Mechanism of action of latex proteases……..,shodhganga.inflibnet.ac.in/bitstream/10603/70176/7/chapter 2.pdf · Latex proteases, irrespective of the plant family and nature of

Mechanism of action of latex proteases……..,

55

Introduction

Hemostasis is the physiological process which maintains the flowing blood in

fluid state within the blood vessel, while it aims at providing thrombotic response

following injury to limit the blood loss (Lundblad et al., 2004). This vital process is

tightly regulated by vascular endothelium, extracellular matrix, blood coagulation

system, platelets and fibrinolytic system (Michiels, 2003; Hoffman and Monroe,

2005; Jenne et al, 2013). Following tissue injury, circulating platelets adhere to

damaged endothelium through exposed ECM proteins. Upon adhesion, the platelets

become activated evident by shape change and activation of receptors, which mediate

aggregation at the site of injury (de Groot et al, 2012). Concomitant to the process of

platelet activation, the blood coagulation cascade is triggered resulting in sequential

activation of proteolytic enzymes, culminating in the formation of fibrin monomers.

The fibrin thus formed undergoes polymerization in the presence of activated factor

XIII. The fibrin mesh stabilizes the aggregated platelets to form a stable hemostatic

plug (Hoffman and Monroe, 2005; Szántó et al., 2012). In addition to physiological

factors, external factors influence hemostasis.

Latex is an important plant based component, which is a widely employed

hemostatic agent in traditional system of medicine because of its ability to stop

bleeding from fresh wounds (Thanakamma, 2003). The use of topical hemostatic

agents from natural sources is gaining importance in wound care and management,

owing to the efficacy and safety of naturally derived hemostatic agents (Samudrala,

2008). In spite of its usage in traditional medicine, very few reports indicate the latex

bio-active components involved and the mechanisms involved in exhibiting

hemostatic effect. The property of latex to stop bleeding is mostly attributed to

proteases, and partly to secondary metabolites (Rajesh et al., 2005). Proteases from

latices of Apocyanaceae, Asclepiadaceae and Euphorbiaceae exhibit procoagulant

effect, irrespective of the nature of proteases. In continuation, the cysteine proteases

from Asclepiadaceae plant latices exhibited ‘thrombin-like’ activity and facilitated the

formation of clot even in the absence of Ca2+

ions. (Shivaprasad et al., 2009). But, the

mechanisms of action of latex serine proteases in exhibiting procoagulant effect is not

reported, which needs to be addressed systematically. Procoagulant proteases may act


56

in blood coagulation cascade or affect platelet function to aid process of clot

formation.

The present chapter aims at screening of selected plant latices for proteolytic

activity, nature of proteases and their hemostatic property with special reference to

blood coagulation cascade and platelet function. The interference of latex proteases in

blood coagulation cascade was evaluated using platelet poor plasma (PPP) and

citrated whole blood; whereas, the effect of latex proteases on platelet function was

determined using platelet rich plasma (PRP) and washed platelets.

Materials and methods

Latex yielding plants

Latex yielding plants - Calotropis gigantea (L.), Pergularia extensa (Forssk.)

(Asclepiadaceae), Wrightia tinctoria (R.Br) (Apocyanaceae), Synadenium grantii,

Euphorbia tirucalli (L.) and Euphorbia antiquorum (L.) (Euphorbiaceae), were

selected for the study. Plants were identified and authenticated by Dr. G. Sharanappa,

Associate Professor, Department of Studies in Bioscience, University of Mysore,

Hassan, Karnataka, India. The voucher specimens are deposited in Department of

Studies in Bioscience, University of Mysore, Hassan, Karnataka.

Human blood

The experiments involving human samples were carried out in accordance to

the protocols reviewed by the Institutional Human Ethical Committee, University of

Mysore, Mysore (Sanction order No. IHEC-UOM No.40/Ph.D/2009-10).

Citrated whole blood: Blood was drawn from healthy human volunteers (with

consent) and mixed with 3.2% tri-sodium citrate (9 : 1). Citrated whole blood was

used for recalcification experiments using rotational thromboelastometry (ROTEM)

(Pentapharm gmbh, Munich, Germany).

Platelet rich plasma (PRP): Citrated plasma was centrifuged at 22 x g for 15 min. The

clear supernatant obtained was used as platelet rich plasma (PRP) which was used for

platelet aggregation studies. Platelet count – 3.3 lakh/ml; absorbance – 0.82 at 650

nm.


57

Platelet poor plasma (PPP): Citrated plasma was centrifuged at 130 x g for 15 min.

The clear supernatant obtained was used as platelet poor plasma (PPP) for manual

recalcification time assay.

Platelet free plasma (PFP): PRP was centrifuged at 2900 x g for 15 min. The

supernatant obtained was used for calibrating the instrument for aggregation studies.

Platelet count – zero; absorbance – 0.05 at 650 nm.

Washed platelets: Platelets were washed as described by Cazenave et al., (2004).

Briefly, PRP was obtained by mixing fresh blood sample with acid citrate dextrose

solution (ACD) (85 mM sodium citrate; 71 mM citric acid; 111 mM dextrose) in ratio

6 : 1 (blood : ACD), followed by centrifugation at 22g for 15 min in a plastic tube.

The PRP was centrifuged at 2900 x g for 15 min at 37 °C to obtain the platelet pellet.

The pellet was re-suspended in Tyrode’s albumin buffer (145 mM NaCl, 5 mM KCl,

10 mM HEPES, 0.5 mM Na2HPO4, 1 mM MgCl2, 6 mM dextrose, and 0.3% bovine

serum albumin) pH 6.5, containing 10 U/ml heparin and 0.5 μΜ prostacyclin. After

10 min of incubation at 37 °C, the platelets were again washed using Tyrode’s

albumin buffer (3 times). Finally, the platelets were re-suspended in the Tyrode’s

albumin buffer, pH 7.4 containing 0.02 U/ml apyrase. Platelets were counted using a

Neubauer chamber and adjusted to required number of platelets in the final

suspension using Tyrode’s albumin buffer (pH 7.4). Washed platelets were used for

aggregation studies.

Chemicals

Trypsin, papain and pepstatin A (microbial source) were obtained from Sigma

Chemicals (St. Louis, MO, USA). Papain, phenyl methyl sulphonyl flouride (PMSF),

iodo acetic acid (IAA), ethylene diamine tetra acetic acid (EDTA), para- dimethyl

amino benzaldehyde (p-DMAB) were purchased from Sisco Research Laboratory,

Mumbai, India. Neosporin was purchased from GlaxoSmithKline Pharmaceuticals,

Mumbai, India. All the other chemicals used were of highest analytical grade.

Solvents were distilled before use. cytochalasin D, arachidonic acid, ristocetin,

collagen were purchased from Stago Diagnostics, Paris, France.


58

Collection and processing of plant latex

Latex from the selected plants was collected separately in clean and dry test

tubes by breaking the petioles. The collected latex was diluted (1 : 1 for P. extensa

and C. gigantea, 1 : 2 for S. grantii and 1 : 4 for W. tinctoria) with 10 mM sodium

phosphate buffer (pH 7.0) and kept in freezer overnight. The samples were subjected

to subsequent steps of freezing (10 h), thawing and centrifugation (6000 x g) to obtain

clear supernatant. Clear supernatant was dialyzed against 10 mM sodium phosphate

buffer (pH 7.0) with four buffer changes. The processed supernatant is rich in proteins

and is used as the crude enzyme source [W. tinctoria latex proteases (WTLP); S.

grantii latex proteases (SGLP); E. tirucalli latex proteases (ETLP); E. antiquorum

latex proteases (EALP); C. gigantea latex proteases (CGLP); P. extensa latex

proteases (PELP)]. The protein content of this enzyme fraction was estimated

according to the method of Lowry et al., (1951).

Proteolytic activity

Proteolytic activity was carried out according to the method of Murata et al.,

(1963), using fat free casein as substrate. Briefly, 0.4 ml casein (2%) in Tris-HCl

buffer (200 mM; pH 8.5) was incubated separately with different concentrations (2.5,

5, 10 and 25 µg) of WTLP, SGLP, CGLP and PELP for 2 h at 37 °C. The reaction

was terminated by adding 0.44 M TCA, the reaction contents were processed and the

color developed was measured at 660 nm. Enzyme activity was expressed in units;

one unit is defined as the amount of enzyme required to increase the absorbance of

0.01 at 660 nm/h at 37 °C. Similar concentrations of trypsin and papain were used for

the assay as representative serine and cysteine protease respectively.

For inhibition studies, the latex enzyme fractions were pre-incubated with or

without specific protease inhibitors (PMSF, IAA, EDTA, and pepstatin A) prior to the

protease assay.

Recalcification time

Recalcification time was carried out according to the procedure described by

Condrea et al., (1983). Pre-warmed (37 °C) platelet poor plasma (200 µl) was mixed

with 20 µl of Tris-HCl buffer (10 mM; pH 7.4) and incubated for 1 min separately


59

with varying concentrations of WTLP, SGLP, CGLP, PELP, trypsin and papain. The

clot formation was initiated by adding 20µl of 250 mM CaCl2 and the time taken for

visible clot to appear from the time of addition of CaCl2 was recorded in seconds. For

control experiment, Tris–HCl buffer alone was used instead of latex enzyme fraction.

ROTEM analysis for whole blood recalcification time and clot characteristics

Citrated whole blood (300 μl) was mixed with varying concentrations of latex

protein rich fractions. Clotting was initiated by adding 50 μl of 25 mM CaCl2. The

parameters such as clotting time (CT), clot formation time (CFT) and maximum clot

firmness (MCF). CT and CFT were recorded in seconds; MCF was expressed as mm.

Platelet aggregation

Turbidimetric method of Born and Cross (1963) was followed, using a dual

channel Chrono-log model 700-2 aggregometer (Havertown, USA) and Helena

AggRAM (Helena Biosciences, Tyne and Wear, U.K., NE11 0SD Europe). Briefly,

235 μl of PRP or washed platelet suspension was maintained at 37 °C in a siliconized

glass cuvette and pre-incubated with protein rich fractions from different plant latices

and also with reference proteases (trypsin and papain).

Results

Processing of crude latex yielded protein rich fraction: Crude latex was subjected to

processing steps such as dilution, freezing, thawing and centrifugation. Different

dilutions were made for different latices. The dilution is based on the amount of wax

present in the latex. Higher dilutions were made for latex with higher content of wax.

During the steps of dilution, freezing and thawing, wax gets aggregated, which is

removed after centrifugation. The supernatant obtained after dewaxing was dialyzed

against 10 mM sodium phosphate buffer (pH 7.0), using 10 kDa parchment

membrane. During dialysis, low molecular mass components of latex are removed.

The protein rich supernatant thus obtained was used as protease source, which was

used for further experiments. The aliquots were stored at – 4 °C till further use.

Protein rich fraction of latices showed high proteolytic activity towards casein:

Latex protein rich fractions showed moderate to high proteolytic activity towards

casein, with concentration dependent response. WTLP showed highest activity


60

towards casein (22.50±0.90 U/h) at 10 μg concentration, followed by CGLP

(11.50±0.80 U/h), PELP (10.40±1.0 U/h) and SGLP (7.0±0.80 U/h). The activities of

reference proteases – trypsin and papain were 9.0±0.99 and 2.50±0.45 U/h

respectively. The comparative proteolytic activities of protease samples are depicted

in Fig. 2.01a.

Plant latex proteases belong to serine or cysteine superfamily: Nature of proteases

present in latices from selected plants was determined using specific protease

inhibitors. The proteolytic activity of WTLP and SGLP was inhibited by PMSF

(95±2.5% and 90±4.0% respectively), indicating the serine proteases. In case of

CGLP and PELP, the activities were neutralized by IAA (94±3.9% and 95±4.1%

respectively), indicating the cysteine proteases. In all the cases, the proteolytic activity

was unaltered in presence of EDTA (metalloprotease inhibitor) and pepstatin A

(aspartate protease inhibitor), indicating the absence of metallo and aspartate family

of proteases (Fig. 2.01b).

Plant latex proteases exhibited procoagulant effect

Recalcification time using citrated plasma (PPP): The effect of latex proteases on

blood coagulation cascade was determined by recalcification time assay using PPP.

Latex proteases, irrespective of the plant family and nature of proteases, showed

procoagulant effect and facilitated the formation of clot, with concentration dependent

response. At 10 µg concentration, CGLP and PELP showed strong procoagulant

activity with clotting time of 30±4 s and 43±6 s respectively, in comparison to WTLP,

EALP, ETLP and SGLP showed weak procoagulant activity with clotting time of

98±10 s, 95±5 s, 82±5 s and 65±7 s respectively, against control recalcification time

being 195±15 s. CGLP and PELP promoted clotting of PPP even in the absence of

Ca2+

ions, whereas, WTLP, EALP, ETLP and SGLP promoted clotting of PPP only

after supplementing Ca2+

ions. In similar lines, the reference proteases – trypsin and

papain also showed procoagulant effect with clotting time of 105±7 s and 70±9 s

respectively (Fig. 2.02a and 2.02b). For whole blood recalcification assay, two serine

and two cysteine proteases were selected.

Recalcification time using citrated whole blood: The procoagulant effect of latex

proteases was confirmed using citrated whole blood by ROTEM analysis, by


61

evaluating parameters such as CT, CFT and MCF. CT (time from start of

measurement until the initiation of clotting) for latex proteases was 657, 340, 209 and

270 s respectively for WTLP, SGLP, CGLP and PELP, against control clotting time

of 751 s. The finding was in correlation with recalcification time assay using PPP.

Further, CFT (time from initiation of clotting until the clot of 20 mm thickness is

formed) of WTLP, SGLP, CGLP and PELP was 611, 129, 67 and 87 s respectively.

Interestingly, clot formation time of WTLP was prolonged, which was further

supported by decreased MCF (31 and 13 s respectively at 20 and 40 μg respectively).

In contrast, at 30 μg concentration, MCF in case of SGLP, CGLP and PELP treated

blood was 47, 56 and 56 mm respectively, against control MCF of 47 mm, indicating

the efficient clot formation, normal platelet function and clot stabilization by factor

XIIIa. The amplitude of splitting in case of WTLP treated whole blood was very low,

indicating reduced fibrin formation as a result of decreased fibrinogen content. The

splitting of graph may be due to the fibrinogen released from activated platelets. The

observation was confirmed by pre-treatment of WTLP with cytochalsin D, which

inhibited platelet activation, resulting in incoagulable of blood and MCF was 3 mm

(Fig. 2.03; Table 2.01).

Serine proteases from Euphorbiaceae plant latex promote platelet aggregation

Latex protease fractions were subjected to platelet aggregation studies using PRP and

washed platelets. In case of PRP, serine proteases from SGLP (30 μg) strongly

induced platelet aggregation (90.4%) against 3.7% in case of saline. The aggregation

by proteases of SGLP was confirmed by pre-treatment of SGLP with PMSF, which

decreased aggregation by 33%. In similar lines, trypsin also induced platelet

aggregation (76.2%). Contrarily, serine proteases from WTLP, cysteine proteases

from CGLP, PELP and papain did not induce platelet aggregation. Further, SGLP

induced aggregation (82%) in washed platelets. Also, EALP and ETLP induced

aggregation of 73% and 57% respectively, indicating the strong induction of platelet

aggregation by serine proteases of Euphorbiaceae plant latex (Fig.2.04; Tables 2.02

and 2.03)


62

Discussion

Plant latex is a milky white exudate characteristic of certain angiospermic

plant families. Latex plays crucial physiological roles which include storage of

nutrients, water balance and provide defense against invading herbivorous insects

(Agrawal and Konno, 2009). Apart from the role in plant physiology, latex exhibits

range of pharmacological effects, which is attributed to heterogeneous classes of

bioactive constituents which interfere with human/animal physiological processes

(Thakur et al., 2011; Korpenwar, 2012). Bioactive components present in latex

include alkaloids, terpenes, tannins, sterols, glycosides, saponins, flavonoids, and

hydrolytic enzymes such as proteases, invertases, chitinases. An important

pharmacological property exhibited by latex is to stop bleeding from fresh wounds,

followed by wound healing (Thankamma, 2003). Application of topical hemostatic

agents is an important aspect of wound care and management since it serves to

prevent excess blood loss and prevent the entry of opportunistic and invading

pathogenic microorganisms (Samudrala, 2008). Most of the reported pharmacological

properties of latex including hemostatic function is mostly attributed to proteases and

partly attributed to secondary metabolites. In this regard, the present study aims to

provide a comparative account on hemostatic property of plant latex proteases with

special reference to serine proteases and their interference in blood coagulation and

platelet function.

Crude latex collected from selected plants was subjected to processing. During

the processing of latex, interfering molecules such as wax and low molecular mass

constituents (phenolics, alkaloids, terpenes and peptides) are removed. Wax interferes

with the routine in vitro analysis mainly with enzyme assays and spectrophotometric

methods. Low molecular mass components present in latex are reported to be toxic

(Wink, 2009). Hence, processing steps eliminate the interfering molecules; as well

reduce toxicity of the processed latex fractions. The protein rich fraction was used as

source of proteases for studies involving blood coagulation and platelet aggregation.

Plant latex is one of the richest sources of proteases in nature, apart from

snake venoms and microorganisms. Protein rich fractions from plant latices exhibited

moderate to high protease activity towards casein. Studies involving protease


63

inhibitors revealed that selected plant lattices contained either serine or cysteine

proteases, but not both types in given latex. In contrast, any given snake venom

contains both serine and metalloproteases (Paes et al. 2011). Based on the inhibition

of protease activity, presence of only serine proteases in Apocyanaceae and

Euphorbiaceae was confirmed. Further, Asclepiadaceae family latices contain only

cysteine proteases. In support of these findings, earlier reports from Rajesh et al.,

(2006) and Shivaprasad et al., (2009) suggest that any given plant family contains

only one type of protease. Till date, only serine and cysteine proteases have been

reported from plant latex, with only one aspartate protease; ficin from Ficus racemosa

and metalloprotease; cotinifolin from Euphorbia cotinifolia being the exceptions

(Devaraj et al., 2008; Kumar et al., 2011). The unique type of protease present in a

given plant family will be an important tool for chemotaxonomic classification of

plants in cases of ambiguity for assigning plant to a particular family.

Among the pharmacological properties of latex, hemostatic property is

exploited by folk medicinal practitioners and rural population to stop bleeding from

fresh wounds (Thankamma, 2003). This property is attributed to proteolytic enzymes

present in latex (Rajesh et al., 2005). Irrespective of the plant family and the nature of

proteases, latex exhibit procoagulant activity and facilitate the formation of clot, with

an exception of AMP48, an anticoagulant protease from Artocarpus heterophyllus

latex (Shivaprasad et al., 2009; Siritapetawee et al., 2012). The clot thus formed limits

excessive loss of blood following injury and serves as barrier for the entry of invading

and opportunistic pathogens into the wound site (Lawrens et al., 2006). Further, the

clot formed serves as a platform for inflammatory cells and mediators during the

inflammatory phase of wound healing (Stroncek, 2009). In spite of the procoagulant

nature of latex proteases, very few reports explain the mechanism of procoagulant

action. Studies on site specific actions of plant latex proteases interfering in blood

coagulation cascade report the presence of factor X activator - ficin from Ficus carica

and thrombin like enzyme - pergularain e- I from P. extensa latex (Richter et al.,

2002; Shivaprasad et al., 2010). Both these proteases are cysteine proteases. Apart

from these reports, mechanisms of action of many cysteine proteases and most serine

proteases are not known and needs investigation in terms of their interference in blood


64

coagulation cascade. Most of the reported mechanism(s) of action of proteolytic

enzymes from natural source are from in vitro studies carried using PPP.

The results from recalcification experiments using PPP were in correlation

with those of citrated whole blood, using ROTEM. It is an in vitro analysis used for

qualitative and quantitative assessment of functional status of coagulation in citrated

whole blood. It provides information regarding variations in citrated whole blood with

respect to coagulation time, clot formation, strength of clot and clot lysis. Whole

blood recalcification time of SGLP, PELP and CGLP was 340 s, 270 s and 209 s

respectively against control recalcification time of 751 s. Clotting time indicates the

time of initiation of clotting, which depends on factors such as formation of thrombin

and initiation of clot polymerization. Further, the clot formation time of SGLP, PELP

and CGLP was respectively 129 s, 89 s and 67 s, against 275 s in case of control. In

continuation, MCF was 47 mm, 57 mm and 56 mm respectively for SGLP, PELP and

CGLP in comparison to 47 mm in case of control. MCF depends on the increasing

stabilization of the clot by the polymerized fibrin, thrombocytes as well as factor XIII

(Lang et al., 2005). These findings indicate the positive effect of SGLP, PELP and

CGLP in exhibiting procoagulant effect towards citrated whole blood. Further, the

results suggest the normal formation of thrombin, fibrin clot and normal functioning

of factor XIII, in presence of these latex protease fractions.

Similar to latex proteases from S. grantii, P. extensa and C. gigantea, WTLP

decreased the clotting time of PPP and whole blood in dose dependent manner. But,

the clot formation was delayed. The probable reason for reduced clot formation would

be depletion of fibrinogen, which resulted in reduced fibrin formation, which also

resulted in reduced MCF. To support the fibrinogen depleting action of WTLP,

fibrinogen content in PPP was Zero in comparison to 190 mg in PPP. Further, MCF

was found to be 13 mm in contrast to 47 mm in control. The clot firmness results from

the stable fibrin mesh. The reduced clot firmness is due to the depletion of fibrinogen

by WTLP. Further, the depletion of fibrinogen was confirmed by evaluating thrombin

time, where the fibrinogen pre-incubated with WTLP did not clot upon supplementing

thrombin, whereas, purified fibrinogen clotted in 5.3 s after supplementing thrombin.


65

ROTEM analysis is one of the viscoelastic methods for monitoring blood

coagulation using whole blood. The technique is vital in diagnosis of conditions such

as hemorrhage, bleeding and thrombotic disorders (Ferraris et al., 2007). Whole blood

coagulation analyses overcome the limitations of routine coagulation tests using PPP

(Hett et al., 1995; Luddington, 2004). The conditions are similar to physiological

status of blood, which involves interaction among coagulation system, platelets and

RBC’s. The analyses involves monitoring the stages of development and resolution of

clot (clotting time, clot formation time, strength and stability of clot, fibrinolysis).

Unlike plant latex cysteine proteases, the mechanisms of action of serine

proteases on blood coagulation cascade cannot be singled out based on coagulation

analyses in PPP as well as citrated whole blood. To elucidate the mechanism of

procoagulant action, serine proteases from selected plants were subjected to

aggregation studies using PRP and washed platelets. Serine proteases from

Euphorbiaceae plant latices strongly induced aggregation in PRP as well as washed

platelets. In contrast, Apocyanaceae family proteases did not affect platelet

aggregation in either case. Among all Euphorbiaceae latex proteases, SGLP showed

potent aggregation. The role of proteases was confirmed by using PMSF, which

decreased aggregation in PRP by about 30%, indicating the role of serine proteases in

inducing aggregation. In addition, SGLP induced aggregation was decreased by about

35% by GPIIb/IIIa antagonist. Irrespective of the agonist activating the platelet and

the pathway activated, final aggregation of the platelets is mediated through activated

GPIIb/IIIa receptor through fibrinogen (Scarborough et al., 1999). In this regard, the

mechanism of SGLP induced platelet aggregation may be due to thrombin generation

by the action of SGLP on coagulation cascade or direct activation of protease

activated receptors (PARs) by SGLP. The possibility of thrombin generation by SGLP

is ruled out since SGLP induced platelet aggregation even in washed platelets.

Washed platelet preparation is free of coagulation factors including fibrinogen. From

these findings, most probable mechanism of platelet activation by SGLP is activation

of PAR, leading to aggregation of activated platelets. Under physiological conditions,

PARs are activated by thrombin (PAR1 and PAR4 are expressed on platelets) (Tello-

Montoliu et al., 2010). Further, the mechanism of activation of platelets by SGLP has


66

to be elucidated using antagonists for various platelet receptors followed by

monitoring the aggregation.

In conclusion, plant latex, an abundant source of proteases contains serine or

cysteine superfamily of proteases. Irrespective of the protease type, all the proteases

exhibit procoagulant effect and facilitate the formation of clot. Based on the earlier

reports and present investigation, it can be concluded that, cysteine proteases from

plant latex exhibit ‘thrombin-like’ activity; whereas serine proteases from

Euphorbiaceae plant latices are potent inducers of platelet aggregation through which

they exhibit procoagulant effect. The ability of Euphorbiaceae latex proteases to

induce aggregation in washed platelets indicates that they mediate aggregation

through PAR. Further, the mechanisms of action of Apocyanaceae latex serine

proteases in exhibiting procoagulant effect needs further investigation, because of

inconclusive findings with respect to blood coagulation parameters and platelet

function. Plant latex is extensively used to promote healing of wounds apart from

stoppage of bleeding from wounds. Based on the findings of procoagulant nature of

latex proteases, their role in wound healing will be discussed in the following chapter,

along with the underlying molecular mechanisms.


67

Figures and tables

Fig. 2.01: Proteolytic activity of latex proteases and nature of proteases.

a. Dose dependent proteolytic acitivty of latex proteases (0-25 µg) was carried out

using fat free casein as substrate. Results are expressed as Mean±SD (n=5).

b. Latex proteases (10 µg) were separately pre-incubated with specific protease

inhibtors (5 mM each of PMSF and EDTA; 100 µM each of IAA and pepstatin A)

at 37 °C for 30 min prior to the proteolytic assay. The results are expressed as

percentage inhibtion of latex proteases with or without protease inhibitors. The

activity of latex proteases without protease inhibitors was considered as 100%.

a

b


68

Fig. 2.02: Recalcification time analysis of latex proteases using PPP.

a. Varying concentrations (2.5-10.0 µg) of latex proteases, trypsin and papain in

0.01M Tris-HCl buffer (pH 7.4) were separately added to 0.2 ml of PPP and

incubated 37 °C for one min. The clot formation was initiated by supplementing

0.02 ml of 0.25 M CaCl2. The time taken for visible clot formation is recorded. The

results were compared with the clotting time of plasma added with CaCl2 alone.

b. Comparative account on the effect of latex proteases and reference proteases

towards plasma recalcification at 10 µg protein concentration. ** indicates

p<0.005.

a

b


69

Fig. 2.03: Recalcification time analysis of latex proteases using citrated whole

blood. Citrated whole blood (300 μl) was mixed with varying concentrations of

latex protein rich fractions. Clotting was initiated by adding 50 μl of 25 mM CaCl2

and subjected to ROTEM analysis. The parameters such as clotting time (CT), clot

formation time (CFT) and maximum clot firmness (MCF). Clotting time and clot

formation time were recorded in seconds; MCF was expressed as mm.

Table 2.01: ROTEM parameters for whole blood recalcification time of latex

proteases

Sample Clotting time (s) Clot formation time

(s)

MCF

(mm)

Control 751 275 47

WTLP (20 μg)

WTLP (40 μg)

WTLP+CK

657

575

676

611

-----

-----

31

13

3

SGLP (30 μg) 340 129 47

CGLP (30 μg) 209 67 56

PELP (30 μg) 270 89 57


70

Fig. 2.04: Platelet aggregation in PRP.

a1-a3. Aggregation profiles of latex proteases

a1

a2

a3


71

Fig. 2.04. Platelet aggregation in PRP (contd.)

a4. Aggregation profile of reference proteases (AggRAM, Helena Biosciences, UK)

Fig. 2.04: Platelet aggregation in washed platelets.

a5: Aggregation profiles of Euphorbiaceae plant latex serine proteases using washed

platelets in Chrono-log model 700-2 aggregometer (Havertown, USA)

a4

a5


72

Table 2.02: Summary of platelet aggregation study of latex proteases using PRP

Sample Nature of

protease


(%)

PRP alone - 3.7

SGLP

SGLP + PMSF

SGLP + TRAP

Serine 90.4

60.5

55.3

WTLP

WTLP + PMSF

Serine 4.5

7.1

Trypsin Serine 76.2

CGLP Cysteine 1.1

PELP Cysteine 12.5

Papain Cysteine 5.7

Table 2.03: Summary of platelet aggregation study of latex proteases using

washed platelets

Sample Nature of

protease


(%)

Washed platelets

(WP) alone

- -

WTLP

WTLP + Fibrinogen

Serine -

-

SGLP Serine 90*

ETLP Serine 69*

EALP Serine 80*

*- aggregation in the absence of fibrinogen

Documents

Mechanism of action of latex proteases……..,shodhganga.inflibnet.ac.in/bitstream/10603/70176/7/chapter 2.pdf · Latex proteases, irrespective of the plant family and nature of