Activated recombinant CMA-1 (rCMA-1), the chymase inhibitor fulacimstat (BAY 1142524, 1-(3-methyl-2-oxo-1,3-benzoxazol-6-yl)-2,4-dioxo-3-[(1R)-4-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl] pyrimidine-5-carboxylic acid), the plasmin/plasminogen inhibitor BAY 1214237, (10‐chloro‐2‐oxo‐1,2‐dihydropyrimido[1,2‐b] indazol‐4‐yl) piperidinium chloride, rivaroxaban and the fluorogenic substrate for chymase activity, Abz-HPFHL-Lys(Dnp)-NH₂, were obtained from Bayer AG (Wuppertal, Germany). Human purified plasmin was purchased from Innovative Research (Novi, MI, USA).

Blood was collected from healthy donors under a protocol approved by the Comité institutionnel d’éthique de la recherche (CIER) of the Université de Sherbrooke. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Whole blood was collected via venipuncture into sodium citrate (3.2% w/v final), and stored at 4°C for up to 7 days.

Blood clots (50 µl), in the presence or absence of rCMA-1 (1 µM), were generated by adding 31.2 mM of calcium chloride (CaCl₂) to blood samples and incubated at 37°C for 30 min to allow solidification. In another series of experiments towards the development of a reliable biomarker assay, fulacimstat (final concentration of 1 to 100 µM) was added to blood samples. For chymase extraction, clots were homogenized in 10 volumes (v/v) of 20 mM PBS (pH 7.4) supplemented with 0.5 mg/mL of BSA using a glass‐Teflon homogenizer (15), then transferred to a 1.5 ml micro-tube. The homogenates were incubated at 37°C for 30 min, and centrifuged at 18,000xg at 4°C for 30 min, and the supernatants were kept at 4°C until tested for chymase enzymatic activity. Plasmin was purified from clots as previously described (15). Clots were homogenized in 10 volumes (v/v) of 20 mM PBS (pH 7.4) supplemented with 1.5 mg/mL of BSA using a glass‐Teflon homogenizer, then transferred to 1 mL ultracentrifuge tubes. The homogenates were incubated at 37°C for 30 min, and centrifuged at 100,000xg at 4°C for 20 min. Supernatants were kept at 4°C until tested for plasmin enzymatic activity.

A volume of 30 μl of supernatants from clot extracts was treated with either vehicle (0.1% DMSO) or fulacimstat (10 μM) in a white 96‐well plate. In another series of experiments, the plasmin/plasminogen inhibitor, BAY 1214237 (concentration ranging from 0.1 to 50 μM), with blood clots extracts or purified plasmin (1 nM) were added to the plate. A total of 65 to 70 μl of 20 mM PBS (pH 7.4) supplemented with 1.5 mg/ml of BSA (for plasmin) or 0.5 mg/ml of BSA (for chymase) was added to each well for a final volume of 100 μl. Plasmin activity was determined by the hydrolysis rate of 50 μM of the fluorogenic substrate, H-D-Ala-Leu-Lys-7-amino-4-methylcoumarin (Bachem AG, Bubendorf, Switzerland) at 37°C for 1 hour. The fluorescence of the released 7-amino-4-methylcoumarin (AMC) molecule was measured in duplicate with an Infinite M1000 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) with an excitation wavelength (λ_ex) of 370 nm and emission wavelength (λ_em) of 460 nm. Chymase activity was determined by the hydrolysis rate of 10 μM of the fluorogenic substrate, Abz-HPFHL-Lys(Dnp)-NH₂, at 37°C for 1 hour with an λ_ex of 320 nm and λ_em of 400 nm. Increases in fluorescence are expressed as relative fluorescence intensity in arbitrary units per minute (AU/min^-1).

The formation of halo-shaped blood clots was conducted following the methods described by Bonnard et al. (16). To initiate clot formation, 31.2 mM of CaCl₂ was added to blood samples collected from healthy donors. A volume of 20 µl of the CaCl₂-supplemented blood was then carefully added in a circular motion around the edge of wells in a flat-bottomed, transparent 96-well plate, creating a halo-shaped clot through fluidic cohesion, leaving the center of each well clear. The plate was subsequently sealed and incubated at 37°C for 30 minutes to allow the halos to solidify. Initial control experiments showed that rCMA-1 (1 µM) per se did not alter clot stability (results not shown).

Following the formation and solidification of the halo-shaped blood clots as described above, fully formed clots were simultaneously treated with recombinant human tissue plasminogen activator (tPA; 1.5 nM; Cathflo^®, Genentech Inc., SF, USA) and recombinant human chymase (at a final concentration of 0.5 µM or 1 µM) in phosphate-buffered saline (PBS), in the presence or absence of fulacimstat at concentrations of 0.1, 1, or 10 µM (only applied at 1 µM rCMA-1). In another set of experiments, BAY 1214237 (1 µM) was co-administered with tPA, recombinant chymase (1 µM), and fulacimstat (10 µM) onto the pre-formed halos. The plate was sealed and incubated for 2 hours at 37°C with gentle lateral mixing (2 rpm). Absorbance at 510 nm was then measured, and clot degradation was calculated using the formula:

In a different set of experiments, recombinant human chymase (rCMA-1) was incorporated during the initial formation of the halo-shaped blood clots. A volume of 1 µl of activated rCMA-1 was introduced to achieve final concentrations of 0.5 µM or 1 µM, in the presence or absence of 10 µM fulacimstat (used only at the 1 µM concentration of rCMA-1). The chymase was deposited around the bottom edge of the wells, and 20 µl of CaCl₂-supplemented blood was then added in a circular motion to facilitate the halo formation. The plate was sealed and incubated at 37°C for 30 minutes to allow the blood halos to solidify.

Recombinant tPA (Cathflo^®, Genentech Inc., SF, USA) induces blood clot fibrinolysis by activating endogenous plasmin. tPA was diluted to a concentration of 1.5 nM in 80 µl of 10 mM PBS (pH 7.4) and was added into each well. The 96-wells plate was subsequently incubated at 37°C for 30 min. Negative controls (A_total) were obtained from addition of 80 µl of PBS without fibrinolytic agents, and positive controls (A_zero) correspond to wells containing 20 µl of fresh whole blood, non-supplemented with CaCl₂, and 80 μl of 10 mM PBS (pH 7.4). The fibrinolysis rate was measured with a plate reader (Infinite M1000 spectrophotometer (Tecan Group Ltd., Männerdorf, Switzerland)). The degradation of the halo was measured in real time as coverage of each experimental well as the thrombolysed blood increases absorbance at 510 nm. Changes in absorbance were measured every minute at 37°C for 2h, after a 5 second orbital shaking at 195.3 rpm at an amplitude of 3.5 mm. Maximal absorbance measurement was converted to a percentage of degradation according to the formula above mentioned.

In addition to real time monitoring and plotting of blood degradation kinetics, other parameters derived from the same curves, namely: i) The activation time (A_t) required for the induction of fibrinolysis which is defined as the first value of t for which D_x(t)/dt > 1. ii) The time required to obtain 50% lysis (T0.5) is defined as the first value of t which D_x(t) = 50%. iii). The maximum clot lysis rate (CLR_max) corresponds to the maximum value of D_x(t)/dt.

Fresh thrombi obtained from thrombectomy patients undergoing pulmonary embolectomy for intermediate-high risk pulmonary embolism were immediately cooled and transported to the laboratory for processing on the same day. Each thrombus was meticulously dissected into distinct “whitish” and “reddish” sections, each weighing approximately 50 mg. These sections were incubated in 0.5 mL of PBS and stored at 4°C for 18-48 hours to allow the transition of hemoglobin into the buffer. The samples underwent two washes with 0.5 mL PBS and were gently dried. Subsequently, each sample was further subdivided into smaller pieces of around 8 mg. Each 8 mg thrombus piece was then transferred to a 96-well microtiter plate, and 130 µL of PBS was added. A volume of 15 µL of PBS with either vehicle or fulacimstat (1 µM) was added to each well. Chymase activity was determined by the hydrolysis rate of 10 μM of the fluorogenic substrate, Abz-HPFHL-Lys(Dnp)-NH₂, at 37°C for 30 minutes with an λ_ex of 320 nm and λ_em of 420 nm. Increases in fluorescence are expressed as fluorescence intensity in arbitrary units.

All data are presented as mean ± standard error of the mean (SEM). All graphical and statistical analysis were performed on GraphPad Prism 10.4.1 software (GraphPad Software, La Jolla, CA). Normality of data distribution was assessed with the D’Agostino & Pearson test for large samples and the Shapiro-Wilk test for smaller samples. Student’s t-test was used for two-groups comparisons, as the data met the normality assumption. One-way ANOVA followed by Bonferroni’s post hoc test was applied for comparisons between more than tho groups. Two-way ANOVA with Fisher’s LSD test was used for experiments involving two factors. Statistical significance was reached when the P value was < 0.05, and one symbol (*) represents P < 0.05, two symbols (**) represent that P < 0.01, and three symbols (***) represent that P < 0.001.

In a first series of experiments, the plasminogen/plasmin inhibitor BAY 1214237, inhibited, in concentration-dependent fashion, purified- as well as endogenous plasmin extracted from clots formed in vitro using human whole blood, with a half maximal inhibitory concentration (IC₅₀) of 6.488 and 3.718 µM, respectively ( Figures 1A, B ).

Complementary to the inhibitory properties of BAY 1214237 on endogenous plasmin activity shown in Figure 1B , the same inhibitor demonstrated a concentration dependant reduction in the dissolution of halo shaped-blood clots in the presence of 1.5 nM of Alteplase ( Figures 2A ). Furthermore, increasing concentrations of the plasminogen/plasmin inhibitor BAY 1214237 significantly extended activation times and time to 50% lysis (T0.5), while reducing maximal clot lysis rates ( Figures 2C, D ). Additional control experiments investigated the roles of endogenous Factor Xa and plasmin in the induction of coagulation of halo-shaped human blood clots with increasing concentration of the Factor Xa inhibitor rivaroxaban, the chymase inhibitor fulacimstat or the plasminogen/plasmin inhibitor BAY 1214237 (from 0.01 to 100 µM) ( Supplementary Figure S1 ). Rivaroxaban inhibited halo coagulation process with a half maximal inhibitory concentration of 1.249 μM. This effect was not observed with fulacimstat or BAY 1214237, indicating that the latter compounds do not interfere with any coagulation factors. Taken together, these results demonstrate that the halo assay requires the activation of endogenous Factor Xa and plasmin for the induction and dissolution, respectively, of halo-shaped human blood clots.

The chymase inhibitor, fulacimstat (10 μM), exhibited no effect on endogenous chymotrypsin enzymatic activity in whole blood ( Figure 3A ), yet markedly reduced the hydrolytic activity of exogenously administered human recombinant chymase (rCMA-1: 1 µM), extracted from clots formed in vitro using human whole blood. Fulacimstat also demonstrated no effect on plasmin activity in the absence of rCMA-1. In contrast, fulacimstat inhibited the marked reduction of plasmin activity caused by the addition of exogenous rCMA-1 before blood recalcification ( Figure 3B ). In addition, this study showed that fulacimstat inhibited the rCMA-1-dependent degradation of purified human plasmin ( Supplementary Figure S2 ) as previously reported with another chymase inhibitor, TY-51469, by our group (15).

In a next series of experiments, a new assay was developed as a biomarker of fulacimstat activity in human blood clots. Using this assay, it was suggested that plasma free concentrations of fulacimstat may be determined in coagulated blood samples of human subjects, as illustrated in Figures 3C, D .

Considering the significant inhibition of endogenous plasmin hydrolytic activity extracted from blood clot containing rCMA-1, as shown in Figure 3 , the effects of the addition of the latter serine protease on halo fibrinolysis were investigated using the halo assay. rCMA-1 was administered in the presence of a pre-solidified human blood halos. At the highest concentration tested (1 µM), the human recombinant chymase reduced the tPA-induced dissolution of clots by 80% after 2h incubation ( Figure 4 ). The chymase inhibitor (concentrations ranging from 0.1 to 10 µM), administered concomitantly with rCMA-1, restored the tPA-induced fibrinolysis of human blood halos, in a concentration-dependent fashion. Finally, the plasmin/plasminogen inhibitor BAY 1214237 (1 µM), added simultaneously with rCMA-1 (1 µM) and fulacimstat (10 µM), fully reversed the tPA-induced dissolution of human blood halos, as well as the acceleration of halo dissolution in the presence of the chymase inhibitor ( Figure 4 ). Taken together, these results showed that exogenous chymase impaired clot dissolution by plasmin, which was reversed by the addition of fulacimstat in the micro-environment of a pre-formed blood clot.

Next, recombinant tPA (1.5 nM) induced a marked dissolution of human halo shaped blood clots ( Figure 5A ). This halo dissolution was significantly reduced in concentration-dependent fashion by rCMA-1 when added to the blood prior to CaCl₂-induced clotting. In addition, the higher concentration of rCMA-1 (1 µM) significantly reduced activation times (A_t) ( Figure 5B ) and time to 50% lysis (T0.5) ( Figure 5C ), while increasing the maximal clot lysis rates (CLR_max) ( Figure 5D ). All human chymase-dependant effects were sensitive to fulacimstat.

In the last series of experiments, chymase activity was identified in human thrombi obtained from thrombectomy procedures ( Figure 6 ). All clot samples derived from pulmonary embolectomy exhibited detectable chymase activity, which was sensitive to the specific inhibitor fulacimstat. Notably, the whitish sections of thrombi, typically rich in platelets and fibrin, demonstrated a higher chymase enzymatic activity compared to the reddish sections, which are predominantly composed of red blood cells. These findings represent the first direct evidence of active chymase in human thrombi.