Although reversible platelet aggregation observed in response to ADP stimulation in the presence of calcium is a well-known phenomenon, its mechanisms are not entirely clear. To study them, we developed a simple kinetic mass-action-law-based mathematical model to use it in combination with experiments. Light transmission platelet aggregometry (LTA) induced by ADP was performed for platelet-rich plasma or washed platelets using both conventional light transmission and aggregate size monitoring method based on optical density fluctuations. Parameter values of the model were determined by means of parameter estimation techniques implemented in COPASI software. The mathematical model was able to describe reversible platelet aggregation LTA curves without assuming changes in platelet aggregation parameters over time, but with the assumption that platelet can enter the aggregate only once. In the model, the mean size of platelet aggregates correlated with the solution transparency. This corresponded with flow cytometry analysis and with optical density fluctuations data on aggregate size. The predicted values of model parameters correlated with ADP concentration used in experiments. These data suggest that, at the start of the aggregation, when platelet integrins switch “on”, large unstable platelet aggregates are rapidly formed, which leads to an increase in light transmission. However, upon fragmentation of these aggregates, the probability of the post-aggregate platelets’ attachment to each other decreases preventing new aggregation and resulting in the reversible aggregation phenomenon.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin α_IIbβ₃ functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.

Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 μM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPS-induced weak inhibition of platelet activation and the prevention of platelet desensitization.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

The process of clustering of plasma membrane receptors in response to their agonist is the first step in signal transduction. The rate of the clustering process and the size of the clusters determine further cell responses. Here we aim to demonstrate that a simple 2-differential equation mathematical model is capable of quantitative description of the kinetics of 2D or 3D cluster formation in various processes. Three mathematical models based on mass action kinetics were considered and compared with each other by their ability to describe experimental data on GPVI or CR3 receptor clustering (2D) and albumin or platelet aggregation (3D) in response to activation. The models were able to successfully describe experimental data without losing accuracy after switching between complex and simple models. However, additional restrictions on parameter values are required to match a single set of parameters for the given experimental data. The extended clustering model captured several properties of the kinetics of cluster formation, such as the existence of only three typical steady states for this system: unclustered receptors, receptor dimers, and clusters. Therefore, a simple kinetic mass-action-law-based model could be utilized to adequately describe clustering in response to activation both in 2D and in 3D.