Anticoagulant and antiplatelet activities of novel serine protease purified from seeds of Cucumis maderaspatensis

Abstract

In this study, we report the biochemical characterization of a novel serine protease from seeds of Cucumis maderaspatensis, aimed with assessing the anticoagulant and antiplatelet activities. The purified serine protease was obtained by subjecting the seed extract to ammonium sulphate precipitation followed by anion exchange and gel filtration chromatography. Twenty seven-fold purification with the specific activity of 884.2 U/mg of protease activity was obtained. The characterization of the novel protease enzyme activity for optimum temperature, pH and effect of different protease inhibitors and metal ions were measured using caseinolytic assay and casein zymogram. The relative molecular mass of the novel neutral serine protease (CmSP) is ~ 32 kDa. Its anticoagulant was determined by assessing the delay in plasma re-calcification time in both platelet-rich and platelet-poor plasma. The antiplatelet activity of serine protease was demonstrated by inhibition of agonists induced platelet aggregation; it was in the order of Epinephrine > Adenosine tri phosphate. Further studies would decipher the mechanism of action to understand its therapeutic potential as an antiplatelet and anticoagulant molecule.

Introduction

Vascular diseases are the major causes of death worldwide which include cerebrovascular and cardiovascular diseases (stroke, thrombosis, myocardial infraction) (Choi et al. 2013). A fine balance between coagulation/fibrin formation and fibrinolysis/fibrin disintegration are required to maintain the haemostasis. A series of sequential reactions are involved in coagulation cascade involving major component like prothrombinase complex. The prothrombin complex converts prothrombin to thrombin leading to conversion of soluble fibrinogen into insoluble fibrin polymer (clot). The formation of fibrin clot activates zymogen into their active proteases thereby activating the platelet aggregation forming the plug at the site of injury, thus preventing the blood loss (Bennett, 2006). On the other hand to maintain haemostasis pathway, fibrinolysis activates serine protease like plasmin which degrades the fibrin clot (Fenton et al. 1993; Monroe et al. 2002). A commotion in this exquisite balance between hemorrhage and coagulation results in vascular complications posing clinical challenge. Activation of blood coagulation factors and platelets with simultaneous down-regulation of anticoagulant mechanism leads to deposition of fibrin in artery and vein resulting in thrombosis (Machlus et al. 2011). To mitigate the effects of thrombotic complications, anticoagulant fibrinogenolytic enzymes inhibiting the thrombin are proven to be effective (Esmon 2014). Serine proteases such as plasmin, trypsin are studied for their role in clot dissolving property (Alkjaersig et al. 1959).

Proteases are a broad class of enzymes classified under six different classes: Serine, Cysteine, Aspartic, Threonine, Glutamic and Metalloproteases. They are considered as molecular scissors and are involved in many cellular and physiological processes such as hemostasis, blood coagulation cascade and fibrinolysis (Cheronis et al. 1993). Due to their involvement in various patho-physiological conditions, they have wide therapeutic applications (Losso 2008). Earlier studies have shown the involvement of many plant serine proteases as antiplatelet anticoagulant and in hemostatic process (Horl and Heidland 1988). Enzymes or molecules with anticoagulant and antiplatelet activity for commercial applications should be cost effective with no or less side effects like, risk of hemorrhage and gastric bleeding, allergic reactions or any other sort of pharmacological adversaries observed in commercial anticoagulant cardiovascular drugs (Wang et al. 2006 and Ageno et al. 2012). An alternative choice of drugs to treat thrombosis is serine proteases and the search for novel serine protease is incessant.

Plants are good sources to look for antithrombotic and antiplatelet activities that could lead to novel therapeutic enzymes for treating thrombus related complications (Yamamoto et al. 2005 and Antao et al. 2005). Herbal prescriptions of different parts of plants are traditionally used for their anticoagulant potential. Terminalia bellerica, Careya arborea, Allium cepa, Gloriosa superba, Allium sativum, Curcuma longa, all these plants contain bioactive compounds which curbs the coagulation cascade and aid in thinning the clots. Leucas indica is a traditional medicine used to reduce nasal congestion and asthma. The leaf extracts of this plant are reported to possess anticoagulant fibrinogenolytic property due to expression of serine protease (Gogoi et al. 2018). Cucumis maderaspatensis, L belongs to the family Cucurbitaceae which is consumed as vegetable (Gaikwad et al. 2015) in south Indian subcontinent (Fig. 1). The different parts of fruit posses anti-elastase, anti-microbial, anti-inflammatory, anti-proliferative, analgesic and anti-diabetic (Manjula et al. 2014) properties. Some of the proteases have been isolated and studied from the plants belonging to Cucurbitaceae family. An aspartyl protease from the seeds of cucumber (Cucumis sativus) (Wilimowska-Pelc et al. 1983), a metalloprotease from the cucumber (Delorme et al. 2000), and serine proteases from C. ficifolia and C. melo, cucumisin from C. melo, protease D from C. melo, kiwano protease from C. metuliferus (Antao et al. 2005) have been isolated and studied for various applications. Numerous application and commercial value of proteolytic enzyme has led to search novel sources. Studies from our laboratory have discovered the presence of a novel serine protease with anticoagulant and antiplatelet property from the seeds of C. maderaspatensis. To the best of our knowledge, this is the first report to evaluate the biochemical characterization and evaluation of anticoagulant property of the serine protease purified from the seeds of C. maderaspatensis. The purified protease is named as CmSP. The finding of this work suggests the possible therapeutic application of novel serine protease as an anticoagulant and antiplatelet drug.

Fig. 1.

Picture of Cucumis maderaspatensis, L belonging to family Cucurbitaceae. a Plant bearing flower and fruit b fruit c seeds

Materials and methods

Materials

The chemicals used in this study are fat-free casein, Sephadex G-100, bovine serum albumin (BSA), molecular weight markers, Phenylmethylsulphonyl Fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), iodoacetic acid (IAA), mercuric chloride (HgCl₂) and β-mercaptoethanol. All these were obtained from Sigma-Aldrich, St. Louis, MO, (USA). Heavy metal ions, surfactants, were purchased from Merck. All other reagents and chemicals used were of analytical grade. Fresh human blood was collected from healthy donors for the platelet rich plasma (PRP). The ripened fruit was bought from the local market Kushalnagar, India and the seeds were collected.

Preparation and defatting of seed powder

The 100 g of clean, shade dried matured seeds (Fig. 1c) of C. maderaspatensis were subjected to fine powder using blender. The powder was defatted using hexane (1:3 w/v) at 25 ± 2 °C with occasional stirring. The slurry was allowed to settle (1 h) and the process was repeated 2–3 times to remove the total fat. The supernatant hexane was decanted and the residue was air dried at 25 ± 2 °C. The dried defatted fine powder obtained was stored at 4 °C for further use.

Preparation of crude extract

50 g defatted seed flour was suspended in 250 mL of 20 mM phosphate buffer pH 7.2 containing 50 mM sodium chloride (extraction buffer) and kept for stirring on magnetic stirrer for 6–8 h at 4 °C. The homogenate was centrifuged at 10,000 rpm for 20 min, and then supernatant was collected and stored at 4 °C further use.

Ammonium sulfate fractionation

To the crude extract, solid ammonium sulfate was added slowly to get 0–30% and 30–70% saturation. The salt extract mixture was allowed to stand for 1 h in cold. The precipitated protein was collected by centrifugation at 10,000 rpm for 20 min. The pellet was suspended in 5 mL extraction buffer and was dialyzed against the double distilled water for nearly 24 h with 2–3 times water exchange. The protein content and the protease activity were checked in both 0–30% and 30–70% ammonium sulfate fraction. 30–70% ammonium sulfate fraction showed maximum protease activity and was used for further purification.

Purification of enzyme

DEAE-Sephadex A­50 ion exchange chromatography

The dialyzed ammonium sulphate precipitate (30–70%) was loaded on DEAE-Sephadex A-50 (1.5 × 15 cm) anion exchange chromatography column. The column was washed to remove unbound proteins using 100 mL of 20 mM phosphate buffer pH 7.2. Gradient elution was carried out using sodium chloride in 20 mM phosphate buffer pH 7.2 (0–0.5 M) and 2 mL fraction were collected at a flow rate of 0.5 mL/min. Protein content of each fractions were measured at 280 nm and protease activity was assessed using casein as substrate. The proteolytically active fractions eluted were pooled and subjected to Sephadex G-100 chromatography.

Gel filtration chromatography on Sephadex G-100

The proteolytically rich protein sample of DEAE-Sephadex A-50 was pooled and loaded the Sephadex G-100 gel filtration column (1.5 × 120 cm). Phosphate buffer, 20 mM, pH 7.2 was used for both equilibration and elution. The flow rate was maintained at 15 mL/h and fractions of volume 1.25 mL were collected every 5 min. One bed volume of the column was collected as fractions. The protein content was monitored continuously for each fraction at 280 nm. Fractions showing proteolytic enzyme activity were pooled and used for further characterization studies.

Poly acrylamide gel electrophoresis

Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) was performed according into the method of (Laemmli 1970) with some modification. Fifteen microgram protein from each purification step was loaded on 15% SDS-PAGE under denaturing conditions to check the homogeneity. The protein in the gels was visualized by Coomassie Brilliant blue R-250 and silver staining method (Blum et al. 1987).

Proteolytic activity of CmSP

Proteolytic activity was assayed as described by (Heussen et al. 1980) with slight modification. Fat-free casein (0.1 mL, 1% in 50 mM Tris–HCl buffer, pH 8.0) was incubated with 100 µL of purified sample in a total volume of 1 mL for 60 min at 37 °C. Undigested casein was precipitated by adding 0.2 mL of 10% Trichloroaceticacid (TCA) and left to stand on ice for 30 min. The reaction mixture was then centrifuged at 5000 g for 10 min. Sodium carbonate (2.5 mL, 0.4 M) and Folin–Ciocalteu’s reagent (1:2) were added sequentially to 1 mL of the supernatant and color developed was read at 660 nm (Lowry et al. 1951). One unit of the protease activity is defined as the amount of enzyme required to cause an increase in 0.01 unit of absorbance at 660 nm per minute at 37 °C and is expressed as units. The specific activity was expressed as units/mg of protein.

Characterization of CmSP

The optimum temperature of activity for purified protease was determined by conducting the assay at different temperatures in 50 mM Tris–HCl (pH 7.5). The reaction mixture containing enzyme was incubated for 30 min in a water bath at various temperatures (5–90 °C).

The optimum pH of the purified protease was determined by conducting the assay at various pH using different buffers: 50 mM sodium acetate (pH 3–6), 50 mM sodium phosphate (pH 6–7) and 50 mM Tris–HCl (pH 7.0–9.5).

Effect of protease inhibitors (PIs) and metal ions on CmSP activity

The effect of various PIs as phenylmethylsulfonylfluoride (PMSF), 2-iodoacetic acid, mercuric chloride and ethylenediaminetetraacetic acid (EDTA) (2 mM each) on the peptide hydrolyzing activity of purified protease were assessed. For determining the influence of metal ions, the following salts were used: ferric chloride (FeCl₃), potassium chloride (KCl), magnesium chloride (MgCl₂), sodium chloride (NaCl), and calcium chloride (CaCl₂) (5 mM each). The purified enzyme (0.1 mL/5 μg) was pre-incubated with 2 mM of each protease inhibitors or 5 mM of different metal ions were kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0. The reaction mixture was incubated for 30 min, at 37 °C. The residual protease activity was measured as described above. The reactions were performed independently after pre-incubating the sample with appropriate controls.

In-gel activity assay

Casein zymography was performed in polyacrylamide gel (10%) containing casein (1%) as copolymerized substrate, as described by Anson (1938) under non-denaturing and non-reducing conditions. Fifty microgram of protein was loaded. After electrophoresis the gel was washed three times using water, with ten times the volume of gel. The washed gel was incubated in 20 mM phosphate buffer pH 8.0 containing 0.1 M NaCl and 1 mM CaCl₂ for 5 h at 37 °C and then stained with Coomassie Brilliant Blue R-250. The activity band is observed as a clear colorless area depleted of casein in the gel against the blue background.

To check the effect of inhibitors on CmSP, in-gel activity assay was performed. The purified enzyme (0.1 mL/5 μg) was pre-incubated with 2 mM of each protease inhibitors (PMSF, 2-iodoacetic acid, mercuric chloride and EDTA) were kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0 and incubated for 30 min. Undigested casein was precipitated by adding 0.2 mL of 10% Trichloroaceticacid (TCA) and left to stand on ice for 30 min. The reaction mixture was then centrifuged at 5000 g for 10 min. Protein content in the supernatant was checked spectrophotometrically and sample equivalent to 50 µg protein was drawn to perform electrophoresis. Electrophoresis was performed under denaturing conditions on 12.5% SDS-PAGE. The gel was visualized using Coomassie Brilliant Blue R-250.

Anticoagulant activity

Plasma re-calcification time

The plasma re-calcification time was determined according to the method of (Quick et al. 1935). Briefly, different concentration of protease sample (0–100 μg) was pre-incubated with 0.2 mL of citrated human plasma in the presence of 10 mM Tris HCl (20 μL) buffer pH 7.4 for 1 min at 37 °C, 20 μL of 0.25 M CaCl₂ was added to the pre-incubated mixture and clotting time was recorded.

Preparation of platelet-rich plasma and platelet-poor plasma

The method of (Ardlie et al. 1974) was employed for the preparation of human platelet-rich plasma (PRP) and platelet-poor plasma (PPP). The platelet concentration of PRP was adjustedto 3.1 × 10⁸ platelets/mL with PPP. The PRP maintained at 37 °C was used within 2 h for the aggregation process. All the above preparations were carried out using plastic wares or siliconized glass wares.

Platelet aggregation

The turbidimetric method of (Born 1962) was followed using a Chronology dual channel whole blood/optical lumi-aggregation system (Model-700). Aliquots of PRP were pre-incubated with various concentrations of protein sample (0–100 μg) in 0.25 mL reaction volume. The aggregation was initiated independently by the addition of agonists, such as ADP and epinephrine and monitored for 6 min.

Statistical analysis

All analysis was performed using GraphPad Prism 6.01. Data of three sets of independent experiments are expressed as ± mean.

Results and discussion

Isolation and purification of CmSP

Protease inhibitors commonly occur in seed of plants. These are small molecular weight proteins which are expressed to protect the host during insect and pathogen infestation or injury (Ryan 1990). They protect the host by inhibiting the digestive enzymes thereby acting as anti-metabolic proteins. In this study, the seed of C. maderaspatensis belonging to Cucurbitaceae family was evaluated for the expression of protease activity. Ammonium sulphate precipitation, anion exchange and gel filtration chromatography were sequentially performed to purify protease from seed flour of C. maderaspatensis. The protein was isolated from crude extract of defatted seed flour by ammonium sulphate precipitation (0–30% and 30–70%). Ammonium sulphate fraction, 30–70% showed maximum protease activity and hence, after dialysis this was further subjected to DEAE-Sephadex A-50 anion exchange chromatography. The protein fractions with protease activity were pooled and subjected to further purification by loading on to Sephadex G-100 gel filtration chromatography. Twenty seven-fold purification was achieved by subjecting the crude extract to various purification steps. Thirty nine milligram of protein with the specific activity of 844.6 U/mg of protein for protease activity was obtained from 100 g of defatted seed flour at the end of Sephadex G-100 gel filtration chromatography. The fractionation profile of DEAE-Sephadex A-50 anion exchange chromatography and Sephadex G-100 chromatography is shown in Fig. 2a and b, respectively. The total protein (mg), activity (U), specific activity (U/mg of protein) and fold purification from 100 g of defatted seed flour during various steps of protease purification are summarized in purification Table 1. The homogeneity of purified protein during each purification step was checked by loading 15 µg of protein on denaturing SDS–PAGE (15%) and the bands were visualized by silver staining (Fig. 2c). The purified protease is named as CmSP.

Fig. 2.

a Elution profile of CmSP proteases on DEAE-Sephadex A-50 anion exchange chromatography. The partially purified 30–70% ammonium sulfate fraction was loaded onto DEAE-Sephadex A-50 column. The column (3 × 15 cm) was pre equilibrated with 20 mM phosphate buffer pH 7.2. The unbound proteins were washed with same buffer. Gradient elution was carried out using sodium chloride in 20 mM phosphate buffer pH 7.2 (0–0.5 M) and 2 mL fraction were collected at a flow rate of 0.5 mL/min. Protein content of each fractions were measured at 280 nm (---•---) and protease activity (‒‒‒) was assessed using casein as substrate. Fractions showing proteolytic activity were pooled. b Elution profile of CmSP proteases on Sephadex G-100 column chromatography. The proteases from the active fractions of a Sephadex G-100 chromatography was pooled and loaded onto the Sephadex G-100 gel filtration column (1.5 × 120 cm) equilibrated and eluted with 20 mM phosphate buffer pH 7.2. The flow rate was maintained at 15 mL/hr and fractions of volume 1.25 mL were collected every 5 min. The protein content was monitored continuously for each fraction at 280 nm (-----). The proteolytically active fractions (‒‒‒) eluted from Sephadex G-100 chromatography were pooled and stored for further analysis. Inset: Molecular weight marker and protein from Sephadex G-100 c Protein profile of different fractions on SDS-PAGE (15%). CmSP (15 μg in each well) was loaded on SDS-PAGE under denaturing, reducing conditions and the protein bands were visualized by silver staining. Lane MWM-Molecular weight marker (High range), Lane 1—Crude extract, Lane 2–70% ammonium sulphate dialysed fraction, Lane 3—DEAE Sephadex A-50 anion exchange sample, Lane 4—Sephadex G-100 fraction. d Casein zymogram for protease activity (10%, Native PAGE). Lane 1-crude extract, lane 2–70% ammonium sulphate dialysed fraction, lane 3-DEAE A-50 fraction, lane 4-Sephade G-100 fraction, The extracted and purified CmSP were run in 10% native PAGE containing copolymerised casein. The proteins were stained with CBB R-250 and destained with glacial acetic acid/methanol/distilled water (1:3:6). The activity band is observed as a clear colorless area depleted of casein in the gel against the blue back-ground

Table 1.

Purification chart of CmSP (for 100 g of seed flour)

Purification step	Total Protein (mg)	Protease activity (Units)a	Specific Activity (U/mg)	Fold purification	Activity recovery (%)
Crude extract	2935	95,400	32.5	1	100
Ammonium sulfate fractionation (30–70%)	1591	79,250	49.8	1.5	83.1
DEAE Sephadex A-50	196	42,500	216.8	6.6	44.5
Sephadex G-50	39	34,500	884.6	27.2	36.2

^aUnit: One unit of the protease activity is defined as the amount of enzyme required to cause an increase in 0.01 unit of absorbance at 660 nm per minute at 37 °C

The molecular weight of the purified protease was determined using denaturing reducing SDS-PAGE and by plotting the log₁₀ graph of standard molecular weight markers against the relative mobility. High range molecular weight marker ranging from 10 to 245 kDa protein was used to determine the molecular weight of CmSP as shown in inset of Fig. 2b. The relative molecular mass of CmSP was determined to be 32 kDa. The CmSP is a single polypeptide protein as no difference in PAGE band pattern was observed when the sample was run under reducing or non-reducing conditions. Proteases purified from plant exists in the range of 19–110 kDa (Antao et al. 2005) and plant serine proteases isolated from Cucurbitaceae family range from 30 to 70 kDa (Uchikoba et al. 1995). A low molecular weight gelatinolytic serine protease (AG2) had been purified from the seeds of Benincasa hispidaprotein (11.21 kDa) (Atiwetin et al. 2006). Other latex proteases from Euphorbia family range from 25 to 80 kDa (Patel et al. 2007 and Lynn et al. 1988).

To visualize the proteolytic activity of CmSP, casein zymogram was done using copolymerized casein as substrate in polyacrylamide gel under native condition. CmSP hydrolyzes the casein copolymerized in the gel and after staining with Coomassie Brilliant Blue R-250, a clear background is observed against dark background (Fig. 2d) confirming the presence of protease.

Effect of temperature and pH on CmSP activity

Characterization of purified CmSP for optimum activity at different temperature was performed using casein substrate at pH 7.5 in the range 5–100 °C. Figure 3a illustrates the optimum temperature for the proteolytic activity of CmSP. The protease activity increased as the temperature of reaction mixture increased from 5 to 55 °C (Fig. 3a). However, further increase in reaction temperature decreased CmSP activity. The maximum activity of CmSP was observed at 55 °C. With increase in reaction temperature beyond 55 °C loss in protease activity was observed and this is due to thermal denaturation of purified protein leading to decrease in the enzyme activity. The optimum temperature for plant serine proteases vary from 30 °C to 80 °C, but most of the plant serine proteases usually act best in the range 20–60 °C (Antao et al. 2005). Optimum temperature of protease isolated from Cucurbita ficifolia is reported to be at 55 °C (Curroto et al. 1989). The optimum pH of CmSP activity was determined by assaying reaction at different pH range (3–9.5). The optimum activity of CmaSP was found to be at pH 7.4, same as the physiological pH. The protease activity increased with increase in pH of the reaction mixture from 3 to 7.4 (Fig. 3b), and further increase in pH lead to loss in the enzyme activity. Enzyme function depends on its structural integrity (Alici et al. 2018), and loss in structure lead to functional loss of enzyme. Enzymes lose activity beyond their optimal temperature and pH as they undergo structural change leading to inaccessibility of substrate to active site or due to complete structural loss (Patel et al. 2012). Changes in pH beyond optima leads to loss of enzyme activity. The optimum pH of the most plant serine proteases ranges from 7 to 11 (Antao and Maclcata, 2005). The optimal pH of protease isolated from Heliantus annas is reported to be 7.5 (Uchikoba et al. 1995). Similarly, a subtilisin class serine protease isolated from the Trichosantus cucumeroides has optimum pH at 7.5 (Kaneda et al. 1986).

Fig. 3.

a Effect of temperature on enzyme activity. The purified CmSP activity was measured using casein as a substrate at temperature ranging from 5 to 100 °C for 30 min. The residual protease activity of CmSP was measured using casein substrate. b Effect of pH on enzyme activity. The enzyme activity was measured by using casein as a substrate at various pH. The buffers used for 50 mM sodium acetate (pH 3–6), 50 mM sodium phosphate (pH 6–7) and was 50 mM Tris–HCl buffer (pH 7.0–9.5). The residual activity of purified CmSP protease at different pH was measured using casein substrate

Effect of inhibitors and various salts on protease activity

To identify and designate the class of protease to which CmSP belonged to, the purified protease was subjected to proteolytic activity assay in the presence of various inhibitors which are reported to inhibit different classes of protease. The inhibition profile of CmSP in the presence of PMSF, EDTA, HgCl₂ and 2-iodoacetic acid (2 mM each) is shown in Fig. 4a. Among these inhibitors, protease activity was significantly inhibited by PMSF, while other protease inhibitors (except HgCl₂) did not affect CmSP activity. Table 2 shows the percentage of residual activity in the presence of PMSF, EDTA, HgCl₂ and 2-iodoacetic acid. PMSF is a protease inhibitor specific to the class of serine proteases (Gold et al. 1964), while HgCl₂ is a broad spectrum inhibitor. PMSF inhibits CmSP protease activity significantly compared to all other inhibitors tested for the proteolytic activity. The test tube assay results were corroborated by running the same samples on SDS-PAGE as represented in Fig. 4b. The casein protein hydrolysates are the products of casein substrate obtained due to action of uninhibited protease enzyme (CmSP). These are seen as protein bands in Fig. 4b (lane-1, lane-3 and lane-5), while the protease in the presence of PMSF or HgCl₂ is completely inhibited and no or faint protein hydrolysate bands were seen (lane-2 and lane-4 of Fig. 4b). This inhibition profile clearly indicates that CmSP is a serine protease and does not belong to any other class of protease. Proteases isolated from Cucumis trigonus Roxburghi, Benincasa hispidaprotein and Alcaligenes faecalis are reported to be serine proteases (Asif-Ullah et.al. 2006, Thangam et al. 2002 and Atiwetin et al. 2006).

Fig. 4.

a Effect of various inhibitors on CmSP activity. The enzyme (0.1 mL/5 μg) was pre incubated with 2 mM concentration of phenylmethylsulfonylfluoride (PMSF), 2-iodoacetic acid, mercuric chloride and ethylenediaminetetraacetic acid (EDTA) and kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0. The residual activities were measured. Trypsin represents 100% activity. CmSP without any inhibitor is used as control. b Activity assay in SDS-PAGE to determine the effect of inhibitors on CmSP. Lane 1—CmSP (without inhibitor), Lane 2—Sample + phenylmethylsulfonylfluoride, Lane 3—Sample + 2-iodoacetic acid, Lane 4—Sample + mercuric chloride, Lane 5—Sample + ethylenediaminetetraacetic acid. 50 µg of CmSP with or without inhibitor is loaded in each well on SDS-PAGE (12.5%) under denaturing conditions. Protein bands were visualized by CBB R- 250 staining. c Effect of various salts on enzyme activity. The enzyme (0.1 mL/5 μg) was pre incubated with 5 mM concentration of salts: FeCl₃, KCl, MgCl₂, NaCl, and CaCl₂ and kept at 37 °C for 30 min, followed by addition of 1% casein in 50 mM Tris–HCl buffer of pH 8.0. The residual activities were measured. Trypsin represents 100% of the protease activity and CmSP without salt shows the activity of purified CmSP

Table 2.

Effect of various inhibitors on the activity of purified CmSP

Inhibitor (2 mM each)	Residual Protease activity (%)
Trypsin	100
CmSP (No inhibitor)	75
PMSF	2
Mercuric chloride	38.12
IAA	55.33
EDTA	55.50

The effect of metal ions on CmSP enzyme activity was checked in the presence of NaCl, KCl, MgCl₂, CaCl₂ and FeCl₃ salts. The results are shown in Table 3 and Fig. 4c. The proteolytic activity of CmSP was enhanced in presence of divalent metal ions such as Mg²⁺ whereas decrease in the proteolytic activity in the presence of monovalent ion like Na⁺ was observed. The effect of ions on CmSP activity is in the order: Mg²⁺ > CmSP > Ca²⁺ ≈Fe³⁺ > K⁺ > Na⁺. It was observed that presence of monovalent cation Na⁺ and K⁺ shows negative effect on enzyme activity, whereas presence of divalent cations such as Mg²⁺ or Ca²⁺ increase the enzyme activity. It has been reported that serine protease from Bacillus pumilus shows improved enzymatic activity in presence of Ca²⁺ (Alici et al. 2018). Metalloproteases isolated from the Candida kefyr 41 PSB showed increasing activity at 5.0 mM concentration of Mg²⁺, Ca²⁺, Mn²⁺ (Patel et al. 2012).

Table 3.

Effect of metal ions on the activity of purified CmSP

Metal ion (5 mM each)	Residual protease activity (%)
Trypsin	100
CmSP (No salt)	70
NaCl	32
KCl	45
CaCl2	60
MgCl2	75
FeCl3	60

Anticoagulant activity and antiplatelet activity

Serine proteases are key enzymes in anticoagulant and antiplatelet pathway. CmSP being a potent serine protease was evaluated for its antiplatelet and anticoagulant activity. The interference of CmSP in coagulation cascade was checked by monitoring the plasma re-calcification time using human plasma. CmSP showed anticoagulant effect in both platelet-rich plasma (PRP) and platelet-poor plasma (PPP). CmSP delayed the clotting time of PRP from control 195–752 s (Fig. 5a), whereas in case of PPP, the clotting time was extended upto 814 s against 264 s. In both cases of PRP and PPP, the clotting time extended dose dependently upto 80 µg protein and thereafter it remain unchanged. The clotting time of PRP increased from 184 to 407 s, while in PPP, clotting time was 600 s against 242 s in the presence of seed extracts of Macrotyloma uniflorum at 120 µg concentration (Ramachandraiah et al. 2019a). Similarly, the clotting time in PRP is reported to be increased from 210 to 410 s, while in PPP, clotting time observed was from 300 to 600 s when exposed to Cicer arietinum seed extract (Ramachandraiah et al. 2019b). Undariase is a serine protease purified from the edible seaweed, Undaria pinnatifida. Undariase is reported to possess activated partial thromboplastin time of > 180 s, while the prothrombin time was increased by 79.5 and 110 s at 10 and 20 µg, respectively (Choi et al. 2013).

Fig. 5.

a Plasma re-calcification time. CmSP (0–100 µg) was pre-incubated with 0.2 mL of citrated human plasma, platelet rich (PRP) or platelet poor (PPP) in the presence of Tris–HCl, pH 7.4 for 1 min at 37 °C. The clotting time was recorded after adding 20 µL CaCl₂ to the pre-incubated mixture. b Platelet aggregation initiated by adding agonist, ADP. a Traces of platelet aggregation: Trace 1 (ADP 10 µM), Trace 2 (ADP 10 µM + 5 µg CmSP), Trace 3 (ADP 10 µM + 10 µg CmSP), Trace 4 (ADP 10 µM + 15 µg CmSP). b Platelet aggregation (%) c Platelet aggregation inhibition (%). The values represented are from three independent experiments. c Platelet aggregation initiated by adding agonist, Epinephrine. a Traces of platelet aggregation: Trace 1 (Epinephrine 5 µM), Trace 2 (Epinephrine 5 µM + 5 µg CmSP), Trace 3 (Epinephrine 5 µM + 10 µg CmSP), Trace 4 (Epinephrine 5 µM + 15 µg CmSP). b Platelet aggregation (%) c Platelet aggregation inhibition (%). The values represented are from three independent experiments

To confirm that the triggered anticoagulation by CmSP is due to its involvement in coagulation cascade, plasma aggregation test was carried out using PRP. Blood coagulation cascade is a physiological phenomenon that is activated to arrest the bleeding following an injury. However, imbalance due to genetic and environmental factors could alter the normally operating coagulation system that leads to thrombosis, a pathological phenomenon. To study the intervention of CmSP on platelet de-aggregation (antiplatelet) property, a dose-dependent inhibition of ADP and epinephrine-induced aggregation of PRP was demonstrated. CmSP inhibited the agonists such as ADP (Fig. 5b) and epinephrine (Fig. 5c) induced platelet aggregation of about 58 ± 1.4% and 92 ± 2.2%, respectively, at the concentration of 5 μg of protease. Among agonists observed CmSP inhibited in the order of epinephrine > ADP induced aggregation. Seed extract of Cicer arietinum (60 µg) inhibits ADP and epinephrine induced platelet aggregation in PRP by 79–82%, respectively (Ramachandraiah et al. 2019b). Fibrinogenolytic serine protease from Leucas indica is a 35 kDa protein with anticoagulant and thrombolytic activity (Gogoi et al. 2018). Many antiplatelet agents have been characterized from plants and animal sources. Bacethrombase is a 39.5 kDa serine protease purified from Bacillus cereus with anticoagulant and antiplatelet activity (Majumdar et al. 2015). Inhibition of platelet aggregation and intervention in the factors of blood coagulation cascade are the important mechanism by which the proteolytic enzymes bring about their anticoagulant effect. CmSP demonstrated better anticoagulant activity by delaying the clotting time and by inhibiting the ADP/epinephrine induced platelet aggregation. Several physiological agonists namely ADP, epinephrine, thrombin, thromboxane, arachidonic acid and collagen, activates platelets (Kim et al. 2015). These activated platelets plug at the site of injury along with fibrin clot and inhibit blood loss (Sannaningaiah et al. 2018). While, in case of hyper coagulation disorders, there will be burst of activation of platelets too contribute for the formation unusual clot (Majumdar et al. 2015). Thus, antiplatelet agents do play a key role in inhibiting hyper activation of platelets.

Conclusion

Various parts of the plants are considered as the repositories of potent therapeutic compounds used in the treatment of various ailments. In this study, CmSP is purified from the seeds of C. maderaspatensis, L. CmSP, a serine protease is biochemically characterized and elucidated for its pharmacological significance with respect to anticoagulant and antiplatelet activity in vitro. CmSP significantly delayed the clot time and inhibited the ADP and epinephrine induced aggregation of platelets. Studies in vivo are required to vouch this anticoagulant enzyme as the alternate candidate source against thrombosis.

Funding

The authors thank DST-SERB and VGST, GoK India for funding the work.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.