Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-X L , and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.Despite a lot of information being available on triggering factors and pathogenic mechanisms involved in HIT, the causes of thrombocytopenia in HIT are not well delineated. One possibility is that a low platelet count is triggered by the clearance of Ab-coated platelets in the spleen and potentially the liver. The second possibility is that platelets are consumed within thrombi; however, it is unclear if sufficient platelets are incorporated into thrombi to cause thrombocytopenia. The third potential explanation involves platelet disintegration via microvesiculation [6,7], but whether this mechanism is responsible for the low platelet count is unclear. Our recent studies suggest that platelets activated by thrombin undergo fatal dysfunction and cytoplasmic fragmentation clearly distinct from what happens when platelets are activated by adenosine diphosphate (ADP) or collagen [8,9]. Based on these and other findings, we hypothesized that platelets activated in HIT through FcγRIIA undergo activation followed by death, leading to thrombocytopenia with the removal of dead platelets perhaps by neutrophils and macrophages.In this work, we studied the direct effects of PF4-containing HIT-pathogenic immune complexes on isolated human platelets. As a tool, we used two isotype-matched murine anti-human PF4/heparin monoclonal Abs that mimic their human counterparts in vitro [10], and in vivo the pathogenic monoclonal anti-PF4/heparin antibody (KKO) causes HIT in an animal model, while the non-pathogenic monoclonal anti-PF4/heparin antibody (RTO) does not [11]. Importantly, ELISA-positive plasma samples from patients suspected of having HIT contain Abs that show hep...

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses’ furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

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