The role of formyl peptide receptors in the regulation of platelet function

Formyl peptide receptors (FPRs) belong to the family of G protein-coupled receptors (GPCRs) and play crucial roles in the regulation of innate immunity and host defence. FPRs include three family members; FPR1, FPR2/ALX and FPR3. They bind a wide variety of structurally and chemically diverse ligands that can exert various functions. Despite a plethora of research focusing on the role of FPRs in the regulation of immunity, there is a paucity of studies on their roles on the regulation of platelet haemostatic function. Here, we demonstrate the impact of both FPR1 and FPR2/ALX on the modulation of platelet reactivity, haemostasis and thrombosis. By using selective pharmacological inhibitors for FPR1 and FPR2/ALX, and Fpr1- and Fpr2/3-deficient mice, we were able to establish instrumental roles for these receptors in the regulation of the normal platelet haemostatic function. Additionally, we report a crucial role for fMLF in the regulation of platelet function through FPR1 signalling. fMLF exerted a priming effect on platelet activation through inducing distinct functions and enhances thrombus formation under arterial flow conditions. These effects were diminished in the presence of FPR1-selective pharmacological inhibitors and in platelets obtained from Fpr1- deficient mice. In addition, we investigated the role of LL37 in the regulation of platelet function and its modulation on platelet reactivity under pathological conditions, such as psoriasis, via acting through FPR2/ALX. We demonstrate that LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail-bleeding time in mice. Moreover, we report the overexpression of mCRAMP (an LL37 murine orthologue) in affected skin and plasma of a murine [imiquimod (IMQ)-induced] model of human psoriasis and its ability to enhance platelet responses via Fpr2/3. We also report a role for Annexin A1 and its N-terminal peptide, Ac2-26, in the regulation of platelet function through FPR2/ALX. Ac2-26 induced the activation of various platelet functions. Moreover, AnxA1-deficient mice demonstrate enhanced functional responses towards Ac2-26, which may be attributable to the overexpression of Fpr2/3 in these mice. Since both FPR1 and FPR2/ALX and their ligands play critical roles in various pathological conditions, their influence on the modulation of platelet activation and thrombus formation will provide novel insights into the mechanisms that control platelet-mediated complications under various disease settings

Mushroom quality related with various substrates’ bioaccumulation and translocation of heavy metals

Mushrooms are popular due to the nutrition contents in the fruit bodies and are relatively easy to cultivate. Mushrooms from the white-rot fungi group can be cultivated on agricultural biomass such as sawdust, paddy straw, wheat straw, oil palm frond, oil palm empty fruit bunches, oil palm bark, corn silage, corn cobs, banana leaves, coconut husk, pineapple peel, pineapple leaves, cotton stalk, sugarcane bagasse and various other agricultural biomass. Mushrooms are exceptional decomposers that play important roles in the food web to balance the ecosystems. They can uptake various minerals, including essential and non-essential minerals provided by the substrates. However, the agricultural biomass used for mushroom cultivation is sometimes polluted by heavy metals because of the increased anthropogenic activities occurring in line with urbanisation. Due to their role in mycoremediation, the mushrooms also absorb pollutants from the substrates into their fruit bodies. This article reviews the sources of agricultural biomass for mushroom cultivation that could track how the environmental heavy metals are accumulated and translocated into mushroom fruit bodies. This review also discusses the possible health risks from prolonged uptakes of heavy metal-contaminated mushrooms to highlight the importance of early contaminants’ detection for food security

The antimicrobial cathelicidin CRAMP augments platelet activation during psoriasis in mice

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

Palm press fibre and rice straw for cultivation grey oyster mushroom (Pleurotus sajor-caju)

Sawdust (SD) usually sourced from rubber plant is commonly used as substrate to cultivate the grey oyster mushroom Pleurotus sajor-caju in Malaysia. However, the market price of SD is increasing because of its declining availability. Thus, this study aimed to discover an alternative substrate to cultivate grey oyster mushroom. The raw materials used in the substrate were oil palm pressed fiber (PPF), rice straw (RS) and SD, either alone or in combination with different ratios including T1: 90% SD + 10% rice bran (commercial substrate as control); T2: 30% RS + 20% SD + 50% PPF; T3: 50% RS + 50% PPF; T4: 100% RS; and T5: 100% PPF. The suitability of the substrates was determined based on growth, yield, nutrition and mineral content in the mushrooms. The growth and yield of the mushroom fruiting body on the different substrates showed significant differences. The shortest harvesting day was obtained in T4 (100% RS) after 29 days, whereas the longest harvesting day was obtained in T1 (90% SD + 10% rice bran) after 51 days. The highest yield was obtained in T4. Nutrition analysis showed significant difference among the treatments. The highest amounts of protein (26%) and ash (1.29%) were found in T5. Overall, the results showed that mushroom yield performance was good in RS but nutritional content was the highest in PPF. Therefore, RS and PPF have good impact for mushroom growers either in commercial production or in functional food industry to reduce SD dependency

Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage

The endogenous antimicrobial cathelicidin LL37 induces platelet activation and augments thrombus formation

Platelet-associated complications including thrombosis, thrombocytopenia and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis, sepsis and inflammatory bowel disease. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX in platelets, and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, haemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilising a pharmacological inhibitor and Fpr2/3 (an orthologue of human FPR2/ALX)-deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Since the level of LL37 is increased in numerous inflammatory diseases, these results point towards a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases

Ruthenium-conjugated chrysin analogues modulate platelet activity, thrombus formation and haemostasis with enhanced efficacy

The constant increase in cardiovascular disease rate coupled with significant drawbacks of existing therapies emphasise the necessity to improve therapeutic strategies. Natural flavonoids exert innumerable pharmacological effects in humans. Here, we demonstrate the effects of chrysin, a natural flavonoid found largely in honey and passionflower on the modulation of platelet function, haemostasis and thrombosis. Chrysin displayed significant inhibitory effects on isolated platelets, however, its activity was substantially reduced under physiological conditions. In order to increase the efficacy of chrysin, a sulfur derivative (thio-chrysin), and ruthenium-complexes (Ru-chrysin and Ru-thio-chrysin) were synthesised and their effects on the modulation of platelet function were evaluated. Indeed, Ru-thio-chrysin displayed a 4-fold greater inhibition of platelet function and thrombus formation in vitro than chrysin under physiologically relevant conditions such as in platelet-rich plasma and whole blood. Notably, Ru-thio-chrysin exhibited similar efficacy to chrysin in the modulation of haemostasis in mice. Increased bioavailability and cell permeability of Ru-thio-chrysin compared to chrysin were found to be the basis for its enhanced activity. Together, these results demonstrate that Ru-thio-coupled natural compounds such as chrysin may serve as promising templates for the development of novel anti-thrombotic agents

Deletion of annexin A1 in mice upregulates the expression of its receptor, Fpr2/3, and reactivity to the AnxA1 mimetic peptide in platelets

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 (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 recep-tor 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 exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out us-ing 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 inflamma-tory responses via leukocytes, ANXA1 modulates platelet function which may influence throm-bosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings

Isorhapontigenin, a resveratrol analogue selectively inhibits ADP-stimulated platelet activation

Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85µM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100µM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50>100µM) but inhibited collagen induced platelet aggregation at 50µM and 100µM. Isorhapontigenin also inhibited integrin αIIbβ3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100µM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85µM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor

Nutritive Analysis of Five Aquatic Plants at Selected Area in Kelantan, Malaysia

In terms of providing a low cost feeds for animal consumption, the nutrient component for different species of aquatic plants were evaluated. This study emphasize on the nutrition comparative between 5 species of aquatic plants such as Hydrilla verticillata (order: Hydrocharitales), Pistia stratiotes (order: Arales), Chara corallina (order: Charales), Myriophyllum spicatum (order: Haloragidales) and Azolla pinnata (order: Salviniales) were collected at the selected area in Kelantan (6.1254o N, 102.2381oE). The objectives of this study is to differentiate the nutrient component for five different species of aquatic plants. The study shows that the crude fiber values for H. verticillata, P. stratiotes, C. corallina, M. spicatum, and A. pinnata were 15.35±0.35%, 14.10±0.10%, 20.05±0.55%, 25.60±5.10% and 42.95±2.25% respectively. While the percentage of ash content recorded for H. verticillata, P. stratiotes, C. corallina, M. spicatum, and A. pinnata were 18.8%, 31.8%, 20.6%, 28.9% and 12.0% respectively. The organic matter content for H. verticillata, P. stratiotes, C. corallina, M. spicatum, and A. pinnata were 81.20±0.10%, 68.20±0.10%, 79.40±0.20%, 80.80±0.10% and 88.05±1.45% respectively. The crude protein value for H. verticillata, P. stratiotes, C. corallina, M. spicatum, and A. pinnata were 23.18±0.13%, 16.89±0.08%, 22.30±0.13%, 22.78±0.76% and 21.70±0.09% respectively. While the microbial analysis based on total bacterial count for H. verticillata, P. stratiotes, C. corallina, M. spicatum, and A. pinnata were 2.71CFU/mL, 2.88CFU/mL, 2.16CFU/mL, 0.49CFU/mL and 1.68CFU/mL respectively. In conclusion, the nutrient component for five different species of aquatic plants can are differs from each other. So, the nutrient for five different species of aquatic plants are suitable for animal feed