Human blood clotting parameters

Blood was collected from healthy and unmedicated volunteers in polypropylene tubes containing 3.8% sodium citrate (1/10, w/v). The blood sample was centrifuged at 350 xg for 15 min at 25 °C, to obtain platelet-poor plasma (PPP), and PPP was stored at −80 °C until use. The experimental protocols were conducted according to the guidelines of the ethics committee of the Universidade Federal de São Paulo, São Paulo, Brazil.

Activated partial thromboplastin time and prothrombin time determination

aPTT and PT assays were performed as described by Brito et al. [12] with minor alterations. To determine aPTT, we incubated 50 µL (16.3 up to 217.4 µg) of (p-BthTX-I)2 K or 0.15 M NaCl (control) with 50 µL of PPP and 50 µL of aPTT reagent for 2 min followed by the addition of 50 µL of 0.025 M calcium chloride. The clotting time was measured in seconds (sec). PT was assayed by incubating 50 µL of (p-BthTX-I)2 K (108.7–434.8 µg) with 50 µL of PPP for 60 s, followed by the addition of 100 µL of Thromborel S reagent (Siemens Healthineers, Marburg, Germany). Assays were performed in duplicates and the results were expressed in seconds.

Evaluation of (p-BthTX-I)2 in plasma-like activities of kallikrein and factor Xa

Preincubation of 20 µL of human plasma was performed with increasing concentrations of (p-BthTX-I)2 K (5.0–80 µg) or 0.15 M NaCl solution and 10 µL of aPTT activator reagent (actin activated cephaloplastin, Dade Behring, Marburg, Germany) for 10 min at 37 ºC. To determine the hydrolytic activity, 20 µL of this plasma was mixed with 0.7 mM H-D-Pro-Phe-Arg-pNan (substrate for kallikrein-like enzymes) or 2 mM CH3OCO-D-CHA-Gly-Arg-pNa-AcOH (FXa-like enzymes) (Behring, Marburg, Germany) in 0.02 M Tris-HCl, 140 mM NaCl, 5 mM CaCl2, 0.1% BSA (pH 7.4) (FXa) or 0.05 M Tris-HCl, 120 mM NaCl, 0.1% BSA (PKa) and subsequently activated with 10 µL of 0.025 M calcium chloride. Substrate hydrolysis was monitored for 60 min by photometric reading at 405 nm using the SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA, USA).

Measurement of nitric oxide

Nitric oxide (NO) is an important molecule that has been extensively investigated due to its wide-ranging physiological and biological involvement. In biological fluids such as plasma, NO is rapidly converted to oxidation products (NO2− and NO3−), which emit photons into the ground state and these photons can be measured by a photomultiplier tube (PMT). The proportion of NO in the reaction cell is equivalent to the emitted light detected by the PMT [13]. To test NO production, we incubated pooled plasma obtained from healthy donors at COLSAN with increasing concentrations of (p-BthTX-I)2 K (5.0 up to 80 µg) for 2 h, froze the solutions, and stored them at -20 °C until use. For deproteinization, ethanol was added to the plasma sample (1:2 ratio) and incubated for 30 min at 4 °C. Next, samples were centrifuged for 5 min at 17,000 xg at 4 °C. Total protein was quantified in the clear supernatant, and 100 µL aliquots were used to measure total NO oxidation products with a nitric oxide analyzer (NOA, model 280, Sievers Instruments, Boulder, CO, USA) [14]. NO concentrations were expressed as micromoles (µM).

Human platelet aggregation

Platelet aggregation was measured using platelet-rich plasma (PRP) obtained from pooled venous blood of healthy donors collected in conical plastic tubes containing 3.8% trisodium citrate (1:10, v/v). PRP was obtained by centrifugation at 350 xg for 12 min at 25° C. Concentration of 2.5 × 108 platelets/mL was maintained with HEPES Tyrode buffer (pH 7.4) (137 mM NaCl, 2.9 mM KCl,12.0 mM Na2HPO4, 1.0 mM MgCl2, 5 mM HEPES, 5 mM C6H12O6, and 1 mM CaCl2) using a hematological cell counter (Sysmex KX-21 N™, Sysmex, Kobe, Hyogo Japan) [15]. PRP aggregation was performed using the Chrono-Log 490 aggregometer (Chrono-Log, Havertown, PA) with continuous shaking at 37 °C and 160 xg. The agonists used were adenosine diphosphate (ADP) (5 µM) (Chrono-Log, Havertown, PA, USA), collagen (2 µg/mL) (Chrono-Log), and arachidonic acid (1.0 mM) (Chrono-Log). HEPES buffer was used as an experimental control, and the final volume of the reaction was 500 mL. Aggregation was monitored for 5 min and 30 s. The degree of platelet aggregation was defined as the percentage change in light transmittance from PRP (0% light transmission) to PPP (100% light transmission).

To test the effect of the peptide (p-BthTX-I)2 K, we pre-incubated platelets with increasing concentrations of (p-BthTX-I)2 K (7.82–608.7 µg) for 5 min at 37 °C under agitation before adding the agonists. Control of platelet aggregation was performed at the beginning and end of each experiment to confirm platelet viability using 0.1 IU Thrombin (Chrono-Log).

Ex vivo and in vivo experimental models

In order to study the experimental mouse model of arterial thrombosis, we obtained C57BL/6 male adult mice that were 7–8 weeks old and that weighed 20–25 g from the Instituto de Farmacologia e Biologia Molecular Professor Dr. Ribeiro do Vale at Universidade Federal de São Paulo (INFAR-UNIFESP). Animal experimentation protocols were approved by the Committee on Ethics in the Use of Animals (CEUA) process number 2019-7922240819, and all protocols followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

In all experiments, mice were intraperitoneally anesthetized before experiments with ketamine (100 mg/kg), xylazine (20 mg/kg), and morphine (5 mg/kg). The experiments were initiated 10 min after administering anesthesia. During the procedures, the animals were kept warm in a thermal blanket. At the end of the experiment, the animals were euthanized with an excessive dose of ketamine (300 mg/kg) and xylazine (30 mg/kg).

Arterial thrombosis induction

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. Next, (p-BthTX-I)2 K (3.75–20.0 mg/kg) or NaCl (0.15 M) was administered intravenously via retro-orbital injection, and after 10 min, Rose Bengal (50 mg/kg) (3,4,5,6 tetrachloro-2,4,5,7-tetraiodofluorescein, Sigma Aldrich, Saint Louis, MO, USA) was administered. An ultrasonic flowmeter (model MA 0.5 PSB; Transonic System, Ithaca, NY) was then placed around the artery, and a 1.5 mW, 540 nm laser was used to irradiate the carotid artery from a distance of 6 cm. The blood flow rate was monitored continuously for 5 min until it stabilized at 0.05 mL/min or lower.

Bleeding time

The animals were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), and 90 min after administering (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline solution (control), a longitudinal incision of 2 mm was made at the end of mice tail. The incised tail was immersed in saline solution (0.15 M NaCl) at 37 °C. The time until bleeding ceased for more than 30 s was measured; for longer bleeding times, a value of 10 min was adopted [18].

Data analysis

Graphs represent mean ± S. D and were built in GraphPad Prism 8 (GraphPad Software, San Diego, CA). Statistical analysis was performed using Jamovi (The Jamovi project – 2021) version 1.6. Distribution tests were performed using the Shapiro-Wilk test, and group comparisons for non-parametric distributions (bleeding time, PT, aPTT) were performed using the Mann-Whitney test. For samples with a normal distribution, an independent Student’s t-test was performed. In the arterial thrombosis experiment, groups were compared using a two-way analysis of variance test (ANOVA) followed by a Tukey-Kramer post-test.