Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

Pediatric Swine Model of Methicillin-Resistant Staphylococcus aureus Sepsis-Induced Coagulopathy, Disseminated Microvascular Thrombosis, and Organ Injuries.

Sepsis-induced coagulopathy leading to disseminated microvascular thrombosis is associated with high mortality and has no existing therapy. Despite the high prevalence of Gram-positive bacterial sepsis, especially methicillin-resistant Staphylococcus aureus (MRSA), there is a paucity of published Gram-positive pediatric sepsis models. Large animal models replicating sepsis-induced coagulopathy are needed to test new therapeutics before human clinical trials. HYPOTHESIS: Our objective is to develop a pediatric sepsis-induced coagulopathy swine model that last 70 hours. METHODS AND MODELS: Ten 3 weeks old piglets, implanted with telemetry devices for continuous hemodynamic monitoring, were IV injected with MRSA (n = 6) (USA300, Texas Children's Hospital 1516 strain) at 1 × 109 colony forming units/kg or saline (n = 4). Fluid resuscitation was given for heart rate greater than 50% or mean arterial blood pressure less than 30% from baseline. Acetaminophen and dextrose were provided as indicated. Point-of-care complete blood count, prothrombin time (PT), activated thromboplastin time, d-dimer, fibrinogen, and specialized coagulation assays were performed at pre- and post-injection, at 0, 24, 48, 60, and 70 hours. Piglets were euthanized and necropsies performed. RESULTS: Compared with the saline treated piglets (control), the septic piglets within 24 hours had significantly lower neurologic and respiratory scores. Over time, PT, d-dimer, and fibrinogen increased, while platelet counts and activities of factors V, VII, protein C, antithrombin, and a disintegrin and metalloproteinase with thrombospondin-1 motifs (13th member of the family) (ADAMTS-13) decreased significantly in septic piglets compared with control. Histopathologic examination showed minor focal organ injuries including microvascular thrombi and necrosis in the kidney and liver of septic piglets. INTERPRETATIONS AND CONCLUSIONS: We established a 70-hour swine model of MRSA sepsis-induced coagulopathy with signs of consumptive coagulopathy, disseminated microvascular thrombosis, and early organ injuries with histological minor focal organ injuries. This model is clinically relevant to pediatric sepsis and can be used to study dysregulated host immune response and coagulopathy to infection, identify potential early biomarkers, and to test new therapeutics.

Exercise-induced myokines downregulates the ACE2 level in bronchial epithelial cells: Implications for SARS-CoV-2 prevention.

BACKGROUND: The Covid-19 pandemic has emerged as the leading public health challenge of our time (20th century). While vaccinations have finally blunted the death rate, concern has remained about more virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2). METHODS: We utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy. RESULTS: Quantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (p<2.69x10-5) and 41% (p<4.57x10-5), respectively. The high sensitivity of ELISAs showed downregulation of ACE2 and TMPRSS2 protein expression in 16HBE14o cells by 53% (p<0.01) and 32% (p<0.03) respectively with 48 h treated. For rigor, this work was replicated in the human lung cancer cell line A549, which mirrored the downregulation. Proteomic analysis showed dramatic alteration in myokine profile between contracted and uncontracted C2C12 tubules. CONCLUSIONS: The current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise's protective effect against Covid-19. These further strengthen myokines' beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.