Platelet mimetic particles for targeting thrombi in flowing blood.
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ABSTRACT:
SUBMITTER: Doshi N
PROVIDER: S-EPMC3483800 | biostudies-literature | 2012 Jul
REPOSITORIES: biostudies-literature
Publications
Platelet mimetic particles for targeting thrombi in flowing blood.
Doshi Nishit N Orje Jennifer N JN Molins Blanca B Smith Jeffrey W JW Mitragotri Samir S Ruggeri Zaverio M ZM
Advanced materials (Deerfield Beach, Fla.) 20120529 28
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Project description:The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s(-1). At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.
| S-EPMC1895707 | biostudies-literature
Project description:Exposed subendothelial collagen acts as a substrate for platelet adhesion and thrombus formation after vascular injury. Synthetic collagen-derived triple-helical peptides, designated collagen-related peptide (CRP), GFOGER, and VWF-III, can specifically engage the platelet collagen receptors, glycoprotein VI and integrin alpha(2)beta(1), and plasma von Willebrand factor (VWF), respectively. Hitherto, the role of these 3 collagen-binding axes has been studied indirectly. Use of these uniform peptide substrates, rather than collagen fibers, provides independent control of each axis. Here, we use confocal imaging and novel image analysis techniques to investigate the effects of receptor-ligand engagement on platelet binding and activation during thrombus formation under flow conditions. At low shear (100s(-1) and 300s(-1)), both GFOGER and CRP are required for thrombus formation. At 1000s(-1), a combination of either CRP or GFOGER with VWF-III induces comparable thrombus formation, and VWF-III increases thrombus deposition at all shear rates, being indispensable at 3000s(-1). A combination of CRP and VWF-III is sufficient to support extensive platelet deposition at 3000s(-1), with slight additional effect of GFOGER. Measurement of thrombus height after specific receptor blockade or use of altered proportions of peptides indicates a signaling rather than adhesive role for glycoprotein VI, and primarily adhesive roles for both alpha(2)beta(1) and the VWF axis.
| S-EPMC2890152 | biostudies-literature
Project description:BackgroundPlatelets are classically recognized for their role in hemostasis and thrombosis. Recent work has demonstrated that platelets can also execute a variety of immune functions. The dual prothrombotic and immunological roles of platelets suggest that they may pose a barrier to the replication or dissemination of extracellular bacteria. However, some bloodborne pathogens, such as the plague bacterium Yersinia pestis, routinely achieve high vascular titers that are necessary for pathogen transmission.ObjectivesIt is not currently known how or if pathogens circumvent platelet barriers to bacterial dissemination and replication. We sought to determine whether extracellular bloodborne bacterial pathogens actively interfere with platelet function, using Y pestis as a model system.MethodsThe interactions and morphological changes of human platelets with various genetically modified Y pestis strains were examined using aggregation assays, immunofluorescence, and scanning electron microscopy.ResultsYersinia pestis directly destabilized platelet thrombi, preventing bacterial entrapment in fibrin/platelet clots. This activity was dependent on two well-characterized bacterial virulence factors: the Y pestis plasminogen activator Pla, which stimulates host-mediated fibrinolysis, and the bacterial type III secretion system (T3SS), which delivers bacterial proteins into the cytoplasm of targeted host cells to reduce or prevent effective immunological responses. Platelets intoxicated by the Y pestis T3SS were unable to respond to prothrombotic stimuli, and T3SS expression decreased the formation of neutrophil extracellular traps in platelet thrombi.ConclusionsThese findings are the first demonstration of a bacterial pathogen using its T3SS and an endogenous protease to manipulate platelet function and to escape entrapment in platelet thrombi.
| S-EPMC8040536 | biostudies-literature
Project description:To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.
| S-EPMC3532496 | biostudies-literature
Project description:Patients treated with percutaneous coronary intervention receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo.We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at a wall shear rate of 1500 s(-1). The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than in 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator-stimulated phosphoprotein phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group, and only 22.6% of individual values were above the lower normal limit. We found no correlation between volume of thrombus formed on collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function.Measurements of platelet thrombus formation in flowing blood reflects the consequences of antiplatelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk.
| S-EPMC3030676 | biostudies-literature
Project description:Mural thrombi are composed dominantly of platelets and develop under a blood flow. Portions can break off and are carried in the blood flow as emboli. Thrombus growth rates are affected by the velocity of the blood flow, but they do not simply increase with it, they exhibit a maximum, with subsequent decrease. Whereas this variation indicates an interaction of biochemical and physical processes, studies have concentrated widely on understanding only the biochemical processes. Here we show results of simulation of thrombus formation in 3D flows by accounting for the movements of individual platelets. Each platelet follows prescribed rules for interactions while the local flow around the thrombus continuously adjusts to the growing structure of the thrombus, also when embolization occurs. With an activation delay time assigned to each platelet we demonstrate the dependence of thrombus growth rate on blood velocity as found experimentally by Begent and Born [Begent N, Born GV (1970) Nature 227:926-930]. With activated platelets having mutual tensile action sustainable up to a prescribed distance we achieve thrombus growth faster than with shorter maximum distances that make a thrombus less porous; when the prescribed maximum distance is large enough the thrombus shape is not like a "hill" but like a "carpet." We find that thrombus growth rate is enhanced by modest pulsatility but less so when pulsations are amplified in part because of more embolization.
| S-EPMC1859903 | biostudies-other
Project description:This paper presents a rail guided method to apply a Layer-by-Layer (LbL) coating on particles in a microfluidic device. The passive microfluidic approach allows handling suspensions of particles to be coated in the system. The trajectory of the particles is controlled using engraved rails, inducing lateral movement of particles while keeping the axially oriented liquid flow (and the interface of different liquids) undisturbed. The depth and angle of the rails together with the liquid velocity were studied to determine a workable geometry of the device. A discontinuous LbL coating procedure was converted into one continuous process, demonstrating that the chip can perform seven consecutive steps normally conducted in batch operation, further easily extendable to larger cycle numbers. Coating of the particles with two bilayers was confirmed by fluorescence microscopy.
| S-EPMC9758194 | biostudies-literature
Project description:IntroductionMechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions.MethodsWe assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets.ResultsIn studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated.ConclusionsOur studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.
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Project description:Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.
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Project description:Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.
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