of physiological concentrations of GSNO. While wild-type PDI had little effect on platelet aggregation, nitrosylase-biased PDIs such as the CGPC/CGPC and CGRC/CGRC mutant completely inhibited platelet aggregation. These studies show that the prothrombotic oxidoreductase activities of PDI are separable from their antithrombotic nitrosylase activities and that nitrosylase-biased PDI mutants have antiplatelet activity. We next evaluated the effect of PDI nitrosylation on thrombus formation in vivo. Infusion of SNO-PDI into mice inhibited thrombus formation following laser-induced vascular injury of cremaster arterioles. Mice deficient in glutathione-S-nitrosyl reductase (GSNOR) were used to assess the role of endogenous NO in thrombus formation. GSNOR enzymatically reduces GSNO, the main storage form of NO in cells. Platelet accumulation and fibrin formation were hardly detectable in GSNOR-/- mice. Infusion of recombinant WT PDI, but not an enzymatically dead PDI, reversed the defect in platelet accumulation and fibrin generation to levels of WT mice. In order to visualize NO during thrombus formation, the NO-sensitive dye DAF-FM was infused into mice and NO signal in endothelium monitored following laser-induced injury. DAF-FM signal decreased rapidly following laser injury of cremaster arterioles, indicating an activation-induced reduction in endothelial NO in vivo. In conclusion, our studies show that oxidoreductase and nitrosylase activities of PDI are separable and support a model whereby high endothelial NO levels maintain vascular quiescence in part by maintaining PDI as a nitrosylase and blocking its prothrombotic PDI activity. We propose that the reduction of NO levels that occurs with vascular injury or endothelial dysfunction contributes to the conversion of PDI from an anti-thrombotic nitrosylase to a prothrombotic reductase. DisclosuresNo relevant conflicts of interest to declare.

Effect of traditional Uyghur medicine abnormal Savda Munziq extract on rabbit platelet aggregation in vitro and rat arteriovenous shunt thrombosis in vivo

ObjectiveAbnormal Savda Munziq (ASMq) is a standard herbal preparation used in Uyghur traditional medicine to fight chronic diseases including cardiovascular, but its specific effects on thrombosis and platelet aggregation are unknown. MethodsRabbit platelets were incubated with ASMq extract (10, 20, 40µg/ml) for 15, 30, or 60min, and aggregation was induced with ADP and collagen. In vivo, ASMq extract 2.97g/kg, 5.94g/kg, 11.88g/kg per os daily for 15 days were tested on thrombus wet weight in a rat model of arterio-venous bypass thrombosis. Plasma thromboxane B2 (TXB2) and 6-keto-PGF1a (6PG) were measured by radioimmunoassay. Aspirin (12µg/ml and 5mg/kg) and saline were used as control in both experiments. ResultsASMq inhibited ADP and collagen-induced aggregation in vitro in a dose-dependent manner that increased over time, to a maximum of 6.4±1.3% and 21.6±4.0% for ADP and collagen, respectively, at one hour׳s incubation with the highest concentration, whereas the effects of aspirin (34.5±2.2% and 41.9±2.5%, respectively) were stable over time. In vivo, ASMq inhibited thrombus formation dose-dependently, by 70% at the highest dose, compared to 67% with aspirin. ASMq essentially did not change prostaglandin production, compared to the clear inhibition by aspirin. ConclusionAbnormal Savda Munziq extract inhibits dose-dependent platelet aggregation with ADP or collagen in vitro and thrombosis in vivo to values similar to those of aspirin, though unlike aspirin this effect does not seem mediated by an inhibition of cyclo-oxygenase.

Effect of low voltage AC fields on cardiovascular implants.

Coronary Artery Stents have been the preferred form of treatment for vascular occlusive disease, due to the minimally invasive surgical procedure, post-operative recovery time and cost, when compared to open coronary bypass surgery. The cellular response upon applying an AC electric field to type 316LM Stainless Steel stent mimics was investigated in this paper. The highest RBC adhesion was observed at voltages higher than 88 mV and lower than 74 mV. Their unique alignment along the lines of fracture on the stent surface at 88 mV was a phenomenon caused by an increase in electrical conductivity in these regions. Being able to control RBC adhesion may have various clinical implications such as inhibition of thrombus formation, and provide a basis to analyse whether electric fields may be applied to cancer therapy as well.

Thrombotic safety of prothrombin complex concentrate (Beriplex P/N) for dabigatran reversal in a rabbit model

IntroductionIn vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding. Materials and methodsAnesthetized rabbits were treated with initial 0, 75, 200 or 450μgkg-1 dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15min after the start of dabigatran administration, PCC doses of 0, 50 or 300IUkg-1 were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model. ResultsAt the suprapharmacologic dose of 300IUkg-1, PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75μgkg-1 dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300IUkg-1 PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p=0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding. ConclusionsIn this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran.

Abstract 499: Development of a Novel Antithrombotic Therapy Targeting Platelet GPIbα: Assessment of Anfibatide In Vitro and in Phase I Clinical Trial

Platelet adhesion and aggregation are critical for hemostasis and atherothrombosis. Interaction between the GPIb complex and VWF initiates platelet adhesion and contributes to complete vessel occlusion, particularly at sites of stenosis. However, no therapeutic targeting this pathway has been developed. Previously, we demonstrated in murine thrombosis models that Anfibatide, a snake venom-derived GPIbα antagonist, inhibited botrocetin-induced VWF-GPIbα binding and inhibited thrombus formation in vitro and in vivo, without significantly activating platelets or changing bleeding. We subsequently hypothesized that Anfibatide would be a safe and potent anti-thrombotic agent in humans. We tested Anfibatide with human platelets in vitro and conducted the first anti-GPIbα phase I clinical trial with 94 healthy volunteers. In vitro, Anfibatide inhibited ristocetin-induced and low dose thrombin-induced human platelet aggregation. Surface plasmon resonance studies showed that Anfibatide binds to GPIbα and blocks binding of both VWF A1 domain and thrombin. In ex vivo perfusion chambers, Anfibatide strongly inhibited human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface, especially at high shear flow conditions. Importantly, Anfibatide effectively dissolved the preformed thrombi in perfusion chambers. Anfibatide incubation with human whole blood did not significantly change coagulation parameters measured by thromboelastography. After treating volunteers with Anfibatide, no serious adverse events, premature discontinuations due to adverse events, or deaths occurred during the study. Anfibatide bound to 95% of GPIb and inhibited 90% of platelet aggregation without prolonging activated partial thromboplastin time, prothrombin time, thrombin time, or bleeding time. There was no bleeding tendency, no alteration of platelet count, and no detectable anti-Anfibatide antibodies. In conclusion, Anfibatide provides anti-thrombotic effects through inhibition of both GPIbα-VWF and GPIbα-thrombin interactions. These results demonstrate that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.

Effect of ADAMTS‐13 on cerebrovascular microthrombosis and neuronal injury after experimental subarachnoid hemorrhage

Microthrombosis and reactive inflammation contribute to neuronal injury after subarachnoid hemorrhage (SAH). ADAMTS-13 cleaves von Willebrand factor multimers, and inhibits thrombus formation and, seemingly, inflammatory reactions. To investigate the effect of ADAMTS-13 in experimental SAH. A total of 100 male C57/BL6 mice were randomly assigned to four groups: sham (n=15), SAH (n=27), vehicle (n=25), and ADAMTS-13 (n=23; 100μL per 10g of body weight of 100μg of ADAMTS-13 per 1mL of 0.9% NaCl; 20min after SAH). Neurologic performance was assessed on days1 and 2 after SAH. Animals were killed on day2. The amounts of subarachnoid blood, microthrombi, apoptosis and degenerative neurons were compared. The degree of neuronal inflammation and vasospasm was also compared. In five mice each (SAH and ADAMTS-13 groups), bleeding time was assessed 2h after SAH. Systemic administration of ADAMTS-13 achieved significant amelioration of microthrombosis and improvement in neurologic performance. ADAMTS-13 reduced the amount of apoptotic and degenerative neurons. A tendency for decreased neuronal inflammation was observed. ADAMTS-13 did not show any significant effect on vasospasm. The degree of systemic inflammation was not changed by ADAMTS-13 administration. ADAMTS-13 neither increased the amount of subarachnoid blood nor prolonged the bleeding time. ADAMTS-13 may reduce neuronal injury after SAH by reducing microthrombosis formation and neuronal inflammation, thereby providing a new option for mitigating the severity of neuronal injury after SAH.

Semi‐Mechanistic Modelling and Simulation of Inhibition of Platelet Aggregation by Antiplatelet Agents

Antiplatelet agents are a class of pharmaceuticals that decrease platelet aggregation and thus inhibit thrombus formation. We examined the relationships between plasma concentrations of antiplatelet agents (triflusal, clopidogrel and cilostazol) and the platelet aggregation inhibitory effect after dosing. We used triflusal, cilostazol and clopidogrel for the development of a semi-mechanistic PK/PD model. The drugs chosen are used widely and reflect various mechanisms of antiplatelet agents. Time courses of plasma concentrations of the antiplatelet agents and their platelet aggregation effects were analysed using ADPAT V. Pharmacokinetic profiles were fitted to an extended parent-metabolite pharmacokinetic model, based on a two-compartment model, and the pharmacodynamic effects of the agents were fitted to a platelet aggregation effect model that consisted of the following parameters: Ks , the active-form platelet synthesis rate constant; K, the apparent reaction rate constant of the agent and active-form platelets; Kel-PRP , the apparent rate constant of platelets; and ε, an intrinsic activity parameter. This semi-mechanistic PK/PD model described well the relationship between plasma concentrations of antiplatelet agents and platelet aggregation effects. In addition, the estimated parameters were suitable for the explanation of the agents and also have a good correlation with platelet characteristics, such as platelet half-life and platelet aggregation baseline effects. Specifically, we discovered the strong correlations between estimated K parameter and in vitro drug activity. We conclude that this semi-mechanistic PK/PD model explained drug PK/PD characteristics well and will be useful for accurate predictions of antiplatelet effect in the clinical situations.

Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s−1. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

Advances in Biomaterial’s Modification and Thrombosis Inhibition

With the development of modern medical technology, the clinical range of biomaterial applications have gradually expanded. However, bioactivity on the surface of biomaterials may be reduced and induce thrombus formation that will not only cause serious dysfunction of the biological materials, but can also affect the host. Modification of these biological materials can thus help to retain the biological activity on the surface of the material and inhibit thrombus formation, which underlines the importance of their clinical application. The current review will address the modification of biomaterials and inhibition of thrombosis on the surface of biomaterials.

Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, 13) cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface-exposed free sulfhydryls on ADAMTS13 that seemed to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow. A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2 to 8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5 to 8 Thrombospondin type 1 (TSP1) repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation were dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity. Our results demonstrate for the first time that the carboxyl terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol-dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.

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Introduction: Pelvic fractures are a complication of blunt force trauma and are frequently associated with multiple sites of injury. A majority of deaths occur in the first 48 hours as a result of massive hemorrhage into the pelvis, complicated by trauma-induced coagulopathy (TIC). Thromboelastographic (TEG)-guided blood component therapy (BCT) has been theorized to lower mortality and transfusion requirements in patients with TIC. Despite the high incidence of coagulopathy in patients with pelvic fractures, no case studies or guidelines exist for TEG use in these patients. Methods: In this study, we used TEG to guide BCT in 38 patients with pelvic fractures. Clinical data collected included: age, gender, injury mechanism, systolic blood pressure (SBP), calculated base deficit (BD), TEG® Platelet Mapping™ (TEG/PM) parameters, mortality, INR, PTT, Injury Severity Score (ISS), and Glasgow Coma Scale (GCS) not influenced by sedation upon admission in the ED. Results: In 30 of our 38 patients, adenosine diphosphate (ADP) and arachidonic acid (AA) inhibition of thrombus formation and strength were also evaluated by TEG/PM. We demonstrated significant statistical correlation between SBP, BD, maximal amplitude (MA), and massive transfusion (MT). We have also described statistical correlation between ISS, BD ≥ 8, MT, and survivorship. In our studied patient population, the MA predicted MT (MA 50.7 ± 2.2) versus non-MT (MA 59.9 ± 1.5). No correlation was shown between the other TEG parameters. The ratio of BCT administered was 2/2/1 (packed red cells, platelets, and fresh frozen plasma). Conclusions: We have concluded that TEG provides point of care bedside goal-directed BCT for patients with pelvic fractures. Our data support the published criteria that the MA predicts those patients who require MT. TEG/PM also provides guidance for platelet administration.