Background
Dual antiplatelet therapy is a pre-requisite for flow diverter (FD) implantation. The purpose of this study was to assess the thrombogenicity of the p48 FD, coated with the newly developed ...phenox Hydrophilic Polymer Coating (p48_HPC, phenox GmbH, Germany) in comparison with uncoated p48 FDs in an in vitro flow model (Chandler Loop).
Methods
p48 and p48_HPC FDs were implanted into silicon tubes filled with whole human blood and incubated at 37 °C under pulsating flow. After 120 min, platelet count was determined in the blood. Platelet activation markers (PAR1) and formation of microparticles were analyzed in a flow cytometer. Fluorescence microscopy of CD42a positive cells and scanning electron microscopy was used to detect adherent platelets on the wire surface.
Results
Platelets in contact with the uncoated p48 FDs are significantly more activated than those incubated with p48_HPC (73 ± 9% vs. 65 ± 6%,
p
< 0.05) and release more microparticles (1.8 ± 0.5 vs. 1.4 ± 0.4,
p
< 0.05). The platelet count after 120-min circulation in the Chandler Loop was significantly lower for the uncoated p48 compared to the p48_HPC indicating significantly greater adherence of the platelets to the p48 (71 ± 8% vs. 87 ± 5%,
p
< 0.05). SEM and fluorescent antibody imaging revealed minimal platelet adherence to the surface of the p48_HPC compared to the uncoated p48.
Conclusion
The pHPC coating significantly reduces thrombogenicity of the p48 FD. This may help to reduce the risk of thromboembolic complications when using these devices. A reduction in antiplatelet therapy may be possible.
Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue‐factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. ...Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin‐mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin‐dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti‐TF antibodies inhibiting TF‐induced thrombin formation in platelet‐depleted plasma had no effect compared with vehicle‐treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase‐inhibitors bacitracin and anti‐PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin‐stimulated platelets exhibited increased surface‐associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin‐stimulated platelets. Conclusions: Thus, surface‐associated PDI is an important regulator of coagulation factor ligation to thrombin‐stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.
Background: Human neutrophil α‐defensins (HNPs) are important constituents of the innate immune system. Beyond their antimicrobial properties, HNPs also have pro‐inflammatory features. While HNPs in ...plasma from healthy individuals are barely detectable, their level is strongly elevated in septic plasma and plasma from patients with acute coronary syndromes.
Objectives: As thrombosis and inflammation are intertwined processes and activation of human polymorphonuclear leukocytes (PMNL) and subsequent degranulation is associated with full activation of surrounding platelets, we studied the effect of HNPs on platelet function.
Methods: The effect of HNPs on platelet activation parameters and apoptosis was investigated via aggregometry, flow cytometry, confocal microscopy and the ELISA technique.
Results: It was found that HNPs activate platelets in pathophysiologically relevant doses, inducing fibrinogen and thrombospondin‐1 binding, aggregation, granule secretion, sCD40L shedding, and procoagulant activity. HNPs bound directly to the platelet membrane, induced membrane pore formation, microparticle formation, mitochondrial membrane depolarization and caspase‐3‐activity. Confocal microscopy revealed the HNP‐induced formation of polymeric fibrinogen and thrombospondin‐1 amyloid‐like structures, which bound microorganisms. Platelets adhered to these structures and formed aggregates. Blocking of glycoprotein IIb/IIIa (GPIIb/IIIa) markedly inhibited HNP‐induced platelet activation. In addition, heparin, heparinoid, serpins and α2‐macroglobulin, which all bind to HNPs, blocked HNP‐1‐induced platelet activation in contrast to direct thrombin inhibitors such as hirudin.
Conclusions: HNPs activate platelets and induce platelet apoptosis by formation of amyloid‐like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.
Macrophages are an important part of the cellular immune system and play a key role during immune responses. Thus, macrophages are interesting targets in basic and clinical research. Primary ...monocytes or monocyte-derived macrophages do not proliferate on a suitable scale so that their use for functional studies
in
vitro is limited. Immortal proliferating cell lines, such as the human THP-1 monocytic leukemia cell line, are therefore often used instead of primary cells. Transfection is a useful tool to study the function of gene products, but transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages with subsequent differentiation into mature THP-1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. In this study, we describe a simple and rapid approach for efficient transfection of THP-1 monocytes and pre-differentiated THP-1 macrophages using a modified Nucleofection-based approach. The protocol maintains cell viability and functionality, thus allowing efficient transfection of THP-1 cells combined with subsequent differentiation of transfected THP-1 cells into mature macrophages.
ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier ...disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, α2β1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.
ABSTRACT
Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to ...promote thrombus formation in high shear environments. We found that thrombospondin‐1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin‐1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s−1. Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin‐1. Platelet adhesion to thrombospondin‐1 is not mediated via β3‐integrins or GPIa. CD36 partially mediates the adhesion of pre‐activated platelets. We identified GPIb as high shear adhesion‐receptor for thrombospondin‐1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin‐1‐GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.
The design process of rotary blood pumps aims for the lowest rate of hemolysis. We investigated whether the design process should also target minimal effect on platelet activation.
Methods:
Two ...identical rotary pumps with different blade shapes of the diffuser were tested for discrepancies with regard to platelet activation and functional capacity in-vitro. Four times, 0.5 L of human blood from 4 different subjects was used in 2 identical test loops (0.25 L in each) and was pumped under identical conditions over a 4 hours. Blood samples were tested before the start of the pump and after 4 hours.
Results:
There was no difference between the pumps with regard to hemolysis. The ability of the platelets to bind to fibrinogen differed by 65%, expression of CD 62 by 25% and of CD 63 by 27%, the amount of platelet micro-particles by 22%. Among the functional capacity, after stimulation of the PAR-1 receptor of the platelets with 25µM (100µM) thrombin receptor activating peptide (TRAP) the difference was 63% (41%). Stimulation with 5µM ADP showed a difference of 48%. After stimulation with TRAP and ADP the binding to platelets was more pronounced (TRAP, 33%; ADP, 43%). Furthermore, the TRAP-induced expression of granulophysin and P-selectin was 53% and 32% different.
Conclusions:
The degree of hemolysis created by the pumps was not different; there was a huge difference between the pumps with regard to platelet activation and functional capacity. Thus, a low hemolysis rate is not predictive for a low rate of thromboembolic events.
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