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. 2020 Oct 8;11(10):1100–1111. doi: 10.1039/d0md00204f

Peptide-based topical agents and intravenous hemostat for rapid hemostasis

Snehasish Ghosh a, Archana Tripathi b, Paramita Gayen b, Rituparna Sinha Roy b,c,d,✉
PMCID: PMC7651999  PMID: 33479616

graphic file with name d0md00204f-ga.jpgThese article features about peptide based topical and intravenous hemostat.

Abstract

Traumatic coagulopathy due to severe external injury and internal hemorrhage is life-threatening to accident victims and soldiers on the battlefield, causing considerable number of deaths worldwide. Patients with inherited bleeding disorders (such as haemophilia, von Willebrand disease, inherited qualitative platelet defects, and afibrinogenemia) also contribute to the vast number of deaths due to abnormal bleeding, and these patients are difficult to handle during surgery. Platelets and different plasma proteins play an essential role in blood coagulation and in the maintenance of the body's hemostatic balance. The improper function or deficiency of these factors cause abnormal bleeding. To address such bleeding disorders, external clotting agents (such as extracellular protein-inspired natural and synthetic peptide-based sealants and peptide-functionalized polymer/liposome-based sealants) have been developed by different groups of researchers. The primary focus of this review is to provide molecular insights into the existing biologically inspired peptide-based sealants, highlighting the advantages and limitations of such reported designed sealants to handle blood clotting, and also provide insights into the design of improved next-generation surgical sealants.

1. Introduction

Biologically inspired sealants and synthetic sealants have emerged as promising candidates for treating wounds of patients with a traumatic injury or inherent bleeding disorder.1–3 Bleeding disorders can directly or indirectly influence the intrinsic or extrinsic clotting pathways.4 Accident victims and soldiers face massive blood loss or traumatic coagulopathy due to bleeding disorders and lack of fibrinogen. Haemophilia patients face inherited bleeding disorders due to factor VIII deficiency (haemophilia A) or factor IX deficiency (haemophilia B).5 von Willebrand disease is an inherited disease that arises due to the absence of proteins present in plasma, named von Willebrand factor (vWF). This vWF is involved in blood coagulation and contributes to the activation of downstream clotting pathways by stabilizing factor VIII, and thus helping in thrombin formation mediated by factor Xa.6 Afibrinogenemia is an inherited blood disorder in which the blood does not clot normally due to the fibrinogen deficiency.7

To handle surgical or non-surgical bleeding, clinicians use several techniques, including topical sealants, gauze pressure, suture, electrocauterization or laser techniques.8,9 Suturing supports primary wound closure, but fails in soft tissues.8 Topical sealants or agents can be directly applied to the wounded area, and are also known as local hemostats.3,10–12 Their mechanism depends mainly on two factors, the gelation process and the pathway of hemostasis. Gelation property is an important feature of topical agents. It involves weak interaction forces (e.g., H-bond, van der Waals force), as well as strong forces like ionic interaction or covalent conjugation.13 Depending on the method, this gelation process is divided into four types, namely physical, chemical, ionic and enzymatic process. Among the natural fibrous proteins, polysaccharides and synthetic polymers or peptide-based sealants, herein, we have explicitly discussed protein-inspired peptide-based sealants and described their mechanisms with limitations. Topical peptide-based hemostats are collagen-inspired, silk mimetic, elastin-like, zuotin mimetic, fibrin inspired and snake venom-inspired hemostats, whereas intravenous hemostats are blood-derived or synthetic injectable particles like thromboerythrocytes, tissue factor binding peptides and peptide-functionalized platelets/liposomes/polymers.

2. Hemostatic mechanism

Hemostasis is a multi-step process. It is composed of several events, namely vasoconstriction (constriction of blood vessels), platelet adhesion and activation, platelet aggregation, activation of the coagulation cascade, and ultimately the formation of an insoluble fibrin clot.14 Upon injury, a vascular spasm is triggered, followed by vasoconstriction to reduce the blood flow. Next, with a glycoprotein receptor on their surface that can interact with the protein of the extracellular matrix (ECM), the platelets come into contact with the exposed subendothelial matrix layer of the damaged blood vessels containing collagen, fibronectin, and laminin, and can directly bind with this protein through the GPIa/IIa receptor. The vWF, which is itself bound with the exposed subendothelial matrix, mediates this platelet adhesion via two major platelet receptors, GPIb and integrin GPIIb/IIIa.15 As this binding is brief and has a very high dissociation rate, it does not form a stable adhesion. Instead, it helps platelets ‘rolling’ along the damaged endothelium, which further facilitates the additional binding of the platelet receptor to the ECM proteins (like GPVI–collagen).15 GPVI is an immunoglobulin superfamily receptor of platelets that helps to bind with collagen, which leads to platelet activation by triggering the release of adenosine diphosphate (ADP) and thromboxane A2 (TXA2). These released substances further act on the platelets via G proteins (Gq, G12/G13, Gi), leading to the activation of downstream signaling cascades and full platelet activation.15 These activated platelets form pseudopods (arm-like projection), causing clumping and ultimately platelet aggregation.14 Platelet interaction requires platelet–platelet interaction, which is mediated by fibrinogen and/or vWF via activated GPIIb/IIIa complex on two different platelets.16 The most crucial step in hemostasis is the activation of the coagulation cascade (Fig. 1). In the extrinsic pathway, the transmembrane receptor, tissue factor (TF), first binds to its ligand, factor VII/VIIa, and forms the TF: (VII/VIIa) complex, activating factors IX to IXa and X to Xa.17 In contrast, factor XIIa is exposed to the damaged endothelial collagen in the intrinsic pathway, and initiates a cascade of reactions that eventually activates factor X to Xa.14 These two pathways ultimately merge in a common pathway, where activated factor X cleaves prothrombin to form thrombin. This thrombin further activates factor VIII, which converts factor X to Xa. vWF noncovalently binds to factor VIII, preventing its proteolytic degradation, thus stabilizing it and amplifying the thrombin production via the intrinsic pathway.6 In the last step of blood clotting, thrombin mediates the conversion of fibrinogen into an insoluble fibrin network that requires a number of subsequent steps.18 Fibrinogen is a 340 kD homodimeric glycoprotein. This protein comprises two identical subunits and six nonidentical polypeptide chains Aα, Bβ and two γ. The subunits and polypeptide chains are linked by 29 disulphide bridges.19 These six polypeptide chains are assembled in a coiled fashion and form various structural regions, namely a central E region, two distal D regions and two αC regions.19 The central E region comprises the amino terminus of all six polypeptide chains.20 The two distal D regions are formed by the carboxyl group terminus of both Bβ and γ chains. Furthermore, the carboxyl group termini of the Aα chains fold back towards the central E region, and give rise to two interacting αC regions.20 The amino termini of the Aα and Bβ chains have 16 residue fibrinopeptide A (fpA) and 14 residue fibrinopeptide B (fpB), respectively. Thrombin cleaves the Arg–Gly bond of fpA and fpB from the Aα and Bβ chains, leading to the exposure of two pairs of polymerization sites having starting sequences Gly–Pro–Arg and Gly–His–Arg, respectively (Fig. 2).19,20 These two pairs of polymerization sites (knobs) “A” and “B” are located in the central E region, whereas its complementary polymerization sites (holes) “a” and “b” are located in the distal γ and β nodules of the D region, respectively.20 After thrombin cleavage, the exposed knobs “A” and “B” interact with holes “a” and “b” of the neighbouring molecule to form a D : E : D complex. This event leads to the noncovalent polymerization of monomeric fibrin.20,21 The crosslinking of converted polymerized fibrin occurs via N∈(γ-glutamyl)-lysyl bond or isopeptide bond formation mediated by factor XIIIa, a plasma transglutaminase.22,23 The crosslinking occurs between the Lys residue present in the γ chain at the 406th position and the glutamine residue present in the 398th/399th position of the γ chain of the neighbouring fibrin molecule.22,24 The 14-residue sequence (residues 398–411) that forms the fibrin clot is Gln–Gln–His–His–Leu–Gly–Gly–Ala–Lys–Gln–Ala–Gly–Asp–Val.22 Finally, the strong and stable water-insoluble fibrin network reinforces the soft platelet plug formed during the hemostasis process.25

Fig. 1. Process of hemostasis and coagulation cascade (intrinsic, extrinsic, amplification and common pathways) of blood clot formation.

Fig. 1

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Fig. 2. Schematic representation of the fibrinogen molecule with Aα, Bβ and γ chains.

Fig. 2

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3. Peptide-based sealants/hemostats

3.1. Topical sealants/hemostat

Peptide-based topical sealants mimic natural proteins having the ability to clot the blood. Proteins from various animal sources can be used as hemostats, but concerns exist regarding their role in inflammation, batch-to-batch changeability and conceivable disease transfection.26 Hence, the advancement of synthetic nano-biomaterials that can mimic the structure and properties of a particular protein has been developed for promoting blood clotting and wound healing. The advantages and disadvantages of protein mimetic peptide-based sealants are highlighted here, and also summarized in Table 1.

Table 1. Advantages and disadvantages of protein-mimetic peptide-based sealants.

Sl. No. Name of the protein Sequence of peptides Advantage Disadvantage Status of the studies performed
1. Collagen inspired peptide RG(POG)10F, [(Pro–Lys–Gly)4–(Pro–Hyp–Gly)4–(Asp–Hyp–Gly)4] Biocompatible, biodegradable, effective hemostat Lower adhesion strength Physicochemical, in vitro and ex vivo characterization29–31
2. Silk fibroin PEG-silk, C12-GAGAGAGY Biocompatible, biodegradable, high mechanical strength Inflammation Physicochemical characterization33
3. Elastin like peptide [[VPGVG]4IPGVG]14 Biocompatible, biodegradable, effective hemostat and wound sealing material Adverse effect on human is unknown In vitro and in vivo (rat model) studies41
4. Yeast protein KLD12, EAK16, (RADA)4 Biocompatible, biodegradable, effective hemostat Adverse effect on human is unknown In vitro and in vivo (mouse model) studies. RADA is in preclinical phase46–52
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3.1.1. Collagen inspired peptide-based hemostats

Collagen-mimetic peptides have emerged as an alternative approach in hemostasis, instead of whole collagen protein. Collagen proteins derived from various animal sources may cause infection, disease transmission and inflammation. Synthetic collagen-inspired peptides can mimic the structural and functional properties of collagen, and are also cost-effective. The trademark triple helical structure of collagen is the result of the repetitive amino acid arrangement (Xxx–Yyy–Gly)n, where the Xxx and Yyy typically represent proline (Pro, 28%) and 4-hydroxyproline (Hyp, 38%), respectively. Different studies have demonstrated that n = 10 results in a stable triple helix, and n = 5 results in an unstable structure.27 Triple helix stability increases on incorporating different types of interactions, like electronegative interactions, H-bond mediated stabilization, π–π interaction, and cation–π interaction.27,28 Chen et al. designed and synthesized a collagen-mimetic model peptide having sequence Arg–Gly–(Pro–Hyp–Gly)10–Phe, which eventually self-assembled in a head-to-tail manner to give a nanofiber-like structure via strong cation–π interactions between the cationic Arg and aromatic Phe residues. This study demonstrated that the cation–π interactions play a crucial role in the self-assembly of small collagen-related peptides.29 Hartgerink and coworkers have designed a collagen-mimetic peptide, named KOD and having sequence (Pro–Lys–Gly)4–(Pro–Hyp–Gly)4–(Asp–Hyp–Gly)4, as a hemostatic agent (Fig. 3a). This triblock peptide forms a multi-hierarchal self-assembly like natural collagen. As this peptide has the (Pro–Hyp–Gly) repeating unit of collagen in the central domain, it facilitates triple helix formation. Furthermore, the incorporation of salt-bridged hydrogen bonds between the Lys and Asp residues stabilizes the triple helix in a sticky-ended assembly.30 Later studies of this group showed that these collagen-mimetic peptide-based nanofibrous materials adhere with platelets, activating the platelets to clot blood and plasma similar to natural collagen. They also demonstrated that KOD acts as a fluid barrier in the bleeding zone to stop hemorrhage with minimal pro-inflammatory cytokine production and hemolytic rate.31

Fig. 3. Protein-inspired peptide-based hemostats: (a) collagen mimetic,30,31 (b) elastin mimetic,33,41 and (c) yeast protein-inspired peptides46,52 as topical hemostatic agents.

Fig. 3

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3.1.2. Silk protein mimetic peptide-based sealants

Diverse silk fibers produced by silkworms, spiders, scorpions, bugs and flies play an important role in prey catching or laying eggs. Table 2 lists the peptide sequences present in different kinds of silk proteins obtained from various sources, and they have different tensile strengths. Silk fibers obtained from arachnids and silkworms are lightweight, amazingly strong and elastic, biocompatible, biodegradable and display mechanical properties such as high tensile strength and elasticity.32–34 The quality of such silk is far better than the best synthetic fibers designed and synthesized today by our cutting-edge techniques. In the medicinal field, silk is utilized for safe, biodegradable sutures, and even for microsurgery. Kaplan and co-workers have reported a new class of PEG-silk based sealant.32 This sealant is prepared by crosslink formation within seconds by means of a chemical reaction between the thiols and maleimides present on the PEG molecules that possibly form a β-sheet.32 These systems have excellent biological, physical and mechanical properties like sealants and hemostats.

Table 2. Different kinds of silk proteins and their motifs32–34 .
Sl. No. Key motif Source Functional property
1 [(AEAEAKAK)2AG(GPGQQ)6GS]9 Dragline spider silk Rearrangement of segments from α helices to β strands within the peptide chain
2 (GPGXX)n or (GGX)n or (GPGGX)n X = any amino acids Flageliform silk Spiral β turn which provides elasticity
3 (AAQAASAA)n, (AAQAA)n, (AASQAA)n Tubuliform spider silk Hydrophilic with low elasticity
4 (AAAAAA)n Spider silk Crystalline β sheet domain
5 (QPGSG)n, (NVNVN)n Fiber coating (glue) proteins Adhesiveness
6 (GAGAGS)n Silk fibroin Crystalline β sheet domain
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Serica Technologies Inc. (Medford, Massachusetts) is marketing FDA approved silk for soft tissue repair. Another FDA approved protein silk, fibroin, is used for drug delivery, surgical suture and tissue engineering.35 Other market-available silk-based sutures are Surusil®, Covidien, Suru and Sofsilk™.36 Silk proteins have several applications. Minagawa et al. reported the production of recombinant fibrinogen (rFib) using a transgenic silkworm, signifying an excellent approach for the industrial production of fibrinogen that eliminates the risk of bloodborne infection and disease transmission.37

3.1.3. Elastin like peptide-based sealants

Elastin, a highly elastic protein of connective tissue, is required for the maintenance of elasticity of various tissues and organs over a lifetime, including skin, lungs and blood vessels.38 Elastin has a covalent crosslinking network of topoelastin (TE) monomers, which have both hydrophobic and hydrophilic domains. The hydrophobic domain is enriched with Gly, Leu, Val and Pro residues, whereas the hydrophilic domain has Lys–Ala and Lys–Pro motifs.39 The hydrophobic domain of topoelastin forms the aggregate, whereas the hydrophilic domain takes part in crosslinking. The crosslinking reaction initiates when the lysyl oxidase (LOX) and LOX-like enzymes catalyze the oxidative deamination of the ε-amino group of Lys residues, and form the highly reactive aldehyde (α-aminoadipic acid-δ-semialdehyde (allysine, Lya)). After the oxidation process, covalent crosslinking occurs either between two reactive aldehydes (Lya residues) by aldol condensation, forming allysin aldol, or by the reaction of a reactive aldehyde (Lya residue) with the ε-amino group of another Lys residue via Schiff base reaction.39,40 The crosslinking between two reactive aldehydes or one reactive aldehyde and ε-amino group of another Lys residue is a non-enzymatic process. Purification of the indigenous elastin is problematic because of its insoluble nature due to the presence of the hydrophobic domain having Gly, Leu, Val and Pro residues. Elastin-inspired peptides (ELPs) are flexible, thermo-responsive, biocompatible and are inspired by the hydrophobic domain of topoelastin (TE) with the pentapeptide repeat: (Val–Pro–Gly–Xxx–Gly)n, where Xxx is any amino acid residue other than proline and n is the number of repeats (Fig. 3b).41 In elastin protein, these hydrophobic pentapeptide domains are called elastomeric domains. Researchers developed elastin-inspired peptide (ELPs)-based sealants that photo-crosslink by UV-visible light without further modification of the amino acid residues in the protein sequence.41,42 They incorporated a Cys residue at the end of the sequence, which helps to retain the elastic and mechanical properties of the actual protein. So far, they tested this material in vivo in a rat liver bleeding model. Better hemostasis was observed for the test subject than for the control.41 Senior et al. found that ELPs having Val–Gly–Val–Ala–Pro–Gly hexapeptide repeats caused a proliferation of fibroblast cells and upregulated collagenase production, indicating the involvement of this peptide in collagen remodeling and wound healing.43 Treatment of keratinocytes with 10–6 or 10–5 M of this hexapeptide resulted in the suppression of cell growth, and increased the expression of involucrin and transglutaminase-1. These increased markers of terminal differentiation of keratinocytes indicated the promotion of the re-epithelialization step of wound healing.43

In another study, Soon and coworkers prepared a double-temperature responsive, mixed micelle containing two ELP block copolymers, of which one of the blocks contained the N-terminal fibrinogen binding tetrapeptide Gly–Pro–Arg–Pro. Upon micelle formation, this construct was able to bind fibrinogen at ambient temperature, but not at physiological temperature. The modular nature of this system can be used for developing in vivo depot systems that will only be triggered to release the drug in situ upon specific stimuli.44 Thus, this system can be used for targeted delivery of clot promoting drugs, like batroxobin.

3.1.4. Zuotin-mimetic peptide-based hemostats

Zuotin-mimetic peptide-based hemostats mimic the yeast protein zuotin (zDNA binding protein), and have alternating positively and negatively charged amino acid residues, e.g., +–+–+–+– or ++––++–– etc.45 A class of self-assembling peptides has been developed, termed as self-complementary ionic peptides, termed as self-complementary ionic peptides, like RADA-16 having sequence (Arg–Ala–Asp–Ala)4, peptide KLD12 having sequence AcNH–Lys–Leu–Asp–Leu–Lys–Leu–Asp–Leu–Lys–Leu–Asp–Leu–COONH2 and peptide EAK16 having sequence (Ala–Glu–Ala–Glu–Ala–Lys–Ala–Lys)2.46,47 These peptides having alternating hydrophilic and hydrophobic amino acid residues adopt a β-structure, and also form hydrogels due to changes in the ionic strength or pH (by addition of salts or buffers).47 Ellis-Behnke and co-workers previously reported the utilization of this kind of hydrogel to accomplish the complete hemostasis with less time.48,49 Additionally, these are viable, biocompatible, easy to prepare, and less expensive. These materials create nanofiber boundaries to accomplish the hemostatic process promptly when applied at different injured sites, like the brain, liver or skin of vertebrates. An amphiphilic self-assembling peptide RADA-16, having sequence (Arg–Ala–Asp–Ala)4 (Fig. 3c), has shown to be exceedingly viable in in vivo blood clotting after it has been applied directly to the wound area.50,51 The utilization of (RADA-16/HA)200 [((Arg–Ala–Asp–Ala)4/HA)200] (HA – hyaluronic acid)-coated gauze on porcine skin wounds illustrates that these scaffolds can quicken hemostasis in vivo, and show a promising way of making a cost-effective, biodegradable, biocompatible and strong hemostatic bandage.52

3.1.5. Fibrin-inspired peptide-based sealants

S. Ghosh et al. have developed fibrin-inspired peptide-based sealants, described in Table 3. Table 3 also describes three ionic sealants made of peptides having complementary charges. Sealant 2 was formed by an equimolar mixture of peptides 2 and 3 in the presence of transglutaminase (TG) and Ca2+ ions at pH 7.4. Peptide 2 has 12 Asp residues and 2 Gln residues at the 6th and 13th positions, whereas peptide 3 has 14 Lys residues.53 The Gln residues were incorporated in peptide 3 to engineer intermolecular isopeptide linkages with the Lys residue of peptide 2. Such an intermolecular isopeptide bond is also observed in fibrin. Sealant 3 has a protease-stabilized DAla residue. Sealant 3 was studied to examine the effect of protease-resistance in the designed sealant, and also to study how the positional changes of the isopeptide linkages can affect the molecular self-assembly in sealant 3 compared to sealant 2. FE-SEM, TEM and AFM data confirm the fibrin-like networked structure in the designed sealants (Fig. 4).53 The ionic sealant, sealant 2, has exhibited much higher nano-mechanical force (22 ± 2.7 nN) compared to fibrin (13.6 ± 1.3 nN). The ex vivo Hayem method demonstrates that the designed sealant 2 (∼28 s) exhibits much faster sealing ability than natural fibrin (∼56 s) and entraps blood corpuscles, like fibrin, and is biodegradable (Fig. 4). The presence of the isopeptide bond was confirmed by two short model peptides by LC-ESI MS/MS studies. Isopesptide bonds between two sealant peptides can have both cis and trans orientations. Based on the isopeptide bond forming cross-linking sites, the pattern of the molecular self-assembly can differ among the sealant systems (Fig. 5).53 Such designed enzymatically modified peptide-based self-assembled sealant is biocompatible, as well as biodegradable, and has enormous potential to be translated in the clinics.

Table 3. Sealants and their nano-mechanical force values53.
Sealant code no. Peptide code no. Sequences Nano-mechanical force (nN)
Sealant 1 Peptide 1 H–Gln–Gln–His–His–Leu–Gly–Gly–Ala–Lys–Gln–Ala–Gly–Asp–Val–OH 12.3 ± 4.2
Sealant 1a Peptide 1a Ac–Ala–Lys–Ala–Gln–His–Val–NH2 8.2 ± 0.45 (S. Ghosh unpublished data)
Sealant 2 Peptide 2 H–Asp–Asp–Asp–Asp–Asp–Gln–Asp–Asp–Asp–Asp–Asp–Asp–Gln–Asp–OH 22 ± 2.7
Peptide 3 H–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–Lys–OH
Sealant 3 Peptide 4 H–DAla–Asp–DAla–Gln–DAla–Asp–DAla–Asp–DAla–Asp–DAla–Gln–DAla–Asp–OH 18.6 ± 1.3
Peptide 5 H–DAla–Lys–DAla–Lys–DAla–Lys–DAla–Lys–DAla–Lys–DAla–Lys–DAla–Lys–OH
Fibrin Fibrinogen protein 13.6 ± 1.3
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Fig. 4. FE-SEM images (scale bar, 2 μm) of (a) blood corpuscles, (b) fibrin with blood corpuscles, (c) sealant 1 with blood corpuscles, (d) sealant 2 with blood corpuscles and (e) sealant 3 with blood corpuscles. Two different ex vivo clotting time methods are shown here: (i) Hayam method and (ii) thrombin clotting time. Photographic images in the inset show the ex vivo clotting time of (i) sealants with indigenous fibrin-free blood corpuscles by Hayem method and (ii) sealants with plasma by thrombin clotting time method. FE-SEM images (scale bar, 1 μm) (f) show the gradual disruption of the sealant 2 network in the presence of trypsin at different time points (0 min, 10 min and 240 min). These figures (a–e) are reproduced from ref. no. 53 with permission from Springer Nature, copyright 2017 (https://creativecommons.org/licenses/by/4.0/).53.

Fig. 4

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Fig. 5. The orientations (a) of two possible isopeptide bond formations between the two peptide molecules: (i) cis and (ii) trans. Cross-linked sealant systems (b): (iii) sealant 1-cis formed as the peptide 1 dimer, (iv) sealant 1-trans formed as the peptide 1 trimer, (v) sealant 2-trans type 1 formed as a peptide trimer (two peptide 2 molecules and one peptide 3 molecule), (vi) sealant 2-trans type 2 formed as a peptide trimer (two peptide 3 molecules and one peptide 2 molecule), (vii) sealant 3-cis type 1 formed as a peptide trimer (two peptide 4 molecules and one peptide 5 molecule) and (viii) sealant 3-cis type 2 formed as a peptide trimer (two peptide 5 molecules and one peptide 4 molecule). These figures are reproduced from ref. no. 53 with permission from Springer Nature, copyright 2017 (https://creativecommons.org/licenses/by/4.0/).53.

Fig. 5

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3.1.6. Snake venom-inspired peptide-based hemostat

Snake venoms are complex mixtures of biologically active proteins and peptides. Many of them strongly interact with diverse proteins of the blood coagulation cascade, and affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting the endothelium.54,55 Thus, it has become an exciting area of developing drugs with promising hemostatic activities. Kumar et al. developed a peptide-based nanofibrous snake venom-based hemostat SB50.56 It is composed of a self-assembling peptide hydrogel (termed SLac) that, on its own, can act as a physical barrier to blood loss. SLac is a multidomain peptide (MDP) having sequence Lys–Ser–Leu–Ser–Leu–Ser–Leu–Arg–Gly–Ser–Leu–Ser–Leu–Ser–Leu–Lys–Gly–Arg–Gly–Asp–Ser. SLac has a cell adhesion sequence (Arg–Gly–Asp–Ser), as well as an MMP-2 cleavage site having sequence (Leu–Arg–Gly). The cell adhesion sequence (Arg–Gly–Asp–Ser) in the hydrogel facilitates cell adhesion. The MMP-2 cleavage site having sequence (Leu–Arg–Gly) facilitates proliferation through the extracellular matrix (ECM). They loaded SLac with snake venom-derived batroxobin (50 μg mL–1) peptide, and prepared a drug-loaded hydrogel (SB50). Unlike thrombin, the clotting ability of batroxobin was not inhibited by heparin. The nanofibers could be syringe-loaded and delivered via needle or transcatheter. Batroxobin-loaded hydrogels rapidly stopped bleeding (within 20 s) in both normal and heparin-treated rats in a lateral liver incision model. SLac loaded with drugs has shown excellent local and systemic biocompatibility, rapidly infiltrating with host cells that secrete native matrix, and resolve over a period of 2–3 weeks in vivo.

3.2. Peptide-based intravenous hemostat

Managing internal bleeding specifically at the site of injury is far more challenging than the external ones. Several groups have demonstrated that injectable peptide-based sealants, blood-derived or synthetic injectable particles, and peptide-functionalized liposome or polymer-based particles can facilitate internal blood clotting.57,58 Such particles should enhance the body's own coagulation system by interacting with the coagulation cascade. Many research groups have tried to handle compressible (injury site where direct pressure or tourniquet can be applied) and non-compressible hemorrhages (an internal bleeding site where direct pressure cannot be applied) by using injectable particles that can interact easily with platelets or clotting cascades only at the site of injury (Fig. 6).

Fig. 6. Strategies for designing intravenous hemostats.

Fig. 6

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3.2.1. Thromboerythrocytes

Coller et al. first reported the use of the thromboerythrocytes type of particles for promoting rapid hemostasis.59,60 Thromboerythrocytes are an RGD sequence (Ac–Cys–Gly–Gly–Arg–Gly–Asp–Phe–NH2) containing fibrinogen-mimetic peptide-functionalized erythrocytes. Erythrocytes were functionalized with such peptides instead of fibrinogen to avoid the risk of infectious materials. Such RGD-containing peptides are smaller than the fibrinogen protein, which indicates that significant multivalent interactions will be observed in the case of peptide-functionalized erythrocytes (∼0.5–1.5 × 106 peptides per erythrocyte) compared to fibrinogen-functionalized erythrocytes (∼58 fibrinogen per erythrocyte).59,60 It is reported that nearly 80 000 GPIIb–IIIa receptors are there per platelet, and approximately 40 000 fibrinogens can reportedly bind to activated platelets. Activated platelets have a stellate shape, and can bind to any of the three peptide domains in fibrinogen (RGD motifs: RGDF, RGDS; H12 sequence: His–His–Leu–Gly–Gly–Ala–Lys–Gln–Ala–Gly–Asp–Val).59,60 The significant contribution of this work is that this group has demonstrated that RGD-functionalized sealants have the potential to specifically perform on-demand sealing of the blood at the site of any internal injury.

3.2.2. Platelets

Platelets are the first candidate to initiate the hemostatic mechanism upon vascular injury, and platelet substitutes exist as a significant class of intravenous hemostats. Although platelet transfusion has remained a therapeutic strategy to cease bleeding, the short shelf life (less than a week) of platelets limits their function. To address the issue, platelets isolated from whole blood by differential centrifugation can be lyophilized and further injected intravenously for the treatment of internal hemorrhages. In the pig liver injury model, this showed improved hemostasis and reduced blood loss in comparison with the control group.61 In the ear bleeding model of a thrombocytopenic rabbit (rabbit with low platelet count), this showed no improved efficacy compared to the control group.62 To overcome the associated risk of disease transmission and limited efficacy of the lyophilized platelets, the concept of a synthetic platelet was introduced.

Bertram et al. prepared synthetic platelet peptide conjugates for intravenous hemostasis.63 They have synthesized polymer PLGA (poly(lactic-co-glycolic acid))-based platelets and conjugated them with the RGD (Arg–Gly–Asp) peptide sequence. The rat femoral artery model showed that the bleeding time for synthetic particles was effectively less than the control saline. They also showed a prominent amount of clot excised from the injured area after cessation of bleeding by scanning electron microscopy images. They injected 20 mg mL–1 doses, which showed no complications for up to 7 days. The Lavik group reported on synthetic platelets using poly(lactic acid), instead of PLGA (poly(lactic-co-glycolic acid)) and the RGD (Arg–Gly–Asp) peptide as a binding domain.64,65 This material is stable at temperatures up to 50 °C, and for 7 days without compromising its hemostatic ability. Anselmo et al. used platelet-like nanoparticles (PLN) using spherical polystyrene nanoparticles, and coated them with four bilayers of poly(allylamine hydrochloride) (PAH) and bovine serum albumin (BSA). PLNs functionalized with wound-specific ligands showed high adhesion to collagen and vWF. Again, PLN functionalized with fibrinogen-interacting peptides having sequence Gly–Arg–Gly–Asp–Ser exhibited high specific adhesion. They also found that compared to the spherical and rigid discoidal counterpart, PLNs exhibited increased surface binding.66 Brown et al. reported a fully synthetic platelet-like particle (PLP) composed of ultralow crosslinked (ULC) poly(N-isopropylacrylamide-co-acrylic acid) microgels with a molecular recognition motif that increased clotting in vitro, and wound-triggered hemostasis was observed with decreased bleeding time.67 Nandi et al. also prepared ULC poly(N-isopropylacrylamide-co-acrylic acid) microgels to mimic the morphology of activated platelets; they found that PLPs influenced the wound healing mechanism in a murine model having dermal injury.68

3.2.3. Liposomes

Liposomal nanoparticles are primarily used as a drug delivery agent. Okamura et al. formulated a nanoparticle named H12-(ADP)-liposome. Here, they conjugated an ADP-containing liposome with a fibrinogen mimetic dodecapeptide H12 having sequence His–His–Leu–Gly–Gly–Ala–Lys–Gln–Ala–Gly–Asp–Val. This nanoparticle showed better efficacy to cease the bleeding in thrombocytopenic (deficiency of platelets in the blood) rat and rabbit bleeding models. The H12 dodecapeptide facilitates platelet aggregation and hemostatic plug formation, whereas the ADP-encapsulated liposome mediates the aggregation-dependent controlled release of ADP at the site of injury to further enhance the blood clot.69,70 Dyer et al. have developed SynthoPlate liposomes, which are made of collagen and functionalized with von Willebrand factor-binding peptides (to mimic platelet adhesion) and fibrinogen-mimetic peptide (to promote platelet aggregation).71 This synthetic platelet (SynthoPlate) has reduced blood loss compared to the control in a mouse liver model with non-compressible intraperitoneal hemorrhage (internal bleeding in the peritoneal cavity, which cannot be controlled by compression. The peritoneal cavity is a small area of space located between the internal abdominal organs and the inner abdominal wall).

3.2.4. Tissue factor (TF)-targeted peptide-based amphiphiles

To stop noncompressible bleeding in a targeted way and to circumvent the undesirable off-target response, Morgan et al. covalently conjugated self-assembling peptide amphiphiles to the tissue factor binding sequence RTL (Arg–Thr–Leu–Ala–Phe–Val–Arg–Phe–Lys) and administered it intravenously.72 The TF binding sequences were derived from the linear sequences of factor VII. They have demonstrated that peptide amphiphiles formed a nanofibrous structure, and the TF targeting peptides initiated platelet aggregation with a significant reduction in blood loss (60%) in the in vivo rat liver injury model.

4. Conclusion and future direction

Over the past several decades, protein-inspired peptide-based designed sealants have been developed. Their physicochemical and functional characterizations have demonstrated their outstanding potential as tissue sealants both in vitro and in vivo. Despite their limited initial success, there are still many challenges. Many such tissue sealants are not clinically safe. They are also not suitable as non-invasive or minimally invasive surgical sealants that can bind strongly to wet tissue at the injured site, and perform very efficient on-demand hemostatic seals. Additionally, different types of tissues have different surface amine contents, different environmental pH in which the tissue resides, different extracellular matrix compositions and different immunological niches. Due to the diversity of tissues and the diversity of wound etiologies of patients, it is not feasible to develop a universal tissue sealant that is suitable for all situations, which could be effective in healing all tissues and also in performing efficient suturing. Suturing is a time-consuming and invasive procedure, and exhibits technical challenges due to tissue penetration and compression. Another challenging aspect is developing a sealant for internal clotting and on-demand sealing without causing any health hazard due to market-available sealing agents. Efficient total wound care requires both blood clotting and wound healing. Millions of people around the world are affected by the poor wound healing of diabetic patients and of wounds created after trauma, surgery or chronic skin disease conditions or burn wounds. Until today, the cellular signaling and molecular mechanism of wound repair and regeneration have been poorly understood. Consequently, efficient clinical care for tissue-specific or patient-specific treatment options is currently missing. Emphasis should be given to understand the detailed wound repair mechanism and signaling pathways of engineered peptides having proangiogenic functionality. Furthermore, a detailed investigation of the pathophysiology of non-healing wounds from tissue sample analysis of patients will facilitate the development of tissue-specific and patient-specific wound care.

Conflicts of interest

There are no conflicts of interests.

Acknowledgments

We thank Prof. Dhananjay Bhattacharyya (SINP) for his approval for reproducing figures from ref. 53 with permission from Springer Nature, copyright 2017 (https://creativecommons.org/licenses/by/4.0/). Authors thank Ms. Kasturee Chakraborty, Dr. Sanchita Mukherjee, Mr. Somnath Jan, Mr. Abhijit Biswas, Mr. Argha Mario Mallick and Mr. Sukumar Mishra for their help, suggestions and insightful discussions. RSR thanks DBT (BT/PR27059/NNT/28/1543/2017), SG thanks CSIR for Ph.D. fellowship, AT and PG thank IISER Kolkata for Ph.D. fellowship.

Biographies

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Snehasish Ghosh

Snehasish Ghosh received his M.Sc. degree in Organic Chemistry from R.T.M Nagpur University in 2010. He recently finished his Ph.D. under the supervision of Dr. Rituparna Sinha Roy at Indian Institute of Science Education and Research Kolkata. He has worked on designing peptide-based sealants for facilitating blood clotting.

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Archana Tripathi

Archana Tripathi received her M.Sc. degree in Human Physiology from the University of Calcutta in 2017. She is currently pursuing her Ph.D. under the supervision of Dr. Rituparna Sinha Roy at Indian Institute of Science Education and Research Kolkata. Her research focuses on designing peptide-based therapeutics against cancer.

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Paramita Gayen

Paramita Gayen received her M.Sc. degree in Biotechnology from Jadavpur University in 2015. She is currently pursuing her Ph.D. under the supervision of Dr. Rituparna Sinha Roy at Indian Institute of Science Education and Research Kolkata. Her research focuses on designing peptide-based therapeutics for faster blood clotting and wound healing.

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Rituparna Sinha Roy

Rituparna Sinha Roy received her Ph.D. from Indian Institute of Science, Bangalore in 2005, followed by postdoctoral research at The Scripps Research Institute, San Diego from 2005 to 2007, and at Harvard Medical School from 2007 to 2011. She is currently an Associate Professor at the Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata. Her research focuses on designing peptide-based regenerative medicine and peptide-based therapeutics against cancer.

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