Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Improved Antithrombotic Activity and Diminished Bleeding Side Effect of a PEGylated αIIbß3 Antagonist, Disintegrin.

Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decrease degradation and reduces renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety and pharmacokinetic profiles and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, there was no effect of PEG-RR in human whole blood coagulation even given at a higher concentration (30 µg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.

A Novel αIIbß3 Antagonist from Snake Venom Prevents Thrombosis without Causing Bleeding.

Life-threatening thrombocytopenia and bleeding, common side effects of clinically available αIIbß3 antagonists, are associated with the induction of ligand-induced integrin conformational changes and exposure of ligand-induced binding sites (LIBSs). To address this issue, we examined intrinsic mechanisms and structure-activity relationships of purified disintegrins, from Protobothrops flavoviridis venom (i.e., Trimeresurus flavoviridis), TFV-1 and TFV-3 with distinctly different pro-hemorrhagic tendencies. TFV-1 with a different αIIbß3 binding epitope from that of TFV-3 and chimeric 7 × 103 Fab, i.e., Abciximab, decelerates αIIbß3 ligation without causing a conformational change in αIIbß3, as determined with the LIBS antibody, AP5, and the mimetic, drug-dependent antibody (DDAb), AP2, an inhibitory monoclonal antibody raised against αIIbß3. Consistent with their different binding epitopes, a combination of TFV-1 and AP2 did not induce FcγRIIa-mediated activation of the ITAM-Syk-PLCγ2 pathway and platelet aggregation, in contrast to the clinical antithrombotics, abciximab, eptifibatide, and disintegrin TFV-3. Furthermore, TFV-1 selectively inhibits Gα13-mediated platelet aggregation without affecting talin-driven clot firmness, which is responsible for physiological hemostatic processes. At equally efficacious antithrombotic dosages, TFV-1 caused neither severe thrombocytopenia nor bleeding in FcγRIIa-transgenic mice. Likewise, it did not induce hypocoagulation in human whole blood in the rotational thromboelastometry (ROTEM) assay used in perioperative situations. In contrast, TFV-3 and eptifibatide exhibited all of these hemostatic effects. Thus, the αIIbß3 antagonist, TFV-1, efficaciously prevents arterial thrombosis without adversely affecting hemostasis.

From Discovery of Snake Venom Disintegrins to A Safer Therapeutic Antithrombotic Agent.

Snake venoms affect blood coagulation and platelet function in diverse ways. Some venom components inhibit platelet function, while other components induce platelet aggregation. Among the platelet aggregation inhibitors, disintegrins have been recognized as unique and potentially valuable tools for examining cell-matrix and cell-cell interactions and for the development of antithrombotic and antiangiogenic agents according to their anti-adhesive and anti-migration effect on tumor cells and antiangiogenesis activities. Disintegrins represent a family of low molecular weight, cysteine-rich, Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing polypeptides, which inhibit fibrinogen binding to integrin αIIbß3 (i.e., platelet glycoprotein IIb/IIIa), as well as ligand binding to integrins αvß3, and α5ß1 expressed on cells (i.e., fibroblasts, tumor cells, and endothelial cells). This review focuses on the current efforts attained from studies using disintegrins as a tool in the field of arterial thrombosis, angiogenesis, inflammation, and tumor metastasis, and briefly describes their potential therapeutic applications and side effects in integrin-related diseases. Additionally, novel R(K)GD-containing disintegrin TMV-7 mutants are being designed as safer antithrombotics without causing thrombocytopenia and bleeding.

Trimucrin, an Arg-Gly-Asp containing disintegrin, attenuates myocardial ischemia-reperfusion injury in murine by inhibiting platelet function.

Trimucrin, a novel small-mass Arg-Gly-Asp (RGD)-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbß3 and phagocyte αvß3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats platelet aggregation was diminished in response to adenosine diphosphate (ADP). We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals received intravenous trimucrin or saline, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity in carotid blood compared with vehicle-treated animals during the same period. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces without affecting platelet counts. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R compared with controls. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function.

Anti-thrombotic agents derived from snake venom proteins.

Snake venoms affect blood coagulation and platelet function in a complex manner. However, two classes of venom proteins, snaclecs and disintegrins have been shown to specifically target receptors including GPIb, α2ß1, GPVI, CLEC-2 and integrins αIIbß3, αvß3, α5ß1 expressed on platelets, endothelial cells, phagocytes, tumor cells, thus affecting cell-matrices and cell-cell interactions. Here, we focus on disintegrins, a class of low molecular mass Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing, cysteine-rich polypeptide derived from various viper snake venoms. This review describes the potential applications of disintegrins in field of integrin-related diseases, especially arterial thrombosis, angiogenesis, tumor progression and septic inflammation. In addition, a novel RGD-containing disintegrin TMV-7 is being developed as a safer antithrombotic agent with minimal side effects, such as thrombocytopenia and bleeding.