Adhesion Of Human Platelets Research Articles

Self-repairing nonfouling polyurethane coatings via 3D-grafting of PEG-b-PHEMA-b-PMPC copolymer

Long-lasting nonfouling polyurethane coatings via 3D-grafting of a triblock copolymer showed inhibition ability for the adhesion of protein and human platelets.

Vipegitide: a folded peptidomimetic partial antagonist of α2β1 integrin with antiplatelet aggregation activity.

Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10−10 M and 1.5×10−10 M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.

Endothelial retention and phenotype on carbonized cardiovascular implant surfaces

Heart valve disease is an increasing clinical burden for which there is no effective treatment outside of prosthetic replacement. Over the last 20 years, clinicians have increasingly preferred the use of biological prosthetics to mechanical valves despite their superior durability because of the lifelong anticoagulation therapy that is required. Mechanical valve surface engineering has largely focused on being as non-thrombogenic as possible, but despite decades of iteration has had insufficient impact on the anticoagulation burden. In this study, we systematically evaluate the potential for endothelialization of the pyrolytic carbon surface used in mechanical valves. We compared adsorbed adhesion ligand type (collagen I, fibronectin, laminin, and purified adhesion domain fragments GFOGER and FN7-10) and concentration on endothelial adhesion rates and adhesion strength on Medtronic-Hall prosthetic valve surfaces. Regardless of ligand type or concentration, endothelial adhesion strengthening was insufficient for their intended ultra-high shear stress environment. We then hypothesized that microfabricated trenches would reduce shear stress to tolerable levels while maintaining endothelial access to the flow stream, thereby promoting a confluent and anticoagulant endothelial monolayer. Computational fluid dynamics simulations predicted an empirical relationship of channel width, depth, and spacing that would maintain interior surface shear stress within tolerable levels. Endothelial cells seeded to confluence in these channels retained a confluent monolayer when exposed to 600 dyn/cm2 shear stress for 48 h regardless of applied adhesive ligand. Furthermore, sheared EC expressed a mature anti-coagulant profile, including endothelial nitric oxide synthase (eNOS), VE-cadherin, and significantly downregulated plasminogen activator inhibitor-1 (PAI-1). As a final test, channeled pyrolytic carbon surfaces with confluent EC reduced human platelet adhesion 1000-fold over pyrolytic carbon alone. These results advance a promising biohybrid approach to enable active moderation of local coagulative response in mechanical heart valves, which could significantly extend the utility of this important treatment for heart valve disease.

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.

The First In Vitro and In Vivo Assessment Of Anfibatide, a Novel Glycoprotein Ib Antagonist, In Mice and In a Phase I Human Clinical Trial

BackgroundPlatelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial. MethodsAnfibatide was purified from venom of the Agkistrodon acutus snake and its purity was analyzed by mass spectrometry. The effect of Anfibatide on murine platelet function was assessed by in vitro platelet aggregometry, ex vivo perfusion chamber, and two complementary in vivo intravital microscopy models. The effects of Anfibatide on human platelet aggregation and thrombus formation were studied in vitro, and thrombealastography (TEG) was also performed. Most importantly, we evaluated the safety and efficacy of Anfibatide on platelet function and coagulation in a total of 94 healthy human volunteers in a phase I clinical trial. ResultsMALDI-TOF mass spectrometry of Anfibatide showed only one peak and the mass to charge ratio is 29799.7. Anfibatide specifically inhibited ristocetin-induced human platelet aggregation. Interestingly, Anfibatide was not able to inhibit botrocetin-induced murine platelet aggregation in plate-rich plasma (PRP), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. We found Anfibatide did not affect ADP-, TRAP- or collagen-induced aggregation in PRP, suggesting its specificity to GPIb. In ex vivo perfusion, Anfibatide strongly inhibited murine and human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface at both high and low shear flow conditions although it is far more sensitive at high shear. Importantly, Anfibatide effectively dissolved the preformed thrombi when we continuously perfused Anfibatide-treated whole blood through perfusion chambers, demonstrating its potential as an anti-thrombotic therapy. In the mesenteric arteriole thrombosis model, Anfibatide strongly inhibited platelet adhesion, thrombus formation, and prevented vessel occlusion in response to FeCl3 injury (P<0.05). At sites of laser-injured cremaster arterioles, Anfibatide also dramatically inhibited platelet accumulation and thrombus growth. Anfibatide did not cause significant murine platelet activation in vitro and had no significant change in coagulation parameters in TEG when we treated human whole blood with Anfibatide, suggesting it had minimal side effects. In the phase I clinical trial, results showed that Anfibatide can occupy approximately 95% of GPIb and inhibit up to 90% of ristocetin specific platelet aggregation. The inhibitory effect was undetectable four hours after Anfibatide was withdrawn. There were no serious adverse events, or deaths that occurred during the study. Anfibatide did not significantly prolong bleeding time, activated partial thromboplastin time (APTT), prothrombin time (PT), or thrombin time (TT). There was also no spontaneous bleeding or bleeding from blood collection sites. Anfibatide did not significantly affect platelet count and no anti-Anfibatide antibodies were detected in the subjects, suggesting that Anfibatide is well-tolerated in healthy individuals. ConclusionThese comprehensive studies in mice and human subjects and in the first clinical trial clearly demonstrated that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy. Disclosures:Hou:Lee's pharmaceutical holdings limited: Research Funding. Lei:Lee's pharmaceutical holdings limited: Research Funding. Zhao:Lee's pharmaceutical holdings limited: Research Funding. Shen:Lee's pharmaceutical holdings limited: Research Funding. Zhou:Lee's pharmaceutical holdings limited: Research Funding. Wang:Lee's pharmaceutical holdings limited: Research Funding. Marshall:Lee's pharmaceutical holdings limited: Research Funding. Ni:Lee's pharmaceutical holdings limited: Research Funding.

Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures:No relevant conflicts of interest to declare.

Tangeretin Regulates Platelet Function Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling

Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Dietary α-linolenic acid increases the platelet count in ApoE−/− mice by reducing clearance

AbstractPreviously we reported that dietary intake of alpha-linolenic acid (ALA) reduces atherogenesis and inhibits arterial thrombosis. Here, we analyze the substantial increase in platelet count induced by ALA and the mechanisms of reduced platelet clearance. Eight-week-old male apolipoprotein E knockout (ApoE−/−) mice were fed a 0.21g% cholesterol diet complemented by either a high- (7.3g%) or low-ALA (0.03g%) content. Platelet counts doubled after 16 weeks of ALA feeding, whereas the bleeding time remained similar. Plasma glycocalicin and glycocalicin index were reduced, while reticulated platelets, thrombopoietin, and bone marrow megakaryocyte colony-forming units remained unchanged. Platelet contents of liver and spleen were substantially reduced, without affecting macrophage function and number. Glycoprotein Ib (GPIb) shedding, exposure of P-selectin, and activated integrin αIIbβ3 upon activation with thrombin were reduced. Dietary ALA increased the platelet count by reducing platelet clearance in the reticulo-endothelial system. The latter appears to be mediated by reduced cleavage of GPIb by tumor necrosis factor-α–converting enzyme and reduced platelet activation/expression of procoagulant signaling. Ex vivo, there was less adhesion of human platelets to von Willebrand factor under high shear conditions after ALA treatment. Thus, ALA may be a promising tool in transfusion medicine and in high turnover/high activation platelet disorders.

Measurement of the effect of platelet activators and heparin on adhesion and spreading of human platelets using impedimetry

Purpose: Thrombogenesis has been recently focused in cardiology. Numerous methods exist for the assessment of platelet aggregation, but there is no well standardized and generally accepted technique for the isolated measurement of platelet adhesion. Impedimetry is a real-time technique for the dynamic measurement of cell adhesion, proliferation, viability and cytotoxicity. In this work we propose this novel method as a candidate for the measurement of platelet adhesion. Methods: The effect of activators (epinephrine, ADP and collagen) and unfractionated heparin on human platelets isolated from healthy individuals under no current drug therapy was investigated. For these experiments the xCELLigence SP system was used, where the platelets adhere to golden electrodes placed at the bottom of the wells. The change of electric impedance on the electrodes is in direct correlation with the number and the level of spreading of the adhering platelets. For the measurement of adhesion, platelets were introduced into wells that the drug had already been added to. For the measurement of spreading, platelets were seeded onto the electrodes and the drug was administered afterwards. Results: Both epinephrine and ADP increased platelet adhesion, 10 μM epinephrine caused 5.42 fold (p=0.002), 5 μM epinephrine caused 5.34 fold (p<0,001), 10 μM ADP caused 11.9 fold (p<0.001) and 5 μM ADP caused 9.89 fold (p<0.001) increase in slope; collagen showed no significant effect on platelet adhesion. Unfractionated heparin decreased platelet adhesion on pharmacological concentration, 250 IU/mL unfractionated heparin caused 0.48 fold (p=0.001) decrease in slope, on lower concentrations there was no significant effect. Platelet spreading was increased by ADP: 10 μM ADP caused 16.12 fold (p=0.011) and 5 μM ADP caused 10.89 fold (p=0.007) increase in slope. Epinephrine and collagen had inhibitory effects on the spreading of platelets. Conclusions: Platelet adhesion proved to be qualitatively and quantitatively well detectable by impedimetry. Characteristic slopes of data measured have a good correlation with the effect of substances used as laboratory reference and drugs of medical therapy. The novel method is offered as a useful technique for the more detailed evaluation of platelet function and the following of antithrombotic drug therapies.

Platelet responses to dynamic biomaterial surfaces with different poly(ethylene glycol) and polyrotaxane molecular architectures constructed on gold substrates

Four different dynamic biomaterial surfaces with different molecular architectures were prepared using two hydrophilic polymers: poly(ethylene glycol) and polyrotaxanes containing α-cyclodextrin. Either one or both terminals of the poly(ethylene glycol) or polyrotaxanes were immobilized onto a gold substrate via Au-S bonds, resulting in poly(ethylene glycol)-graft, polyrotaxanes-graft, poly(ethylene glycol)-loop, and polyrotaxanes-loop structures. Human platelet adhesion was suppressed more effectively on the graft surfaces than on the loop surfaces for both poly(ethylene glycol) and polyrotaxanes due to the high mobility of graft polymer chains with a free terminal. Moreover, the platelets adhered to the polyrotaxane surfaces much less than the poly(ethylene glycol) surfaces, possibly because of the mobile nature of the α-cyclodextrin molecules that were threaded on the poly(ethylene glycol) chain. Actin filament assembly in adherent platelets was also greatly prevented on the poly(ethylene glycol)/polyrotaxanes-graft surfaces in comparison with the corresponding loop surfaces. A clear correlation between the numbers and areas of adherent platelets on these surfaces suggests that platelet adhesion and activation were dominated by the platelet GPIIb/IIIa-adsorbed fibrinogen interaction. These results indicate that both of the different modes of dynamic features, sliding/rotation of α-cyclodextrin and polymer chain mobility, effectively suppressed platelet adhesion in spite of the similar hydrophilicity. This research affords a novel chemical strategy for designing hemocompatible biomaterial surfaces.

In vitro effects of serotonin and noradrenaline reuptake inhibitors on human platelet adhesion and coagulation

BackgroundAlthough several studies show that there is an increased risk of bleeding events during antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs), few studies show direct effects in vitro of SSRIs on hemostasis. MethodsThis study was undertaken to investigate the effects on platelet adhesion and plasma coagulation (APTT and PT) of two common SSRIs, citalopram and sertraline, the selective noradrenaline reuptake inhibitor reboxetine, and the serotonin and noradrenaline reuptake inhibitor venlafaxine. ResultsNone of the compounds affected plasma coagulation significantly but all compounds except for venlafaxine inhibited platelet adhesion by approximately 50% or more at the highest concentration (100μg/l, p<0.01). The potency of respective compound to inhibit platelet adhesion to both collagen and fibrinogen surfaces was in the following order; citalopram > sertraline > reboxetine. In contrast, venlafaxine caused a weak but statistically significant increased platelet adhesion to fibrinogen. ConclusionThis study showed that sertraline, citalopram and reboxetine direct and acutely decrease platelet adhesion to both collagen and fibrinogen in vitro. These results also indicate that increased risk for bleeding complications in antidepressant users may not only be explained by depletion of serotonin in platelets.

Biocompatibility and anti-cracking performance of perfluorocarboxylic acid ionomer membranes for implantable biosensors

Perfluorosulfonate ionomer (PFSI) membranes can be used to protect biosensors in biological environment. Perfluorocarboxylate ionomer (PFCI) has similar structure to PFSI, and is thus expected to be applicable in vivo. In this study, biocompatibility and anti-cracking performance of PFCI membranes were first investigated. Among various kinds of PFCI membranes, the Na+-type exhibited best blood compatibility, as evidenced by the human platelet adhesion and hemolysis rate experiments. When cultured on Na+-type PFCI membranes, endothelial cells spread and proliferated to colonize the entire surface, indicating good cell adhesion activity of the membranes. The Na+-type membranes were also subcutaneously implanted into guinea pigs, whose incisions were healed after operation without significant rejection phenomenon. After incubation in cell culture medium for 12 weeks, PFCI membranes kept intact only with trace amount of calcium phosphate deposition. XRD and SAXS results demonstrated that PFCI membranes possessed higher crystallinity and smaller ion cluster size than PFSI membranes, thereby endowing the membranes with much better anti-cracking performance. The excellent biocompatibility and anti-cracking performance of Na+-type PFCI membranes make them promising implantable biomaterials and protective layers in biosensors.

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

AbstractIntegrin α2β1–mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3KβKD), but occurred normally in PI3KγKD platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)–knockout mice, and we found that intracellular Ca2+ linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca2+. Whereas activation of Pyk2 occurred normally in PI3KβKD platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1–mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.

Flavonoids Purified from Parsley Inhibit Human Blood Platelet Aggregation and Adhesion to Collagen under Flow

Blood platelets are directly involved in both haemostatic and pathologic thrombotic processes, through their adhesion, secretion and aggregation. In this study, we investigated the effect of genins (aglycone flavonoids without sugar group) isolated from parsley (Petroselinum crispum) leaves in vitro on human platelet aggregation and adhesion to a collagen-coated surface under physiologic flow conditions. The aggregation and adhesion studies were monitored after pre-incubation of platelets with genins. Genins inhibited dose dependently aggregation induced by thrombin, ADP and collagen. The strongest effect was observed in collagen induced aggregation (IC50 = 0.08 ± 0.01 mg/ml). The HPLC identification of genins compounds revealed the presence of keampferol, apigenin and other not identified compounds. The aggregation tests showed that these compounds have anti-aggregating activity. In addition, adhesion of human platelets to collagen was greatly decreased (over 75 %) by genins (0.3 mg/ml). While the mechanism by which genins act is unclear, we suggest that these compounds may interfere with a multiple target step in the haemostasis process. These results show that genins isolated from parsley has a potent antiplatelet activity. It may be an important source of beneficial antiplatelet compounds that decrease thrombosis and cardiovascular diseases.

Imer Blocks Phosphorylation of the β3 Integrin, Decreasing Platelet Activation Responses and Protecting Mice From Arterial Thrombosis

Abstract 189 Background:Growth Arrest Specific gene 6 (Gas6) signaling through platelet-surface Tyro3/Axl/Mer (TAM) receptors leads to platelet activation and thrombus stabilization. This occurs via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, stimulating tyrosine phosphorylation of the β3 integrin. This process amplifies outside-in signaling via αIIbβ3, which is necessary for stable aggregate formation. iMer is a truncated form of the extracellular domain of the Mer receptor tyrosine kinase, produced by alternative splicing, that inhibits Gas6/TAM signaling, likely by acting as a decoy receptor for Gas6. Objectives:We hypothesized that inhibiting the Gas6/TAM pathway with a novel Gas6-sequestering protein would decrease platelet activation responses. We therefore evaluated iMer's inhibition of Gas6 signaling in human and murine platelets in vitro and in vivo. Methods:We measured the inhibitory effect of iMer on platelet activation using laboratory evaluations of platelet function and a murine carotid artery thrombosis model. In vitro studies included aggregometry, adhesion to collagen in a flow chamber, and platelet spreading. These platelet activation responses were tested in human platelets in the presence or absence of the inhibitor and also in wild type (WT) and Gas6/TAM −/− murine platelets. A ferric-chloride model of carotid artery injury was used to compare susceptibility to thrombosis between littermate C57BL/6 mice treated with either iMer or vehicle. Platelet aggregation data was evaluated by the Wilcoxon Signed Rank Test, and times to occlusion following carotid artery injury were compared using the Mann-Whitney Rank Sum Test. Results/Discussion:Western blot analysis demonstrated decreased β3 integrin phosphorylation in iMer-treated human platelets after addition of human Gas6 when compared to controls, consistent with decreased Mer signaling in the presence of iMer.iMer-treated human platelets exhibited significant decreases in ADP- and collagen-induced platelet aggregation. ADP-stimulated samples treated ex vivo with iMer showed an aggregation mean of 74% (SD= +/− 3%), compared to 86% aggregation (+/− 3%) in controls (p=0.016). Collagen-stimulated samples treated ex vivo with iMer exhibited a mean of 70% aggregation (+/− 8%), compared to 88% aggregation (+/−2%) in controls (p=0.004).Electron micrographs of adhered human platelets revealed that iMer delayed, but did not permanently abrogate, platelet spreading on fibrillar collagen (100 μg/mL).Flow cytometric analysis of human platelets showed reduced expression of platelet-surface activation markers (P-selectin and PAC-1) despite stimulation with fibrillar collagen (1 μg/mL).Microfluidic flow assay demonstrated that adhesion of untreated human platelets to collagen at a wall shear rate of 100s−1 resulted in 21.3% (SD=+/− 8%) mean surface area coverage, while ex vivo iMer-treated samples showed only 1.1% (+/− 0.9%) coverage. These results are consistent with those of WT mice compared to that of Gas6/TAM −/−mice in preliminary studies using the same system.Following ferric chloride injury to the carotid artery, 71% of vehicle-treated control mice (n=7) had initial occlusions that remained stable, and only 14% remained patent. In contrast, only 25% of the iMer-treated mice (n=8) formed initial occlusions that remained stable, while 50% remained patent. The iMer treated mice also had a significant decrease (p=0.02) in the duration of first occlusion time (i.e. length of time the initial occlusion lasted), suggesting decreased thrombus stability. Conclusions:iMer is a novel inhibitor of the Gas6/TAM pathway that decreases platelet activation responses and protects mice from arterial thrombosis by decreasing phosphorylation of β3 integrin, which has been shown to be necessary for thrombus stabilization. This compound may, therefore, have translational applications as a novel anti-platelet agent. Disclosures:No relevant conflicts of interest to declare.

Investigation of human platelet adhesion under low shear conditions in a rotational flow chamber

Even though blood pumps have come into clinical usage, thrombo-embolic complications still pose a major problem, and they have not yet been clarified and quantified. However, it is known that the basis of thrombus formation is platelet adhesion, which is thought to be closely associated with the shear rate. Therefore, our current interest focuses on the effect of shear conditions on platelet adhesion. We have designed and carried out an experimental setup allowing fluorescent microscopy of whole blood within a rotational viscometer under controllable shear conditions. A small area of the bottom plate was coated with type I collagen, which provided a model of the injured vessel as a target for platelet adhesion. Using this setup, the time course of platelet adhesion under several different shear rates, ranging from 127 to 723 s−1, was studied. Platelet adhesion increased along with shear rates up to 283 s−1, followed by a gradual decrease when the shear rate exceeded 346 s−1. The adhesion amounts were statistically significant between 283 and 173 s−1 (p = 0.02), 173 and 127 s−1 (p = 0.035), and 283 and 503 s−1 (p = 0.03), respectively. This result suggests that there is an optimal shear condition around 300 s−1 for platelet adhesion to type I collagen.

Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines

Background -The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.Methods -The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.Results -Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.Conclusion -These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

Endothelial C-reactive protein increases platelet adhesion under flow conditions

While data regarding the pathogenetic role of C-reactive protein (CRP) in atherothrombosis are accumulating, it is still controversial whether local CRP secretion is of any pathobiological significance. The present study examined whether endothelial-derived CRP modulates autocrine prothrombotic activity. Endothelial cells were isolated from hearts of mice transgenic to human CRP and grown in primary cultures. Human CRP expression was confirmed in these cells compared with no expression in cultures derived from wild-type congenes. Adhesion of human platelets to endothelial cells was studied in the "cone and plate" flow system. Platelet adhesion to cells expressing CRP was significantly increased compared with that in controls (n = 6, P < 0.01). The proadhesive effect of CRP was significantly suppressed in mouse heart endothelial cells and in human umbilical vein endothelial cells following treatment with small interfering RNA for human CRP. Adhesion was modulated by an increase in P-selectin. P-selectin expression correlated with a proadhesive phenotype, and blocking P-selectin with neutralizing antibody significantly decreased the adhesion of platelets to CRP-expressing cells (40.4 ± 10.5 to 9.4 ± 6.9 platelets/high-power field, n = 5 to 6, P < 0.01). In conclusion, human CRP that is locally produced in endothelial cells increases platelet adhesion to endothelial cells under normal shear flow conditions. These findings indicate that CRP exerts a local effect on endothelial cells via P-selectin expression, which promotes platelet adhesion and subsequent thrombus formation.

Hemocompatibility of pyrolytic carbon in comparison with other biomaterials

The present generation of mechanical heart-valves prostheses offers significantly improved blood compatibility, combined with appropriate physical and mechanical properties and durability; however, thromboembolism persists as a crucial clinical complication because of the key role played by adsorbed plasma proteins and adherent platelets in determining hemocompatibility. In the present work human plasma protein adsorption and platelet adhesion and activation were evaluated on four materials, selected according to their different physico-chemical and morphological properties: pyrolytic carbon (PyC), titanium alloy Ti–6Al–4V (Ti), tissue culture polystyrene (TCPS) and polystyrene sterilized by γ -irradiation (StPS). Morphological and chemical properties of surfaces were assessed by Time of Flight Secondary Ion Mass Spectrometry (Tof-SIMS), X-ray Photoemission Spectroscopy (XPS), Atomic Force Microscopy (AFM) and Contact Angle (CA) analysis, while protein adsorption and platelet adhesion and activation were evaluated by colorimetric and immunofluorescence tests and Scanning Electron Microscopy (SEM), respectively. Among the analyzed materials, PyC induced the lowest level of protein adsorption and platelet adhesion and activation. In particular, immunofluorescence analysis of fibronectin (FN), fibrinogen (FG), von Willebrand factor (vWF), Hageman factor (FXII) and albumin (ALB) showed that PyC surface was characterized by high levels of FG adsorption, low levels of FN, ALB and vWF and the absence of FXII. Finally, when analyzing the biological response as a function of surface properties, it was found that protein adhesiveness increased with increasing contact angle values. To the contrary, platelet activation related to the total amount of adherent proteins was poor while cell activation was possibly dependent on the detailed composition of the underlying protein layer. Furthermore, differences in surface roughness of the examined materials did not seem to influence the biological response in terms of platelet activation and protein adsorption.

Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.