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- HEK cells (1)
- antiâPF4/heparin antibodies (1)
- atomic force microscopy (1)
- cell elasticity (1)
- interaction (1)
- length (1)
- nanoindentation (1)
- platelet factor 4 (1)
- stiffness (1)
Antibodies recognizing complexes of the chemokine platelet factor 4 (PF4/CXCL4) and polyanions (P) opsonize PF4-coated bacteria hereby mediating bacterial host defense. A subset of these antibodies may activate platelets after binding to PF4/heparin complexes, causing the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). In autoimmune-HIT, anti-PF4/P-antibodies activate platelets in the absence of heparin. Here we show that antibodies with binding forces of approximately 60â100 pN activate platelets in the presence of polyanions, while a subset of antibodies from autoimmune-HIT patients with binding forces Z100 pN binds to PF4 alone in the absence of polyanions. These antibodies with high binding forces cluster PF4-molecules forming antigenic complexes which allow binding of polyanion-dependent anti-PF4/P-antibodies. The resulting immunocomplexes induce massive platelet activation in the absence of heparin. Antibody-mediated changes in endogenous proteins that trigger binding of otherwise non-pathogenic (or cofactor-dependent) antibodies may also be relevant in other antibody-mediated autoimmune disorders.
Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no
previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated plateletactivation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet
defects.
Abstract
Mechanical characteristics of individual cells play a vital role in many biological processes and are considered as indicators of the cellsâ states. Disturbances including methylâβâcyclodextrin (MβCD) and cytochalasin D (cytoD) are known to significantly affect the state of cells, but little is known about the realâtime response of single cells to these drugs in their physiological condition. Here, nanoindentationâbased atomic force microscopy (AFM) was used to measure the elasticity of human embryonic kidney cells in the presence and absence of these pharmaceuticals. The results showed that depletion of cholesterol in the plasma membrane with MβCD resulted in cell stiffening whereas depolymerization of the actin cytoskeleton by cytoD resulted in cell softening. Using AFM for realâtime measurements, we observed that cells mechanically responded right after these drugs were added. In more detail, the cell´s elasticity suddenly increased with increasing instability upon cholesterol extraction while it is rapidly decreased without changing cellular stability upon depolymerizing actin cytoskeleton. These results demonstrated that actin cytoskeleton and cholesterol contributed differently to the cell mechanical characteristics.
Abstract
Background
Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparinâinduced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6â, 8â, 10â, and 12âmer size and a hypersulfated 12âmer (S12âmer).
Methods
We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and antiâPF4/heparin antibodies.
Results
The synthetic heparinâlike compounds display stronger binding characteristics to PF4 than animalâderived heparins of corresponding lengths. Upon complexation with PF4, 6âmer and S12âmer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8â, 10â, and 12âmer heparins. AntiâPF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins â¤Â 8âmer than with complexes formed between PF4 and heparins âĽÂ 10âmer. Addition of one sulfate group to the 12âmer resulted in a S12âmer, which showed substantial changes in its binding characteristics to PF4.
Conclusions
We provide a template for characterizing interactions of newly developed heparinâbased anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
