The interactions between specific snake venom toxins and muscle constituents are the major cause of severe muscle damage that often result in amputations and subsequent socioeconomic ramifications for snakebite victims and/or their families. Therefore, improving our understanding of venom-induced muscle damage and determining the underlying mechanisms of muscle degeneration/regeneration following snakebites is critical to developing better strategies to tackle this issue. Here, we analysed intramuscular bleeding and thrombosis in muscle injuries induced by two different snake venom toxins (CAMP-Crotalus atrox metalloprotease (a PIII metalloprotease from the venom of this snake) and a three-finger toxin (CTX, a cardiotoxin from the venom of Naja pallida)). Classically, these toxins represent diverse scenarios characterised by persistent muscle damage (CAMP) and successful regeneration (CTX) following acute damage, as normally observed in envenomation by most vipers and some elapid snakes of Asian, Australasian, and African origin, respectively. Our immunohistochemical analysis confirmed that both CAMP and CTX induced extensive muscle destruction on day 5, although the effects of CTX were reversed over time. We identified the presence of fibrinogen and P-selectin exposure inside the damaged muscle sections, suggesting signs of bleeding and the formation of platelet aggregates/microthrombi in tissues, respectively. Intriguingly, CAMP causes integrin shedding but does not affect any blood clotting parameters, whereas CTX significantly extends the clotting time and has no impact on integrin shedding. The rates of fibrinogen clearance and reduction in microthrombi were greater in CTX-treated muscle compared to CAMP-treated muscle. Together, these findings reveal novel aspects of venom-induced muscle damage and highlight the relevance of haemostatic events such as bleeding and thrombosis for muscle regeneration and provide useful mechanistic insights for developing better therapeutic interventions.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

1,8-cineole, a monoterpenoid is a major component of eucalyptus oil and has been proven to possess numerous beneficial effects in humans. Notably, 1,8-cineole is the primary active ingredient of a clinically approved drug, Soledum® which is being mainly used for the maintenance of sinus and respiratory health. Due to its clinically valuable properties, 1,8-cineole has gained significant scientific interest over the recent years specifically to investigate its anti-inflammatory and antioxidant effects. However, the impact of 1,8-cineole on the modulation of platelet activation, thrombosis and haemostasis was not fully established. Therefore, in this study, we demonstrate the effects of 1,8-cineole on agonists-induced platelet activation, thrombus formation under arterial flow conditions and haemostasis in mice. 1,8-cineole largely inhibits platelet activation stimulated by glycoprotein VI (GPVI) agonists such as collagen and cross-linked collagen-related peptide (CRP-XL), while it displays minimal inhibitory effects on thrombin or ADP-induced platelet aggregation. It inhibited inside-out signalling to integrin αIIbβ3 and outside-in signalling triggered by the same integrin as well as granule secretion and intracellular calcium mobilisation in platelets. 1,8-cineole affected thrombus formation on collagen-coated surface under arterial flow conditions and displayed a minimal effect on haemostasis of mice at a lower concentration of 6.25 µM. Notably, 1,8-cineole was found to be non-toxic to platelets up to 50 µM concentration. The investigation on the molecular mechanisms through which 1,8-cineole inhibits platelet function suggests that this compound affects signalling mediated by various molecules such as AKT, Syk, LAT, and cAMP in platelets. Based on these results, we conclude that 1,8-cineole may act as a potential therapeutic agent to control unwarranted platelet reactivity under various pathophysiological settings.

Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor (37,43)Gap27 on Cx40(-/-) mouse platelets and of the Cx40 inhibitor (40)Gap27 on Cx37(-/-) mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate hemostasis of victims through effects on platelets, vascular endothelial, and smooth muscle cells. In this study, we have isolated and functionally characterized a snaclec that we named "rhinocetin" from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13 kDa, respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in a dose-dependent manner but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP-, or thrombin-induced platelet activation. Rhinocetin antagonized the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen-induced platelet functions such as fibrinogen binding, calcium mobilization, granule secretion, aggregation, and thrombus formation. It also inhibited integrin α2β1-dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios, including hemostasis, thrombosis, and envenomation.

Platelet-associated complications including thrombosis, thrombocytopenia, and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis. Although several mechanisms that may contribute to the dysfunction of platelets during inflammatory diseases have been reported, knowledge on the primary molecules/mechanisms that underpin platelet-associated complications in such conditions is not fully established. Here, we report the significance of the mouse antimicrobial cathelicidin, mouse cathelicidin-related antimicrobial peptide (mCRAMP) (an orthologue of LL37 in humans), on the modulation of platelet reactivity during psoriasis using Imiquimod-induced psoriasis in mice as an inflammatory disease model for psoriasis vulgaris in humans. The activation of platelets during psoriasis is increased as evidenced by the elevated levels of fibrinogen binding and P-selectin exposure on the surface of platelets, and the level of soluble P-selectin in the plasma of psoriatic mice. The skin and plasma of psoriatic mice displayed increased levels of mCRAMP. Moreover, the plasma of psoriatic mice augmented the activation of platelets obtained from healthy mice. The effect of mCRAMP is partially mediated through formyl peptide receptor 2/3 (Fpr2/3, the orthologue to human FPR2/ALX) in platelets as a significant reduction in their activation was observed when FPR2/ALX-selective inhibitors such as WRW4 or Fpr2/3-deficient mouse platelets were used in these assays. Since the level of antimicrobial cathelicidin is increased in numerous inflammatory diseases such as psoriasis, atherosclerosis, and inflammatory bowel disease, the results of this study point towards a critical role for antimicrobial cathelicidin and FPR2/ALX in the development of platelet-related complications in such diseases.

Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer's disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.