Injury to the tissue activates the proteins of the coagulation cascade which is vital to stop blood loss from the site of injury followed by the restoration of normal tissue by clearing dead tissue debris. Platelets, underlying epithelial cells, coagulation factors, fibrinolytic systems, and blood vessels maintain the coagulation system. Bleeding disorders are caused by any slight imbalance of any of the processes in the cascade and can lead to complications such as hemorrhage or thrombosis. The lack of coagulation factors resulting in excessive blood loss. Currently, bypass agents, replacement, and recombinant factors are used to treat the bleeding disorders which is not recommended for a longer period due to the production of inhibitory antibodies and anaphylactic reactions.

Plant-based treatment is gaining importance for its safety and efficacy. Plant latex proteases are one of the major sources of therapeutics that are used for treating various conditions. Many proteases are characterized by their pro-coagulant activity and stop bleeding due to the presence of thrombin-like enzymes. Thrombin-like latex proteases aid the clotting of factor VIII-deficient plasma. Also, they play a major role in clotting by enhancing platelet aggregation by activating protease-activated receptors 1 and 4. The action of latex proteases is of the broad spectrum. It is essential to check the specific targets and assess their complete pharmaco-characterization to make them possibly better molecules with fewer side effects and enhanced efficiency. This can be an inexpensive and better alternative strategy to treat external wounds in bleeding disorders than the existing strategies

• Hemostasis

• Coagulation cascade

• Platelets

• Clotting factors Bleeding disorders

• Plant latex proteases

Sarkar S, Urs AP, Vishwanath BS (2024) Plant Latex Proteases as a Better Alternative Treatment Recourse to Treat External Wounds in Bleeding Disorders. J Chronic Dis Manag 8(1): 1039.

Aptt: Activated partial thromboplastin time; CBC: Complete Blood Count; DDAVP: Desmopressin; E: Epinephrine; ER: Epinephrine Receptor; GP aIIbβIII: Glycoprotein aIIbβIII; GP Ib: Glycoprotein Ib; HMWK: High Molecular Weight Kininogen; NR: No Reports; PAR 1: Protease-activated Receptor 1; PAR 4: Protease-activated Receptor 4; PDGF: Platelet-derived Growth Factor; PLPs: Plant Latex Proteases; PT: Prothrombin Time; S: Serotonin; SR: Serotonin Receptor; TF: Tissue Factor; TPA: Tissue Plasminogen Activator; TXA2: Thromboxane 2; TXA2R: Thromboxane 2 Receptor; VEGF: Vascular Endothelial Growth Factor; Vwd: von Willebrand disease; vWF: von Willebrand factor

Hemostasis is a critical phenomenon in the physiological system that maintains the normal flow of blood in a liquid state and responds to various injuries or any vascular insults that can lead to blood loss through clotting, which simply means the arrest of bleeding [1,2]. The coagulation cascade is a mechanism that involves various factors that bring about the clotting of blood, when necessary, removal of dead and damaged tissues and also replacement of the area with new tissues, and restoration of normal functions of the vasculature [2]. If any of the processes malfunction, it will result in excessive blood loss that can also be fatal and hamper tissue healing [3]. If the clotting factors are excessive or over-functional, it can lead to thrombophilia; if they are deficient or hypo-functional, it will result in bleeding disorders [4-7].

Coagulation Cascade

The importance of the coagulation cascade was observed in the early 1960s [8]. Hemostasis is a dynamic and innate process of arresting bleeding complications [9]. The platelets, coagulation and fibrinolytic systems, and blood vessels maintain this. The hemostatic system is responsible for maintaining blood in a fluid state, free from the aggregation of platelets and thrombus formation with the help of prostacyclin, antithrombin III, and nitric oxide (NO) within the endothelial cells [10]. These naturally occurring substances found in the blood assist in the prevention of clots by causing the conversion of plasminogen to plasmin to promote fibrinolysis [11]. Damage or injury to the endothelium will initiate a cascade of events in an attempt to control bleeding. It is classified into primary and secondary hemostasis [12]. Blood consists of various hemostatic factors which are thrombogenic which aid clotting and non-thrombogenic which lead to the inhibition of the clotting process beyond the injury site along with the dissolution of clots [13,14]. There is a delicate balance between the procoagulants and the anti-coagulants [15]. A slight imbalance of any of the processes in the cascade can lead to complications such as hemorrhage or thrombosis [16]. Most of the coagulation factors are synthesized in the liver, except factors III, IV, and VIII [17]. These factors undergo post-translational modification through vitamin K-dependent γ-carboxylation of glutamate that allows these factors to bind to the divalent cations such as Ca+2 to initiate coagulation [17,18]. The endothelial cell lining is generally anti-thrombogenic due to the presence of negatively charged heparin-like glucose-amino-glycans (GAGs), neutral phospholipids, and the synthesis and secretion of platelet inhibitors such as NO and prostacyclins [19]. Various other factors that maintain normal blood flow and avoid unnecessary thrombus formation are heparin sulfate and antithrombin III which inhibit factors IIa, IXa, and Xa [20]. Thrombomodulin holds the thrombin in a bound form that further activates proteins C and S [21]. Activated protein C alleviates factors Va and VIIIa and activated protein S inhibits the activity of factor IXa [22,23]. The physiological representation of the normal blood flow maintained through endogenous moieties and mechanisms present in the vascular system are shown in Figure A.

Figure A: Pictorial representation of the hemostatic mechanisms and maintenance of normal blood flow in the blood vessels due to the intricate balance maintained between the thrombotic and the anti-thrombotic elements. Blood is composed of plasma and formed elements. The anti-thrombotic elements present are heparinlike GAGs, neutral phospholipids, anti-thrombin-3, protein C, protein S, and thrombomodulin. The endothelium secretes platelet inhibitors like NO and prostacyclins. Heparin sulfate and anti-thrombin-3 inhibit factors IIa, IXa, and Xa. Thrombomodulin inhibits thrombin, further activating protein C and protein S. Activated protein C inhibits factors Va and VIIIa whereas activated protein S inhibits factor IXa. (ECM- Extracellular matrix)

The sub- endothelial layer is thrombogenic due to the presence of collagen,von Willebrand Factor (vWF), laminin, thrombospondin, and vitronectin [24]. That is why, the exposed endothelial layer to the sub-endothelial layer leads to the activation of the coagulation cascade. Vascular insult leads to arteriolar vasospasm that is mediated by reflex neurogenic mechanisms through the noise/ pain receptors to internalize endothelin and thromboxane2 (TXA2) [25]. In brief, disruption of the endothelium will first cause local vasoconstriction to occur, limiting blood flow to the area. Primary hemostasis is initiated by platelets with the release of vWF, a large plasma glycoprotein made and stored in endothelial cells and megakaryocytes. Platelets and vWF will combine to form a plug at the site of injury. Circulating vWF continues to bind with collagen and factor VIII as well as other endothelial substances, allowing the platelet plug to adhere to the area of injury [26]. Through activation of the clotting cascade and secondary hemostasis, this initial platelet plug will be reinforced to a sturdy fibrin clot. The clotting cascade operates through a dual process system in which various clotting factors become activated which results in the formation of a fibrin strand or clot at the site of tissue injury [27]. A deficiency of any of the essential clotting factors will result in difficulty forming a fibrin clot, resulting in excessive bleeding [24,28].

In conclusion, primary hemostasis is a complex interaction between platelets, vessel walls, and adhesion molecules that leads to the formation of the platelet plug [26,29]. Secondary hemostasis is when the temporary platelet plug is secured firmly by the fibrin mesh formation through the activity of thrombin [30]. Fibrinogen is cleaved into fibrin strands that form a mesh- like structure that stabilizes the clot formed through the platelet plug [27,31].

Platelets

Platelets are an important moiety of the blood coagulation cascade. It is a disc-shaped enucleated cell, derived from megakaryocytes [32]. They form the initial hemostatic plug through aggregation that further provides a surface for the assembly of the activated clotting factors for fibrin stabilization and clot retraction [33]. In any incident of vascular assault, platelets adhere to collagen and vWF in the sub-endothelial region through the glycoprotein Ib (GPIb) receptors on the vWF [34]. Thrombin acts on the protease-activated receptors 1 and 4 (PAR 1 and 4) present on the platelet membrane and initiates a series of downstream signaling cascades such as activation of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) and phosphorylation of P38. PI3K further activates protein kinase B (PKB) also known as Akt and cytosolic phospholipase A2 (cPLA2) which ultimately leads to the release of TXA2. PLC, in turn, activates and releases inositol triphosphate (IP3) and diacylglycerol (DAG) through phosphatidylinositol 4,5- bisphosphate (PIP2). IP3 increases the intracellular Ca2+ while DAG activates protein 2+ IX. Factor IXa then triggers the activation of factor VIII which ultimately forms a tenase complex on the phospholipid (PL) surface to activate factor X [46]. Extrinsic and intrinsic pathways subsequently come together in the common pathway where factor Va, tissue PL, and Ca2+ form the protease complex to activate thrombin from its zymogen form, prothrombin [47]. Thrombin finally acts on fibrinogen to form a fibrin mesh [48]. The covalent cross-linking fibrin mesh helps stabilize the platelet plug. Apart from forming the fibrin mesh, thrombin also activates factor XIII which acts as the fibrin stabilizing agent [49]. The coagulation cascade involving the extrinsic and the intrinsic pathways is shown in Figure B. 2.

Figure B 1: Pictorial representation of hemostasis after vascular insult. a. Stages of hemostasis and in-detail mechanism of the platelet activation and aggregation leading to platelet plug formation. 2. Activation of the coagulation cascade results in firm clot formation through fibrin formation over the platelet plug due to the presence of the negatively charged phosphatidyl serine. The coagulation cascade is categorized into extrinsic and intrinsic pathways and ultimately leads to the formation of fibrin mesh over the platelet plug from fibrinogen through thrombin. The clot is further stabilized by factor XIIIa. (ER- Epinephrine receptor, SRSerotonin receptor, TXA2R- Thromboxane A2 receptor).)

The above-mentioned coagulation cascade has been modified and the modern version considers that the intrinsic pathway is not parallel to the extrinsic pathway [48]. Instead, the augmented thrombin generation is primarily initiated by the extrinsic pathway [47].

Wound Healing and Wound Debridement

The platelet plugs with the overall clot formed, undergo retraction, pulling the two ends of the damaged endothelium, and kinase C (PKC). Ca  and PKC help the degranulation process.

The platelets release two types of granules, α and σ or dense granules. α granules consist of P-selectin, fibrinogen, fibronectin, factor V, factor VIII, adenosine triphosphate (ATP), platelet factor IV, platelet-derived growth factor (PDGF), and transforming growth factor-α (TGF-α). Whereas, dense/ σ granules contain adenosine diphosphate (ADP), Ca+2, serotonin, histamine, and epinephrine. This forms the surface for further activation of other coagulation factors [35-37]. Upon stimulation, platelets release TXA2 and ADP, a stimulus for initiating activation of other nearby platelets, which further leads to the platelet plug formation that temporarily seals the vascular injury. Platelet to platelet interaction is through the glycoprotein αIIbβIII (GP αIIbβIII) [38]. The aforementioned mechanism is represented in Figure B. 1. As TXA2 is pro-coagulatory, to regulate hemostasis, prostacyclin acts as the endogenous antagonist of platelet aggregation [39]. Thus, a balance between prostacyclin and TXA2 is maintained to carry out normal blood low and coagulation processes. Through this regulatory mechanism, the extension of the clot is prevented and localized only in the injured site [32].

Clotting Factors

Other moieties involved in the coagulation cascade are clotting factors. Clotting factors are present in the bloodstream as zymogens [40]. These proteins bind to Ca2+ to initiate clotting when required [41]. Clotting factors are involved in the extrinsic and intrinsic pathways that come together with the common pathways leading to blood clotting [42].

The extrinsic pathway is mainly a plasma-mediated hemostasis that is initiated by tissue factor (TF) expressed by the sub-endothelial cells [43]. Contact between TF with factor VIIa and Ca2+ then activates factor X, representing the extrinsic pathway [44]. Parallel to the extrinsic pathway, the intrinsic pathway is initiated through the action of factor XIIa to activate thrombin [45]. Further, factor XIIa, high molecular weight kininogen (HMWK), prekallikrein, and factor XIa activate factor PDGF and vascular endothelial growth factor (VEGF) result in the replacement and renewal of the worn-out tissues that aid in wound healing [50]. As the process of wound healing is important, so is the process of wound debridement. The fibrinolytic system helps in wound debridement and runs parallel to the coagulation process. It is the enzymatic process of dissolving and digesting the fibrin clot through the action of plasmin [51]. The process of clot retraction and fibrinolysis leading to wound healing and wound debridement is indicated in Figure C.

Figure C: Representation of the clot retraction and repair mechanisms ultimately leading to wound debridement through fibrinolysis. The platelet plug formed starts retracting and pulls the damaged endothelium together through contraction. VEGF and PDGF released through the platelet granules help in tissue regeneration and healing. Tissue plasminogen activator (TPA) activates plasmin from plasminogen. Plasmin cleaves the fibrin mesh. The entire process results in the renewed and replaced tissues in the injured area.

Thus, the process of hemostasis being an intricate physiological cascade, regulates the fluidity of the blood, bleeding, wound, and wound healing through the delicate balance between the thrombogenic, anti-thrombogenic, and fibrinolytic mechanisms [52]. Imbalances in the aforementioned pathways lead to various thrombotic and bleeding disorders [53]. Hence physiology should be deeply understood, especially due to the numerous factors included, to predict pathological and clinical consequences before implementing any pharmacological interventions towards any coagulation complication [54].

Bleeding Disorders

The body possesses innate mechanisms to control bleeding in the setting of an injury. An understanding of these basic physiological processes is critical to aid in the identification and diagnosis of bleeding disorders. Bleeding disorders are a group of conditions that result when the blood does not clot. In normal clotting, platelets stick together and form a plug at the site of an injured blood vessel [55]. Clotting factors in the blood then interact with the platelet plug to form a fibrin clot, essentially a gel plug, which holds the platelets in place and allows healing to occur at the site of the injury while preventing blood from escaping the blood vessel. Too much clotting can lead to thrombotic conditions resulting in heart attacks and strokes. The inability to form clots can be dangerous, which can result in excessive bleeding or hemorrhage. Bleeding can result from either too few or abnormal platelets, abnormal or low amounts of clotting proteins, or abnormal blood vessels [56]. Individuals with a family history of bleeding disorders should focus on detection and treatment of any such conditions.

Hemophilia is a rare genetic bleeding disorder that slows down or prolongs the coagulation of blood during an injury. The formation of poor clots due to the missing clotting factor is the main reason for continuous bleeding [57]. It mainly affects males and females are only carriers. There are three types of hemophilia namely, hemophilia A, hemophilia B, and hemophilia C. Hemophilia A also termed classic hemophilia, is caused due to the deficiency of factor VIII [58]. Hemophilia B arises due to the deficiency of factor IX and is also termed Christmas disease [59]. Hemophilia C, on the other hand, is caused due to the deficiency of factor XI [60]. All three clotting factors, i.e., factors VIII, IX, and XI are a part of the intrinsic pathway of the coagulation cascade [61]. There is a variant of hemophilia B termed hemophilia B Leyden where the affected individuals show excessive and frequent bleeding in their childhood that reduces after puberty due to unexplained reasons. Both hemophilia A and B show similar signs and symptoms but differ in genetic mutations of factor VIII and factor IX respectively [62,63].

The other prominent bleeding disorder after hemophilia is von Willebrand disorder (vWD) which results when the blood lacks functioning vWF, a protein that helps the blood to clot and also carries another clotting protein, factor VIII [64]. Hemophilia is perhaps the most well-known inherited bleeding disorder, although it is relatively rare [65]. Many more people are affected by vWD, which is the most common inherited bleeding disorder [66]. vWD is classified into three different types, Types 1, 2, and 3 based on the levels of vWF and factor VIII activity in the blood [66-68]. Individuals with bleeding disorders show symptoms that include heavy bleeding from minor injuries, easy bruising, and excessive bleeding following dental procedures or any type of surgery [69]. Hence proper and specific detection, management, and treatment are advisory to avoid life-threatening situations during an injury that leads to uncontrolled bleeding [69].

Diagnosis

Diagnosis of bleeding disorders is through analyzing the patient history, clinical evaluation, and laboratory tests that include complete blood count (CBC), coagulation tests that include prothrombin time (PT), activated partial thromboplastin time (aPTT), international normalized ratio (INR), bleeding time, differential diagnosis, and measurement of individual clotting factors for abnormal concentrations [70]. Approximately one- third of the cases that arise are due to spontaneous mutation and do not predicate on any specific race [71]. Usually, in the case of hemophilia, aPTT is prolonged while PT, INR, bleeding time, and platelet count remain normal [72]. However, the prognosis cannot be based only on these tests. Hence molecular genetic testing is carried out that identify the exact mutation of a particular gene and also assess if the individuals are carriers [73].

Management and Treatment

It is well known that any factor deficiency disorder has no cure. The only prognosis includes management of the disorder by medical or genetic counseling and supplementation of the deficient factor and the patients are always taken care of by a professional healthcare team [74]. The replacement of the deficient factor is the current treatment protocol for managing bleeding disorders. As of now, recombinant factors VIII and IX concentrates are available [70]. Mild conditions require “on- demand therapy” which is the supplementation of factors only during the bleeding episodes. In the case of severe bleeding, factors are infused at regular intervals to avoid spontaneous bleeding episodes and hence termed “prophylactic treatment” [75].

During severe injuries since immediate treatment is essential, infusion of replacement factors is found to be expensive and complicated. As these drawbacks limit the success of the treatment or management of bleeding disorders, new alternative approaches have to be put forward in a better or cumulative way than the existing treatment options that can also be cost-effective [76]. In this case, as proteases from plant latex already play a vital role in coagulation and wound care management, they can also be a potentially better and inexpensive alternative recourse to treating bleeding disorders [77]. However, current research provides insights only on the topical or external cut wound applications of the plant latex proteases that are restricted to the local anomalies of the system during an injury. The activities represented by the plant latex proteases include platelet aggregation, fibrinogenolytic/ thrombin-like, fibrinolytic/ plasmin-like, and mitogenic/ cell proliferating activities. A better understanding and identification of the potential purified moieties from the plant latex proteases acting on a specific step of the hemostatic mechanism can provide an enhanced insight into the utilization of the plant latex protease to treat bleeding disorders.

The Proteases (3.4.21) are the enzymes that catalyze the peptide bond cleavage. It is found in all organisms and accounts for up to 2% of the human genome [78,79]. There are 7 families of proteases depending upon the amino acid they contain in their active site namely, arginine, aspartate, cysteine, serine, glutamate, threonine, and metalloproteases [80]. Serine, cysteine, and matrix metalloproteases are important types of proteases that play a major role in wound healing or clotting [81]. Proteases are also important industrial enzymes that account for 60% of the enzyme market. The major advantage of proteases is that they are easy to harvest, process, store, and use. They also exhibit broad specificity with stability in a wide range of pH, temperature, and other factors [82]. Protease activity is not restricted only to protein digestion and turnover, instead, they are involved in trypsinogen activation as first illustrated by Davie and Neurath [83]. Later Davie Ratnoff and Mactarlane showed that proteases play an important role in clotting as well [84]. One of the important sources of proteases from plants is latex, which has also been used in wound healing or as a procoagulant, for the past 20 years industrially, which accounts for 20% of the latex used as a therapeutic [85]. Nonetheless, the history of the use of lattices in wound care management dates back centuries. Traditionally different parts of plants like bark, stem, leaves, and roots are used in wound care management and other interventions of the coagulation cascade [86]. In India, latex-producing plants widely used in ancient medicinal practices such as Ayurveda, Siddha, Homeopathy, Unani, and Folk are the majority of them are listed in the Date Base ‘Foundation for Revitalization of Local Health Traditions (FRLHT) and Traditional Knowledge Digital Library (TKDL) created by Ministry of Ayurveda, Yoga, Naturopathy, Unani, Siddha and Homeopathy (AYUSH), Government of India.

Lattices are the plant exudates produced from the specialized laticifer cells. Over 20 different families of angiosperms such as Altingiaceae, Amaranthaceae, Ascleceae, Asteraceae, Caricaceae, Dipterocarpaceae, Lamiaceae, Papaveraceae, Plumbaginaceae, Solanaceae, Euphorbiaceae, Asclepidaceae, Apocynaceae, Moraceae, and Sapotaceae etc, produce latex through various parts of the plant such as leaf, stem, or root depending on the type of predators, pests or infectious agents present [87]. The latex- producing plants are usually widespread in arid regions and can either be herbs, shrubs, or a tree. The major role of latex is to protect the plant against pathogens, pests, or predators through recognition and induction of defensive mechanisms against the identified threat, storing and transporting nutrients, regulation of water balance, growth, development, germination, circadian rhythm, apoptosis, and so on. It also provides a certain level of drought resistance to the plants and therefore, latex-producing plants are usually not present in tropical or cold regions. Nonetheless, the specific role of latex remains elusive due to the broad range of actions exhibited differently in different plant species [88]. Lattices are a mixture of secondary metabolites and bioactive components such as acetogenins, alkaloids, resins, phytosterols, tannins, terpenoids, cardiac glycosides, lignans, cannabinoids, proteins, carbohydrates, amino acids, waxes, phosphatides, and proteins (enzymes/ proteases). Inorganic components of the lattices are predominantly Ca2+ and Mg2+ along with Mn2+, Fe2+, Cu2+, and Zn2+.

Plant latex proteases, the major fraction of the lattices, remain the major target for therapeutics. Though many plant latex proteases are used widely in ancient medicine, the exact mode of their actions is not well documented. Plant latex proteases from almost 2,000 different plants are used in wound healing and therapeutics as procoagulants, wound clearers, anti-cancerous, anti-inflammatory, anti-proliferative, vasodilatory, anti-oxidant, anti-microbial, anti-parasitic, anti-helminthic, and insecticidal molecules [89]. Plant latex proteases can either be involved in constricting blood vessels to reduce blood loss during an occurrence of the wound aid platelet aggregation or induce fibrin mesh formation. The procoagulant activity is mainly attributed to the thrombin-like enzymes that exhibit clotting factor-like activity from the latex [90], and the wound debridement activity is due to the presence of the fibrinolytic/ plasmin-like enzymes.

For instance, some of the latex protease can aid clotting in factor VIII deficient plasma showing that the protease bypasses the factors and helps in coagulation [87,91]. Nonetheless, even if a certain factor is absent in the physiological system, the use of plant latex protease to bring about coagulation can be the most advantageous because these proteases act on various processes of the coagulation cascade from the initial to the final stages. Thereby, even if a particular factor is absent, plant latex protease would bring about coagulation by acting at the final step leading to clot formation [88]. Some plant latex proteases also promote the growth of the damaged tissue. This signifies that the latex proteases can also act as a growth factor, or growth-inducing molecule [92]. The involvement of the latex proteases is in hemostasis (blood coagulation) through fibrinogenolysis, wound healing, and wound debridement through fibrinolysis, mitogenic activities are in general represented in Figure D. 1 [91]. The role of various latex proteases either purified or partially purified, interfering in different stages of hemostasis and healing are shown in Figure D. 2. Figure D. 2 also emphasizes the plant latex proteases that are isolated and studied in our laboratory and also in other laboratories studied for various properties including procoagulant, thrombin-like, plasmin-like, and mitogenic activities.

Figure D: Role of plant latex proteases in different levels of hemostasis and wound healing. 1. Generalized representation of latex proteases involved in multiple physiological processes during hemostasis and wound healing. [91,108]. 2. Various plant latex proteases used as partially purified extracts and purified forms that act on different stages of hemostasis and wound healing. Plant latex proteases represented in black are the proteases studied in our laboratory. Plant latex proteases represented in red are other purified proteases.

The other major advantages of the latex protease are, that they are non-pathogenic to either humans or animals, very easy to harvest, and a highly inexpensive source of essential therapeutic substances [93]. As mentioned earlier, proteases from natural zoological sources have pathogenic potency creating immunological complications along with low yield. Therefore, the plant latex proteases are the rational targets due to their major pharmacological and therapeutic importance and hence can be used to treat various defective blood clotting disorders, especially in treating patients with external cuts or external bleeding complications, because the latex proteases are restricted to the topical application [92].

Crude latex extracts can be less effective in exhibiting therapeutic properties due to the presence of gums and waxes which makes it essential for the purification of the bio-active components from the latex to exploit the important therapeutic properties [94]. The purification is usually carried out using the freeze-thaw method and chromatographic techniques. Even experimental validation of certain plant latex proteases is carried out in rodents and many are even available in the market for human use in terms of excision and incision cuts and burns [95].

In one of our previous studies, plant lattices from 47 different genera from 12 different families were procured, partially purified, and evaluated for their role in hemostasis, wound healing, and wound debridement, out of which 33 of them showed potential results [91]. It is also very well established in our laboratory that many latex proteases exhibit thrombin and plasmin-like activity and bypass various factor-deficient plasma to induce clotting and wound debridement respectively. It is also shown that various plant latex proteases have potential growth factor- like or growth-inducing activities that aid tissue regeneration.

The brief tabulation of the research carried out in our laboratory that concerns plant latex proteases as a hemostatic factor, wound debridement agent, and tissue regenerating molecules are represented in Table A.

Table A: List of the plant latex proteases purified/ studied in our laboratory.

The latex proteases are used either in partially purified or purified forms. The partially purified extracts or the purified proteases exhibited various activities from the initial phases to the final phases of hemostasis and wound healing. The proteases involved in platelet aggregation, possess thrombin-like or fibrinogenolytic activities, plasmin-like or fibrinolytic activities, and mitogenic or cell proliferation properties. Hence the proteases are considered procoagulatory or aid wound healing or wound debridement processes.

All the latex proteases isolated, purified, and characterized, are mentioned in Table. A were identified and authenticated by the DoS in Botany and also assessed for the listings in FRLHT, TKDL, and AYUSH as aforementioned. Also, the animal and human blood samples used from healthy volunteers were obtained and used as per the approved protocols from the Institutional Animal Ethical Committee (IAEC) and Institutional Human Ethical Committee (IHEC) respectively. (The reference numbers of IAEC and IHEC are in the respective pre-published research articles cited). The protocols were in accordance with the guidelines of the committees [87,88, 91, 92,96-102]. Out of all the plant latex proteases screened in our laboratory, only Calatropis gigantea latex protease was shown to exhibit toxic effects at higher doses [91]. In summary, the plant latex proteases isolated from various plants such as Pergularia extensa (Jacq.) N.

E. Br, Tabernaemontana divaricate (L.) R. Br. ex Roem. & Schult, Vallaris solanacea Kuntze, Asclepias curassavica L, Calatropis gigantea R. Br, Cynanchum pauciflorum R. Br, Wrightia tinctoria R. Br, Oxystelma esculentum (L. f.) Sm, and Carica papaya L, which have been studied in our laboratory either in purified, partially purified, or as crude latex extracts, show procoagulant, mitogenic, wound healing, and wound debridement activities, Thus, assessing the overall endogenous effects and pathophysiology needs to be studied in detail for potentiating the use of plant latex protease as a procoagulant.

Other noteworthy instances are, Papain and Chymopapain (Caricapapaya L) which are clinically used for wound debridement [103,104]. In one particular study, a protease from the latex of Ficus carica L was found to interfere with hemostasis by acting as a factor X activator. Other commonly and clinically used purified plant latex proteases are Plumerin-R (Plumeria rubra L), Curcain (Jatropha curcus L), Purgularain e I (Pergularia extensa (Jacq.) N. E. Br), etc. These components exhibit early wound healing by enhancing wound debridement, neovascularization, and improved organization of collagen and elastin. These latex proteases act on various phases of wound healing by mimicking the functionality of the clotting factors [88,95,105]. For instance, Ficin (Ficus carica L), mimics factor X and plays a major role as a hemostatic agent, Papain mimics factor XIII a and thrombin, Plumerin-R and Curcain are used for wound healing, Pergularaine e I and Plumerin-R are also used for collagenation, Components like CMS2MS2 from Carica candamarcensis Hook. f, are mitogenic and Cg24-I from Cryptostegia grandiflora Roxb. Ex. R. Br, are anti-microbial [88,91,95,101,105]. Many other such proteases are available and well-documented to be used as a therapeutic, especially in terms of hemostasis. Thrombin-like enzymes present in the latex proteases act as the bypassing agent in factor-deficient conditions and are also involved in primary hemostasis by acting on the PAR-1 present on the platelets leading to aggregation [99]. For example, Papain clots factor VIII deficient plasma. Though thrombin and Papain contain cys 25 and his 159 in their catalytic diad, they show no sequence or structure similarity except functionality. Mostly they interfere with the common pathway of the coagulation cascade [103]. Aspirin is a known anti-platelet aggregation component that is supplemented for patients with cardiovascular disorders (CVD). The major drawback of using aspirin is, that during an injury, clotting is hindered due to improper platelet aggregation and clot formation [106]. Plant latex proteases are also known to inhibit this process. The advantage of the use of latex protease is that they act both on the initial phase and final phase of hemostasis, due to their pro-coagulatory and thrombin-like activity, and fibrinolytic activity respectively. In the initial phase, it acts as a hemostatic agent, whereas in the final phase, it acts against inflammation along with gelatinolytic and collagenolytic activities that contribute towards wound debridement [86]. They are simpler and cheaper to obtain and purify as well which is another major advantage [101].

Although the plant latex proteases show potent procoagulatory activity and have no potential for pathogenicity to humans or animals, they are still not considered to be a drug class have very low clinical importance, and are restricted only for topical application. The molecular mechanisms underlying the role of latex protease in hemostasis in vivo are not completely elucidated [93]. Though the mechanism remains elusive, it can be exploited due to the promising procoagulatory activity along with enhanced wound care management that has been proved in many cases [107]. Therefore, the search for novel proteases from medicinal plants holds extreme importance globally as the current treatments pose problems. Nonetheless, purification, and characterization of physiological, pharmacological, and toxicity studies are highly essential to evaluate their therapeutic capability as an alternative recourse to bleeding disorders.

Folk medicinal practitioners worldwide utilize various medicinal plants as remedies for different ailments. Although these plants are effective, scientific validation for their use is often lacking. For centuries, tribal and rural communities in India and around the globe have employed plant latices to halt bleeding and promote wound healing. Blood coagulation and fibrinolysis processes are crucial during the cessation of bleeding and wound healing. These processes are tightly regulated by specific proteolytic enzymes. Numerous plant latices contain proteolytic enzymes, but only a few reports suggest their involvement in blood coagulation and fibrinolysis that are well-established. However, there is limited information regarding the biochemical basis of the observed pharmacological activities of these plant latices. Therefore, the plant latex protease can be a potential agonistic molecule alongside the current conventional treatment mechanisms until the in-detail mechanisms are completely understood and utilized as a supplement or an independent alternative to hemostatic disorders. A systematic study about the occurrence and properties of proteases correlated to plant families, and knowledge about the chemical properties can aid in chemotaxonomic studies. Further, the biochemical characterization of the latex protease, the study of their substrate specificities, and the ability to alter the human physiological system might be beneficial. The plant latex protease positively affects the hemostatic and wound healing processes but is mostly restricted to topical application rather than endogenous administration in the pharmaceutical industry. Thus, this review aims to evaluate the contribution of various latex proteases that facilitate blood coagulation and fibrinolysis and establish the biochemical basis and mechanism of their action. Overall, plant latex proteases bring about platelet aggregation, and clots factor deficient plasmas, consist of thrombin-like and plasmin- like activity, aid fibrinolysis, help in wound debridement and wound healing, also contribute to tissue regeneration due to the growth factor-like moieties present or induction of growth. Various discoveries and studies suggest that latex proteases may have specific sites of action in blood coagulation and fibrinolytic reactions. Proteases exhibiting specific actions, such as factor V activators, factor VII activators, prothrombin activators, thrombin-like enzymes, protein C activators, plasminogen activators, and plasmin-like enzymes, etc., hold potential therapeutic applications in treating bleeding disorders, especially in the external bleeding complications in the patients to induce clot formation as an alternate or a specific recourse.

The authors are grateful to Dr. H. V. Shivaprasad and Dr. V. N. Manjuprasanna P to have provided their valuable suggestions.P. Naveen and P. Pramod Kumar helped with minor software and reviewing. Supriya Sarkar is grateful for the Department of Science and Technology- Innovation in Science Pursuit for Inspired Research (DST-INSPIRE). The authors also acknowledge Spandan Nayaka for other minor aid with figures.

The study did not receive any specific grant from funding agencies in the public, commercial, and not-for-profit sections.

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