ANTICOAGULANT AND ANTIPLATELET ACTIVITIES OF JACKFRUIT (ARTOCARPUS HETEROPHYLLUS) SEED EXTRACT

  Objective: The current study focuses on the anticoagulant and antiplatelet activities of aqueous seed extract of Jackfruit (AqSEJ).Methods: Anticoagulant effect of AqSEJ was tested using plasma recalcification time, mouse tail bleeding time, Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Antiplatelet activity was examined by platelet aggregation studies using agonists such as ADP, Collagen and Epinephrine.Results: The AqSEJ enhanced the clotting time of citrated human plasma from control 200±10 s to 740±14 s. The anticoagulant activity of AqSEJ was further strengthened by in-vivo mouse tail bleeding assay. The i. v. injection of AqSEJ significantly prolonged the bleeding time in a dose dependent manner. The recorded bleeding time was>10 min (P<0.01) at the concentration of 30 μg against the PBS treated control of 1.48±0.10 min with the IC50 values 37.5 μg/ml and 47.5 μg/ml respectively. Interestingly, AqSEJ specifically prolonged the clot formation process of only APTT but not PT, revealing the anticoagulation triggered by the extract could be due to its interference in an intrinsic pathway of the blood coagulation cascade. Furthermore, AqSEJ inhibited the agonists such as ADP, epinephrine and collagen induced platelet aggregation of about 66.7%, 39.2% and 37.0% respectively at the concentration of 200 μg.Conclusion: AqSEJ showed anticoagulant and antiplatelet activities. Hence, it may serve as a better alternative for thrombotic disorders.Â

The action of Echis carinatus and Naja naja venoms on human neutrophils; an emphasis on NETosis

Background: Neutrophils are the first line defense cells of the innate immunity. As a final defense, they discharge their de-condensed chromatin/DNA fibers, the NETs (Neutrophil Extracellular Traps), by a process called NETosis. Two types of NETosis have been currently described: the suicidal/delayed/classical-type, which is ROS dependent that results in the ejection of nuclear DNA, and the vital/rapid/early-type, which may or may not require ROS but, eject nuclear/mitochondrial DNA or both. Thus, Echis carinatus and Naja naja venoms are comparatively studied for their NET inducing property. Methods: Formation of NETs, cell viability, ROS, and Ca2+ levels are estimated. An in vivo toxicity study and possible cellular signaling have been addressed using immunoblots and pharmacological inhibitors. Results: E. carinatus and N. naja venoms respectively induce suicidal and vital NETosis. E. carinatus venom induces NETosis by activating NOX and PAD-4 enzymes in a ROS dependent manner via PKC/ERK/JNK signaling axis, while N. naja venom does it by activating PAD-4 enzyme, but independent of ROS requirement and as well as PKC/ERK/JNK activation. Conclusion: For the first time our study demonstrates the distinct action of E. carinatus and N. naja venoms on the process of NETosis. NETosis being a newly explored area in snake venom pharmacodynamics, it is important to study its impact on the various pathophysiological properties induced by snake venoms. Significance: Understanding the varied actions of snake venoms on neutrophils/blood cells and the role of DNase are likely to provide insights for better management of snakebite pathophysiology

Research into the Causes of Venom-Induced Mortality and Morbidity Identifies New Therapeutic Opportunities.

Snakebite primarily affects rural subsistent farming populations in underdeveloped and developing nations. The annual number of deaths (100,000) and physical disabilities (400,000) of snakebite victims is a societal tragedy that poses a significant added socioeconomic burden to the society. Antivenom therapy is the treatment of choice for snakebite but, as testified by the continuing high rates of mortality and morbidity, too many rural tropical snakebite victims fail to access effective treatment. Here, we advocate for more basic research to better understand the pathogenesis of systemic and local envenoming and describe how research outcomes can identify novel snakebite therapeutic strategies with the potential to be more accessible and affordable to victims than current treatment

Aggregation is impaired in starved platelets due to enhanced autophagy and cellular energy depletion

<p>Platelet hyperactivity is the hallmark of thrombosis and hemostasis disorders including atherosclerosis, diabetes, stroke, arthritis, and cancer causing significant mortality and morbidity. Therefore, regulating platelet hyperactivity is an ever growing interest. Very recently, basal autophagic process has been demonstrated to be essential for normal functioning of platelets. However, autophagy can be elevated above basal level under conditions like starvation, and how platelets respond in these settings remains to be elucidative. Therefore, in this study we demonstrate a substantial autophagy induction (above basal level) by starvation, which decreases platelet aggregation responses to various agonists. The decreased aggregation in starved platelets was restored in combination with autophagy inhibitors (3-methyladenine and NH₄Cl) and acetate supplementation. Starved platelets also showed decreased calcium mobilization, granule release, and adhesive properties. Furthermore, <i>ex vivo</i> platelets obtained from starved rats showed increased autophagy markers and decreased aggregation responses to various agonists. Our results distinctly explain that enhanced autophagy and cellular energy depletion are the cause for decreased platelet activation and aggregation. The study emphasizes the cardinal role of starvation and autophagy in the management of diseases and disorders associated with platelet hyperactivity.</p

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance

Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 mu g/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance

Jackfruit (Artocarpus heteophyllus) seed extract exhibits fibrino(geno)lytic activity

Objective: The current study assesses the fibrinogen and fibrin clot hydrolyzing activities of aqueous seed extract of Jackfruit (AqSEJ). Methods: The protein banding pattern of AqSEJ (100 μg) was analyzed on SDS-PAGE. The proteolytic activity of AqSEJ was confirmed by spectrophotometer and zymography experiments. Fibrinogen, fibrin and plasma protein hydrolyzing activities of AqSEJ were analyzed on SDS-PAGE under reduced conditions. Plasminogen activation and indirect hemolytic activities was analyzed using spectrophotometer. The non-toxic property of AqSEJ was tested by edema, hemorrhage in experimental mice. Results: AqSEJ exhibited proteolytic activity and the specific activity was found to be 1.04 units/mg/min. Furthermore, AqSEJ non-specifically hydrolyzed Aα, followed by Bβ and γ chains of human fibrinogen and specifically hydrolyzed α polymer and α chain of partially cross linked human fibrin clot without affecting β chain and γ-γ dimer even up to the tested dose of 30 µg for the incubation period of 8 hours. Importantly, AqSEJ did not hydrolyze other plasma proteins and devoid of plasminogen activation property. The proteolytic activity of AqSEJ was completely neutralized by PMSF and IAA, while EDTA, EGTA, 1,10-Phenanthroline did not, suggesting the presence of serine and cysteine family proteases. Moreover, AqSEJ did not cause edema and hemorrhage in experimental mice up to the tested dose of 200 µg and nontoxic to RBC cells. Conclusion: AqSEJ hydrolyzes fibrinogen and fibrin clot and non-toxic in nature. Hence, this work showcases the potential applications of Jack fruit seed proteases in the treatment of thrombotic disorder