Dissecting thrombus-directed chemotaxis and random movement in neutrophil near-thrombus motion in flow chambers.

BACKGROUND: Thromboinflammation is caused by mutual activation of platelets and neutrophils. The site of thromboinflammation is determined by chemoattracting agents release by endothelium, immune cells, and platelets. Impaired neutrophil chemotaxis contributes to the pathogenesis of Shwachman-Diamond syndrome (SDS). In this hereditary disorder, neutrophils are known to have aberrant chemoattractant-induced F-actin properties. Here, we aim to determine whether neutrophil chemotaxis could be analyzed using our previously developed ex vivo assay of the neutrophils crawling among the growing thrombi. METHODS: Adult and pediatric healthy donors, alongside with pediatric patients with SDS, were recruited for the study. Thrombus formation and granulocyte movement in hirudinated whole blood were visualized by fluorescent microscopy in fibrillar collagen-coated parallel-plate flow chambers. Alternatively, fibrinogen, fibronectin, vWF, or single tumor cells immobilized on coverslips were used. A computational model of chemokine distribution in flow chamber with a virtual neutrophil moving in it was used to analyze the observed data. RESULTS: The movement of healthy donor neutrophils predominantly occurred in the direction and vicinity of thrombi grown on collagen or around tumor cells. For SDS patients or on coatings other than collagen, the movement was characterized by randomness and significantly reduced velocities. Increase in wall shear rates to 300-500 1/s led to an increase in the proportion of rolling neutrophils. A stochastic algorithm simulating leucocyte chemotaxis movement in the calculated chemoattractant field could reproduce the experimental trajectories of moving neutrophils for 72% of cells. CONCLUSIONS: In samples from healthy donors, but not SDS patients, neutrophils move in the direction of large, chemoattractant-releasing platelet thrombi growing on collagen.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

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.

Theoretical Explanation for the Rarity of Antibody-Dependent Enhancement of Infection (ADE) in COVID-19.

Global vaccination against the SARS-CoV-2 virus has proved to be highly effective. However, the possibility of antibody-dependent enhancement of infection (ADE) upon vaccination remains underinvestigated. Here, we aimed to theoretically determine conditions for the occurrence of ADE in COVID-19. We developed a series of mathematical models of antibody response: model Ab-a model of antibody formation; model Cv-a model of infection spread in the body; and a complete model, which combines the two others. The models describe experimental data on SARS-CoV and SARS-CoV-2 infections in humans and cell cultures, including viral load dynamics, seroconversion times and antibody concentration kinetics. The modelling revealed that a significant proportion of macrophages can become infected only if they bind antibodies with high probability. Thus, a high probability of macrophage infection and a sufficient amount of pre-existing antibodies are necessary for the development of ADE in SARS-CoV-2 infection. However, from the point of view of the dynamics of pneumocyte infection, the two cases where the body has a high concentration of preexisting antibodies and a high probability of macrophage infection and where there is a low concentration of antibodies in the body and no macrophage infection are indistinguishable. This conclusion could explain the lack of confirmed ADE cases for COVID-19.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status.

INTRODUCTION: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. MATERIALS AND METHODS: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. RESULTS AND CONCLUSIONS: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.