Improving the understanding of plasma kallikrein contribution to arterial thrombus formation using two plant protease inhibitors

Abstract

The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a K of 1.3 × 10 M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 μM and 15.4 μM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.

Inventory of human-edible products from native Acacia sensu lato in Africa, America, and Asia: Spotlight on Senegalia seeds, overlooked wild legumes in the arid tropics

“…The 70 kDa peptide, for instance, may be associated with the albumin fraction (Boye et al, 2010;Murray et al, 1978) and chymotrypsin, human immunodeficiency virus-1 reverse transcriptase, and breast tumor inhibitors, as was reported already for Acacia confusa seeds (Lam & Ng, 2010). The 15 and 22 kDa peptides have been identified as Kunitz-type inhibitors of serine proteinases (including trypsin, chymotrypsin, coagulation cascade proteinases, immune complement activators), potent antithrombotic agents (Lopes et al, 2009;Odei-Addo et al, 2014;Salu et al, 2018), fungicides (Lopes et al, 2009), and insecticides (Babu & Subrahmanyam, 2010;Machado et al, 2013). Besides, Zahid et al (2017) isolated a high molecular weight arabinogalactan protein (411 kDa) from S. Senegal seeds and showed it modulates skin innate immune responses.…”

Unveiling the bioactivity and bioaccessibility of phenolic compounds from organic coffee husks using an in vitro digestion model

“…Activated partial thromboplastin time is an assessment of intrinsic clotting factors, whereas PT is used to characterize extrinsic clotting factors. 19 The results show the anticoagulant properties of the coffee husk extracts obtained in the study before and after simulated gastrointestinal digestion in vitro. In general, all extracts showed prolonged antithrombotic activity compared with NaCl (0.15 M), except for the EP gastric digestion samples.…”

“…Both determinations were performed in a dual-channel semiautomated coagulometer designed for low-volume laboratories (BF II coagulometer, Dade Behring, Glasgow, Delaware, USA), according to the procedure described by Salu et al 19 Plasma for the assays was obtained from whole blood of three healthy volunteers, collected in 3.8% (w/v) trisodium citrate (1/10), and centrifuged at 1726 × g for 15 min (25 °C). Assays were performed in duplicate, and results were expressed in minutes.…”

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time

“…Arterial thrombosis was induced by photochemical damage to the endothelium as described [16], with minor modifications as described by Salu et al [17]. The left carotid artery was isolated, and a mid-cervical incision was made with the help of a microscope.…”