SMTP (Stachybotrys microspora triprenyl phenol) enhances clot clearance in a pulmonary embolism model in rats

We assessed structure-activity relationships of SMTPs to select a congener to be studied in detail. SMTP congeners identified so far are roughly classified into two groups. One is the single-unit congener, consisting of the core triprenyl phenol unit and an N-linked side-chain, which is a side-chain of an amino acid (Figure 1A). The other group is the two-unit congener, consisting of two core units bridged by a diamine (Figure 1A). Among the 8 congeners tested, two-unit congeners (SMTP-7, -7D, -8, and -8D) were more active than single-unit congeners (SMTP-4D, -5D, -6, and -6D) in enhancing u-PA-catalyzed plasminogen activation (Figure 1B). α-Tocopherol, which has a structure resembling the core unit of SMTP (Figure 1A), was inactive (Figure 1B). To characterize further, 3 additional two-unit congeners, SMTP-9, -30, and -31 (Figure 1A), were synthesized (additional file 1). SMTP-9 and -31, which had two carboxyl groups, were less active than congeners with one carboxyl group (Figure 1B). SMTP-30, which had no carboxyl group, was essentially inactive (Figure 1B). Thus, the number of carboxyl group affects the activity of two-unit SMTP congeners. Based on these results, SMTP-7 was selected for detailed characterization.

SMTP-7 enhanced t-PA-catalyzed plasminogen activation as well as the activation catalyzed by u-PA (Figure 2A). In the kinetic determinations of u-PA-catalyzed plasminogen activation, SMTP-7 markedly increased V_max and slightly decreased K _m , resulting in a large increase in k_cat (Figure 2B). The increase in plasmin generation was confirmed by SDS-polyacrylamide gel electrophoresis (Figure 2C). Amidolytic activities of u-PA and t-PA were not affected by SMTP-7 (Figure 2D). While SMTP-7 moderately elevated the activity of plasmin (Figure 2D), the magnitude (~3-fold) was smaller than that of plasminogen activation (~100-fold). In size-exclusion chromatography, the molecular elution time of plasminogen was slightly shortened in the presence of SMTP-7 (Figure 2E), suggesting that an increase in apparent molecular volume was induced by SMTP-7. Taken together, these properties of SMTP-7 are consistent with the idea of a plasminogen modulator, which increases plasminogen activation by affecting the conformational status of plasminogen [10].

To assess plasminogen activation in vivo, we determined the level of Pm-AP as an index of plasmin generation. Pm-AP was determined by fibrinogen zymography, in which human and mouse Pm-AP appeared as lysis bands at ~140 and ~130 kDa, respectively (additional file 2). When SMTP-7 was administered to normal mice (5 and 10 mg kg^-1, bolus intravenous injections), the level of Pm-AP in plasma was significantly increased (~1.5-fold, P < 0.05) (Figures 3A and 3B). Both anti-plasminogen IgG and anti-α₂-antiplasmin IgG decreased the intensity of the lysis band, supporting that the lysis band represented Pm-AP (Figure 3C and additional file 2). These results suggest that SMTP-7 enhances plasmin generation in vivo.

SMTP-7 was evaluated further in a rat pulmonary embolism model, in which animals were injected with small particles of ¹²⁵I-labeled plasma clots. The clots predominantly distributed over the lungs, and its clearance was monitored continually as the decay of radioactivity in the thorax. Based on the effects of SMTP-7 on the Pm-AP accumulation in mice (Figure 3), the dose of 5 mg kg^-1 was used. In control animals given saline alone, the clearance of ¹²⁵I-plasma clots occurred slowly (6.4 ± 2.8% per 20 min) (Figure 4A). The treatment with SMTP-7 (5 mg kg^-1, bolus intravenous injection) significantly increased the rate of clot clearance (19.8 ± 2.4% per 20 min) (Figure 4A). This increase was comparable to that (19.8 ± 4.1% per 20 min) brought by scu-PA (250 U kg^-1, bolus intravenous injection) (Figure 4B). The combination of SMTP-7 (5 mg kg^-1) and scu-PA (250 U kg^-1) increased the clot clearance rate to 42.3 ± 4.2% per 20 min (Figure 4B).

Effects of SMTP-7 on bleeding and rebleeding were assessed by a tail amputation assay in normal mice. SMTP-7 was tested at the pharmacological dose (5 mg kg^-1) and a higher dose (30 mg kg^-1). Neither a statistically significant prolongation of bleeding time nor an increase in bleeding volume was observed at both doses (additional file 3).

Human native plasminogen was isolated by lysine-Sepharose affinity chromatography from frozen citrated plasma. The source of the following reagents were: single-chain u-PA (scu-PA; Thrombolyse^®) from Mitsubishi Pharma (Osaka, Japan); high molecular weight u-PA (1.47 × 10⁵ IU mg^-1) from JCR Pharmaceuticals (Kobe, Japan); two-chain t-PA (7.0 × 10⁵ IU mg^-1) from Biopool (Umeå, Sweden); sheep anti-mouse plasminogen IgG from Haematologic Technologies (Essex Junction, VT, USA); rabbit anti-mouse α₂-antiplasmin IgG from Merdian Life Science (Saco, ME, USA); human fibrinogen and human α-thrombin from Sigma (St. Louis, MO, USA); carrier-free Na¹²⁵I from Amersham. Radioiodination of fibrinogen and plasminogen was performed using the iodine monochloride method. Upon trichloroacetic acid treatment, more than 95% of radioactivity in the fibrinogen and plasminogen preparations precipitated with protein. When treated with thrombin, approximately 70% of the radioactivity in ¹²⁵I-fibrinogen was incorporated into the resulting clots.

All the SMTP congeners used in this study were produced by S. microspora IFO 30018. SMTP-4D, -5D, -6, -6D, -7, -7D, -8, and -8D were isolated as described previously [15, 17]. SMTP-9, -30, and -31 were originally isolated as described in additional file 1. In experiments in vitro, SMTPs were dissolved directly in buffer A (50 mM Tris-HCl, 100 mM NaCl, and 0.01% Tween 80, pH 7.4). In animal experiments, SMTP-7 was dissolved in saline by adjusting pH ~9 with dilute NaOH.

Plasminogen activation was determined by measuring the initial velocity of plasmin generation using H-Val-Leu-Lys-p-nitroanilide (Bachem, Bubendorf, Switzerland), a chromogenic substrate for plasmin. A reaction mixture consisting of 50 nM plasminogen, 50 IU ml^-1 u-PA (or 200 IU ml^-1 t-PA) and 0.1 mM of the substrate in 50 μl of buffer A was incubated in a well of a 96-well microplate at 37°C for up to 40 min. Absorbance at 405 nm was measured with an interval of 1 to 2 min. From the slope of the plots of A₄₀₅ nm versus t², the initial velocity of plasmin generation was calculated. In the experiment to determine kinetic parameters, assays were performed with varying concentrations of plasminogen (0.5-2 μM) and a fixed concentration of u-PA (50 IU ml^-1) in the presence or absence of SMTP-7 (100 μM). Since SMTP-7 at 100 μM enhanced plasmin activity toward H-Val-Leu-Lys-p-nitroanilide by 3.26-fold (see Figure 2D), this factor was taken into account in the process of the calculation of plasmin generation velocities.

Plasminogen activation was alternatively assayed by determining the conversion to the two-chain form. ¹²⁵I-Plasminogen (100 nM) was incubated with u-PA (50 IU ml^-1) and aprotinin (100 kallikrein inhibitor units ml^-1) in buffer A at 37°C for 30 min. The mixture was resolved on SDS-polyacrylamide gel electrophoresis under reducing conditions. The gel was stained with Coomassie Brilliant Blue R-250.

Amidolytic activities of plasmin (10 nM), u-PA (1 IU ml^-1), and t-PA (2000 IU ml^-1) were determined at 37°C in buffer A using 10 μM of H-Val-Leu-Lys-7-amino-4-methylcoumarin, t-butyloxycarbonyl-Glu-Gly-Arg-7-amino-4-methylcoumarin, and succinyl-Phe-Ser-Arg-7-amino-4-methylcoumarin, respectively.

Size-exclusion chromatography was performed using a TSK-Gel G-3000SW column (7.5 × 600 mm, TOSOH, Tokyo, Japan) equilibrated with buffer A in the presence or absence of SMTP-7 (120 μM). Plasminogen labeled with Alexa Fluor^® 488 (Molecular Probes, Eugene, OR, USA) (10 μg) was resolved at room temperature at a rate of 1 ml/min. The elution was monitored using a fluorescence detector with an excitation at 495 nm and an emission at 520 nm.

All of the animal protocols were approved by the institutional animal care committee of Tokyo Noko University. Male Wistar rats and male ICR mice were obtained from Japan SLC (Hamamatsu).

Male ICR mice (7 weeks of age) received intravenous SMTP-7 or saline. After 60 min, blood was collected from inferior vena cava in 13 mM sodium citrate. Plasma was rapidly prepared by centrifugation and mixed with one volume of buffer B (125 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (w/v) glycerol, and 0.004% (w/v) bromophenol blue). In some experiments, plasma was pretreated with ant-plasminogen IgG (0.6 mg ml^-1) or anti-α₂-antiplasmin IgG (3 mg ml^-1) for 30 min at room temperature before mixing with buffer B. The mixture (equivalent to 1-2 μl of plasma) was resolved on nonreduced SDS-polyacrylamide gel electrophoresis on a 7.5% gel containing fibrinogen (2 mg ml^-1) at 4°C. As the standard for the calibration of Pm-AP in plasma samples, 1.7 ng of human Pm-AP (prepared by incubating 120 nM human plasmin and 600 nM human α₂-antiplasmin for 30 min at 37°C in buffer A) was resolved on the same gel. After electrophoresis, the gel was cut at the position of ~90 kDa, and the upper half was washed with Triton X-100 (2.5 %, w/v) and incubated in 50 mM Tris-HCl, pH 8.3, and 100 mM glycine for 60 h at 37°C. (The lower half gave a strong lysis band at ~70 kDa, possibly due to plasma kallikrein) Gels were stained with Coomassie Brilliant Blue R-250. The lysis zones due to protease activities appeared as unstained bands on a blue background. Human Pm-AP gave a lysis band at ~140 kDa, while the mouse counterpart afforded a band at ~130 kDa (additional file 2). The scanned image of the stained gel was reversed for presentations. The band intensity was determined using Scion image. The amounts of Pm-AP in test samples were calibrated by comparing intensities of lysis bands of samples with that of the standard, and data were expressed as human Pm-AP equivalent (nM in plasma).

Platelet-poor plasma from male Wistar rats was mixed with ¹²⁵I-fibrinogen (139 μg ml^-1, ~2.5 MBq) and α-thrombin (1 IU ml^-1) in the presence of 44 mM CaCl₂. After incubation at 37°C for 120 min, the resulting plasma clot was washed thrice with saline and powdered in a mortar under liquid nitrogen, followed by homogenization 4 strokes in 2.7 ml saline using a Potter Elvehjem homogenizer with a Teflon pestle (13-mm in diameter). The suspension was left at room temperature for 30 min, and the resulting precipitates were homogenized again. This operation was repeated once more. The combined homogenates were settled for 30 min, and the resulting supernatant was centrifuged at 20 × g for 3 min to obtain pellet consisting of clot particles of 10-100 μm in diameter.

Male Wistar rats weighing ~120 g were kept at 22°C with normal chaw for 1-7 days before the use in experiments. Rats were anesthetized with urethane and chloralose (750 and 65 mg kg^-1, respectively, i.p.), and a probe (equipped with a 10-mm collimator) of a model TCS-163 NaI scintillation survey monitor (Aloka, Tokyo, Japan) was placed above the thorax. A suspension of ¹²⁵I-plasma clot particles (75 μl kg^-1; ~1.2 × 10⁷ Bq per animal) was injected simultaneously with heparin (165 units kg^-1) and NaI (3.3 mg kg^-1) into a caudal vein. The ¹²⁵I-plasma clots predominantly distributed over the lungs [30], and radioactivity over the thorax was measured continually. Saline alone (0.5 ml per animal), saline containing SMTP-7 (5 mg kg^-1), scu-PA (250 U kg^-1), or both SMTP-7 and scu-PA was intravenously injected ~20 min after the embolization. The monitoring of radioactivity was continued further for ~20 min. The fractional change in radioactivity during 20 min after the treatments represented the rate of clot dissolution.

The measurement of bleeding time and rebleeding volume (secondary oozing from the bleeding time wounds) were performed using male ICR mice (6 weeks of age) as described previously [31]. Briefly, mice were anesthetized with 60 mg kg^-1 pentobarbital intraperitoneally and given bolus injection of 5% mannitol (5 ml kg^-1) or SMTP-7 (5 and 30 mg kg^-1) in 5% mannitol via a tail vein. Five minutes after the administration, a 5-mm tail segment was amputated with a razor blade. The tail was immersed immediately in prewarmed saline at 37°C, and the time required to stop visual spontaneous bleeding was determined. To evaluate rebleeding, the tail was then immersed in another 4-mL prewarmed saline (37°C) containing 14 mM trisodium citrate for 60 min. Red blood cells were collected and lysed in water to measure absorbance at 490 nm, from which blood loss by rebleeding was calculated.