Without adaptive immunity, invertebrates have evolved innate immune systems that react to antigens on the surfaces of pathogens. These defense mechanisms are included in horseshoe crab hemocytes’ cellular responses to pathogens. Secretory granules, large (L) and small (S), are found on hemocytes. Once the invasion of pathogens is present, these granules release their contents through exocytosis. Recent data in biochemistry and immunology on the granular constituents of granule-specific proteins are stored in large and small granules which are involved in the cell-mediated immune response. L-granules contain most clotting proteins, which are necessary for hemolymph coagulation. They also include tachylectins; protease inhibitors, such as cystatin and serpins; and anti-lipopolysaccharide (LPS) factors, which bind to LPS and agglutinate bacteria. Big defensin, tachycitin, tachystatin, and tachyplesins are some of the essential cysteine-rich proteins in S-granules. These granules also contain tachycitin and tachystatins, which can agglutinate bacteria. These proteins in granules and hemolymph act synergistically to fight infections. These biomolecules are antimicrobial and antibacterial, enabling them to be drug resistant. This review is aimed at explaining the biomolecules identified in the horseshoe crab’s hemolymph and their application scopes in the pharmaceutical and biotechnology sectors.

1. Introduction

Horseshoe crabs are aquatic arthropods from the Animalia kingdom, Arthropoda phylum, Xiphosura or Xiphosurida order, and Limulidae family. Around 400 million years ago, a horseshoe crab species roamed on the Earth, at least 200 million years older than the dinosaurs [13]. Currently, four horseshoe crabs exist on the Earth though they are restricted to specific regions. Limulus polyphemus is called Atlantic horseshoe crab. The North American continent’s Atlantic coast is home to the Limulus polyphemus. The Tachypleus tridentatus, which is called trispine horseshoe crab, can be found along the Western islands of the Philippines as well as the Northern beaches of Japan and South Vietnam. Carcinoscorpius rotundicauda known as the mangrove horseshoe crab is a crab species found in the northern part of the Bay of Bengal. The Bay of Bengal is where Tachypleus gigas is predominantly found [47]. Figure 1 depicts the global distribution of living and extinct horseshoe crab species.

Mostly, horseshoe crabs are captured for biomedical and pharmacological applications due to their extraordinary blue hemolymph properties. Prosoma, opisthosoma, and telson are 3 parts of horseshoe crab body [2]. There are seven pairs of appendages on the concave ventral side of the prosoma. The first four pairs of walking legs help with the breakdown of food. It has four leaf-like legs stretching out when the appendages are forced against the ground, either pushing sand back or propelling the animal ahead. The ventral side of the abdomen is lined with 6 pairs of membrane appendages. Five pairs of book gills provide the natural surface for gas exchange. As a weapon, the tail spike is used for swimming, correcting, and digging rather than articulating with the posterior abdomen [3]. The horseshoe crab’s distinct body sections are shown in Figure 2.

Amebocytes or granulocytes are special type of cell that are susceptible to bacterial endotoxins found in the horseshoe crab hemolymph [48]. Large (L) granules which are less dense and smaller (S) granules which are dense are two types of granules which fill the cytoplasm of the hemocyte which is shown in the electron micrograph [9, 10]. LPS-induced exocytosis in hemocyte releases granular components into hemolymph plasma. Immunocytochemical examination of these granules revealed three clotting factor including factor C [11, 12] which is a proclotting enzyme [11, 1317]. L- and S-granules store biologically active substances and function as a host defense mechanism against pathogens [10, 18].

Limulus amebocyte lysate and tachypleus amebocyte lysate from horseshoe crab blood are blessings widely used in the pharmaceutical business to determine endotoxin [20]. Bacterial endotoxin test follows the sensitivity of 0.005 EU/ml, which amounts to 0.0005 to 0.001 ng/ml [21]. US Pharmacopeia and FDA have approved the test as an acceptable endotoxin testing strategy for liquid suspensions, biomedical devices [22], drug delivery strategies, and orthodontic procedures [23, 24]. LAL is a widely preferred assay for the testing the endotoxin clinically and environmentally [25]. Furthermore, the rise of pathogens immune to numerous antibiotics has put the health of populations in danger, and horseshoe crabs may be a potential source of antibacterial peptides [26]. To the safe fabrication of immunizations and injectable medications, the horseshoe crabs have become vital for last 40 years [5].

The article is aimed at exploring the remarkable properties of horseshoe crab biomolecules, which are linked to their hemolymph, and their potential applications as biologics and therapeutics in the biomedical and pharmaceutical fields.

2. Source of Information

Information from recent literature (no time restriction) was acquired from the Google Scholar, PubMed, and Scopus databases to choose the material on horseshoe crab and biomolecules of its hemolymph for this review. The horseshoe crab, roaming of horseshoe crab, horseshoe crab’s blood components, immune system of the horseshoe crab, biomedical use of horseshoe crab, LAL kit, TAL kit, COVID-19, and horseshoe crab hemolymph and endotoxin were considered as keywords for the literature search. Protein diagrams have been obtained from Protein Data Bank (PDB), and BioRender and MS PowerPoint have drawn other illustrations.

3. Features of Horseshoe Crab’s Hemolymph

To counter microbial invasion successfully, horseshoe crabs have evolved a new defense system over hundreds of millions of years [27]. The horseshoe crab’s innate immune system is concentrated mainly in the hemolymph, which contains hemocytes or amebocytes [15]. Hemolymph includes two types of hemocytes: granular and nongranular cells [28]. The predominant hemocyte is the amebocyte (Figure 3) [27]. Large and small granules are distinguished by electron microscopy due to their distinctive electron concentration [29]. Table 1 describes the different features of large and small granules.

The hemolymph contains mainly three types of proteins; they are hemocyanin, C-reactive protein, and α2-macroglobulin [30, 31]. Table 2 represents the hemolymph of different species of horseshoe crab.

Additionally, horseshoe crab blood contains plasma and cell-free hemolymph (CFH). The blue hemolymph fluid is called the cell-free hemolymph (CFH). CFH contains hemocyanin (HMC), a predominant (90-95%) protein enriched with blue copper ion which is the reason behind the horseshoe crab’s blue blood. Only the HMC can generate microbicidal reactive oxygen intermediates from prophenoloxidase (proPO). HMC-proPO is converted to PO by extracellular microbial proteases during host-pathogen interactions. The innate immune system effectors are located in the CFH that control humoral antimicrobial defense and bacteria response. Complement elements, CRP isoforms, and lectins are examples of innate immune components with evolutionary roots [27].

4. Coagulation Reaction of Horseshoe Crab’s Hemolymph

Coagulation occurs because of the presence of LPS secreted by the Gram-negative bacteria. It starts with three serine protease zymogens: proclotting enzyme, factor C, and factor B, resulting coagulogen, a clottable protein. Factor C (123 kDa) is an LPS-responsive biosensor [11, 12, 33, 36, 37]. In the presence of LPS or synthetic lipid A analogs, it is autocatalytically converted to an active form, factor C. The activation of factor B (64 kDa) by factor C results in its active form (factor B), turning proclotting enzyme into a clotting enzyme (54 kDa) [35, 3840]. The active clotting enzyme converts coagulogen to an insoluble gel known as coagulin. Although the actual process by which this coagulin gel form is unexplained, a recent compositional analysis of the Tachypleus tridentatus’s coagulogen provided a foundation for understanding the crosslinking mechanism [41]. Sometimes due to fungal infections, bacterial endotoxin test (BET) shows pseudopositive result caused by glucans. A potential beta-D-glucan-sensitive protease zymogen in a hemocyte lysate was discovered in 1981 [42]. Since then, the instability of this protein is known as factor G. This beta-D-glucan-mediated coagulation mechanism may be activated on the surface of fungi [43].

The Limulus intracellular coagulation inhibitors, LICI-1, LICI-2, and LICI-3, are three kinds of serpins identified from the hemocytes [4446]. Each and every LICI belongs to the serpin family and creates complex compounds with the target serine proteases. LICI-1 exclusively inhibits factor C, whereas LICI-2 and LICI-3 suppress factor C, factor G, and activity of the clotting enzyme. LICI-2 has a more substantial inhibitory effect on the clotting enzyme, but LICI-3 favors factor G over other enzymes. One of the antibacterial compounds, big defensin (Figure 4), is copurified with LICI-1 and interacts only with LICI-1, not with LICI-2 or LICI-3 [44].

5. Biomolecules of Horseshoe Crab Hemolymph

Granular hemocytes, which make up 99% of all hemocytes in horseshoe crabs, are responsible for the storage and release of several defensive chemicals, such as the anti-LPS factor, clotting protein coagulogen, protease inhibitors, serine protease zymogens, lectins, and antimicrobial peptides. Table 3 and Figure 5 summarize the defense or biomolecules found in horseshoe crab hemocyte and hemolymph plasma.

5.1. Anti-LPS Factor

Anti-LPS factor (102 amino acid residues) and tachyplesins (17 residues for tachyplesin I) or polyphemusins (18 residues for polyphemusin I) were first found in hemocytes as defensive molecules that negate a range of LPS activities [13, 1517, 4857]. The anti-LPS factor is a molecule in the shape of a disc that has a great charge distribution and is amphipathic. It has a single domain with three α-helices packed against a four-stranded sheet. Gram-negative bacteria cannot grow because they can attach to lipids, which is most likely why this works [49]. Tachyplesin, a peptide with an amphiphilic structure, increases bacteria’s permeability to potassium, including S. aureus and E. coli [50, 51].

5.2. Big Defensin

Big defensin (Figure 6) is a peptide present in both large and small molecules [59]. Gram-negative and Gram-positive bacteria and fungus like Candida albicans are all inhibited by this chemical (Table 4). The isolated molecule, coined “big defensin,” is constructed with 79 amino acid residues [60]. However, big defensin differs in size from mammalian defensins, which typically comprise just 29–34 residues [61].

5.3. Tachycitin

Tachycitin (Figure 7) is a 73-amino acid protein that has five disulfide bridges but no N-linked sugars [63]. Although tachycitin has only mild antibacterial properties on its own, it dramatically enhances the antimicrobial action of large defensin. In the presence of a little amount of tachycitin, the concentration of big defensin required to inhibit the development of Gram-negative bacteria by 50% (i.e., the half-maximal inhibitory concentration [IC50]) is decreased to one-fiftieth of its normal value (Table 4) [64]. Chitin is a crucial protein which is a basic structural unit of fungi as well as the cell wall of bacteria. They may stimulate and accelerate wound healing [58].

5.4. Tachystatins

A novel tachystatin family with extensive antibacterial effectiveness against Gram-negative and Gram-positive bacteria and fungi was discovered by Osaki et al. in 1999. The most potent of these tachystatins is tachystatin C (Table 4). Tachystatin A is homologous to tachystatin B (Figure 8), although the two proteins’ sequences do not share a lot of similarities [65].

5.5. Factor D

Copurified with the separation of horseshoe crab serpins from hemocyte lysates was a novel antimicrobial glycol protein of 43 kDa designated as factor D. Factor D is composed of 394 amino acids and a signal sequence. L-granules of hemocytes contain factor D [67].

5.6. α2-Macroglobulin

In the plasma of the American horseshoe crab, L. polyphemus, they are the third most common protein. Horseshoe crabs have α2M in their plasma and red blood cells [68, 69]. Limulus α2M has a lot in common with mammalian α2Ms [70]. A cDNA that codes for Limulus α2M has a 25 amino acids signal sequence at the NH2-terminus, and the protein it makes is 1,482 amino acid long [71]. It has a similar structure to the human complement factor C8 chain, which is consistent with Limulus α2M playing a role in host defense [58].

5.7. Transglutaminase

Therefore, it is anticipated that a TGase may participate in crosslinking the coagulin gel and in the immobilization of invasive microbes in the horseshoe crab clotting system [72]. The 8.6 kDa and proline-rich proteins are essential in hemocytes’ L-granules, although horseshoe crab TGase is cytosolic. The crosslinking of coagulin or microbial cell walls with other proteins may be facilitated by TGase [58].

5.8. Factor C

Factor C is linked to the amebocyte membrane and activated by LPS in vivo. This causes a signal transduction cascade involving G-protein-coupled receptors (GPCRs), which causes the amebocyte to degranulate and release defensive chemicals, such as the zymogens of the coagulation cascade [73]. It is roughly 38 kDa in size [74]. Higher concentrations of the proclotting enzyme protein and factor C are linked to increased LAL reactivity [75].

5.9. Coagulogen

In horseshoe crabs, the coagulation cascade is made up of a clottable protein coagulogen (Figure 9) and four serine protease zymogens, which include factor C, factor B, factor G, and the proclotting enzyme. LPS and β-1,3-glucans of fungal cell wall components act as biosensors for factor C and factor G, which cause the coagulation factors to be sequentially activated, resulting in the conversion of coagulogen to coagulin [76].

Pathogen-associated compounds in horseshoe crab encourage the rapid formation of a gel generated by the cleavage of coagulogen into coagulin, which then interacts with proxins to build a matrix that immobilizes the pathogen in a network of hemocytes and coagulin polymers [41, 77]. The three-dimensional composition of coagulogen shows a major polymerization mechanism in which the release of the helical peptide C exposes a hydrophobic cove on the “head,” which interacts with the water-insoluble edge or “tail” of another molecule, leading to the formation of a coagulin homopolymer [78].

5.10. CRP

The horseshoe crab, Carcinoscorpius rotundicauda, has a great deal of CRP in its blood [79]. In the species Tachypleus tridentatus from Japan, the CRP families are called CRP-1, CRP-2, and CRP-3 [58, 80]. In the American species Limulus polyphemus, CRP-2 is known as limulin. The CRP functional criteria are met because CRP-1 and CRP-2 bind phosphorylcholine in a way that depends on calcium [79]. Pure horseshoe crabs CRP-1, CRP-2, and CRP-3 did not affect E. coli K12, Enterococcus hirae, Micrococcus luteus, or Staphylococcus aureus 209P in terms of clumping or stopping growth [80]. CRP is essential to the first line of defense against infections. It was found that when CRP is tested with plasma or hemolymph, it binds to a broader range of bacteria than when CRP is tested on its own [79].

5.11. Cystatin

Cystatin may be essential for biological defense mechanisms against invaders and shielding cells from unwanted proteolysis by intracellular and extracellular cysteine proteases. Limulus (L) cystatin, a single-chain protein with 114 amino acids and a molecular weight of 12.6 kDa, was isolated from the Japanese horseshoe crab. It was discovered by immunoblotting to be present in hemocyte’s L-granules. Cystatin formed from L-granules cooperates with other defense compounds produced in response to external stimuli to effectively resist invasive pathogens [58].

6. Impact of Horseshoe Crab Blood on the Biomedical Industry

Horseshoe crab blood has unique qualities that benefit the biomedical sector. That is why it has broad range of use.

6.1. LAL Test

Limulus amebocyte lysate (LAL) is an integral feature of the horseshoe crab’s innate immune system [81]. The activated components of LAL originate in the amebocytes of the horseshoe crab. Active ingredients are released when the amebocytes are broken or lysed. Possessing (1,3)-β-D-glucan lysate can identify Gram-negative bacteria and fungi [42]. There are two main methods for endotoxin detection: the in vitro pyrogen test (IPT) and the test Limulus amebocyte lysate (LAL) [82].

LAL is manufactured by “bleeding” adult horseshoe crabs of both sexes and isolating the amebocytes from the plasma or hemolymph. It is worth noting that commercial LAL can be different depending on the brand or manufacturer. Due to manufacturing variations, there are qualitative and quantitative discrepancies among LAL brands. The assessment can be evaluated in various ways, and the manufacturing technique will vary depending on the type of analysis selected. Levin and Bang introduced the approach [4]. Figure 10 shows the diversified uses of LAL test.

6.1.1. LAL Test in Radiopharmaceutical, Biopharmaceutical, and Pharmaceutical Industry

The LAL test has been shown to be reliable since its establishment. Particularly for radiopharmaceuticals, the LAL test has demonstrated its accuracy. Recombinant drugs are produced through genetic engineering approaches derived from living organisms. LAL is used as a tool to measure the purity of the recombinant drugs because mostly the sources of the drugs are bacteria, fungi, and different types of cell lines where the growth medium, fungi, or bacteria are the potential sources of endotoxin. Gram-negative bacteria and endotoxins easily contaminate water. That is why in the pharmaceutical industry, water is the substance that has the most LAL tests because it is used in all drugs and devices, either as an integral component or as a processing agent. The level of the FDA and the USP permits the level of endotoxin 0.25 endotoxin units (EU) ml-1. Water testing for renal dialysis is a particular type of testing which is followed by the kidney dialysis industry. Concern about endotoxin toxicity is equal to the production of injection water as kidney dialysis water. The water used to make intravenous drugs must already have low levels of endotoxin. Individual components and finished products are tested with LAL to ensure that the intravenous solution and its container meet the endotoxin limit [25].

Pharmaceuticals classified as biologicals are those derived from compounds taken from people and animals, such as clotting factors and insulin. Additionally, biologicals include vaccinations that may contain bacterial or animal components, such as chicken eggs. It is well established that Gram-negative bacteria may easily infect biologicals, resulting in batches containing high levels of endotoxin. Fortunately, biologicals are often given in small amounts and are frequently injected intramuscularly. Nonetheless, in 1976, following severe reactions to a new batch of swine flu vaccination, it was established, in one of the early applications of the LAL test, that the batch of vaccine included an abnormally high quantity of endotoxin that was causing the bad effects [25].

6.1.2. LAL Test in the Biomedical Industry

Syringes, catheters, and other medical devices like needles are frequently made using extremely clean manufacturing processes. Implanted devices, such as porcine heart valves or orthopedic implants with intricate manufacturing processes, might, however, include endotoxins at levels that lead to localized inflammation and ultimately the rejection of the implant. In these circumstances, it is crucial that LAL evaluates the devices [25].

6.2. Utility of Horseshoe Crab Blood in COVID-19

Horseshoe crab blood is essential to the story of scientific progress that spans the 1960s and continues right up to the present day when the globe is coping with a pandemic that has claimed more than 3 million lives. COVID-19 vaccinations that are restoring hope for an end to the epidemic are scientific marvels of the twenty-first century [83]. LAL is used to test each antibody test, each batch of vaccine, and each syringe and vial used to administer the COVID-19 vaccinations [84].

7. Future Aspects of the Biomolecules in the Biomedical and Pharmaceutical Industry

In the past 70 years, horseshoe crab blood has gone from curiosity to vital in pharmaceuticals. Not only bacterial endotoxin tests but also diversified pharmaceutical and biomedical products are dependent on horseshoe crab blood. With the advancement of biological research, more sectors are opening for the utilization of horseshoe crab biomolecules.

7.1. Potential Drug, Antibiotics, and Therapeutic Nature of Hemolymph Molecules

The recent surge in the number of drug and multi-drug-resistant microbial pathogens poses a significant worldwide concern. As a result, identifying novel techniques for developing novel anti-infectives and therapeutic targets is one of the top priorities in global health care. According to a recent study, Gram-positive cocci are the leading cause of nosocomial infections. About 16% infections with Staphylococcus aureus and 14% infections with Enterococcus species take the lead. Invasive fungal illnesses have also posed a life-threatening hazard to people with impaired immune systems because they are difficult to identify, treat, and prevent [85, 86]. However, some living species encounter illnesses that rely on the host’s defensive system or immunity to survive. Vertebrates spontaneously produce antibodies, a kind of security and safety known as innate immunity. Immunity against infectious pathogenesis is mainly developed by ingesting antibodies, commonly known as acquired immunity. Antimicrobial peptides (AMPs) have potent antibacterial action against many bacteria that cause illnesses. As a result, AMPs have lately been regarded as a potential class of antibiotics [66, 87]. Among the invertebrate species, horseshoe crab’s hemolymph can be an enriched source of antimicrobial peptides. Table 2 shows that the antimicrobial activity of this protein is stronger against Gram-negative bacteria than against Gram-positive bacteria [88].

Table 5 describes potential drugs, antibiotics, and therapeutic effects of hemolymph molecules of horseshoe crab. Figure 11 evolves some bases of potential antibiotic components obtained from horseshoe crab’s blue blood.

7.2. Endotoxin Detection Biosensor

The LAL test may have drawbacks like poor stability, high cost, and inconvenience. To address these issues, a variety of novel methods—including electrophoresis, fluorescence, chemiluminescence, and electrochemical methods—have lately been used in the production of endotoxin sensors [99]. Table 6 describes different types of endotoxin detection biosensor.

7.3. Recombinant Factor C

To conserve the horseshoe crab, recombinant factor C (rFC), a synthetic replacement, has been introduced. Factor C molecules from numerous horseshoe crab species have been cloned and thoroughly studied. Using methods of genetic engineering, the recombinant factor C was produced by cloning the DNA of a factor C molecule and serving as a synthetic replacement for the LAL test (rFC). It has demonstrated a broad sensitivity and valid range [5]. The proenzyme activation of rFC is facilitated by minute amounts of endotoxin. The sensitivity of endotoxin detection increases from 0.005 EU ml-1 to 0.001 EU ml-1 when the amount of rFC is increased from 10 g to 80 g. When compared to commercial LAL under the same test conditions, rFC showed a lower background reading and a more sensitive reactivity to endotoxin [105].

Table 7 includes multiple researches on the usefulness of rFC as bacterial endotoxin testing on various samples. The rFC test is quantitative, whereas the LAL test is qualitative or semiquantitative, and the rFC-based assay was equivalent to the LAL test for detecting Gram-negative bacterial endotoxin. LAL is triggered by peptidoglycan from Gram-positive bacteria, exotoxins from group A streptococci, and simple polysaccharides like dithiols, yeast mannans, and bacterial dextrans, which might result in false positive results. Surprisingly, the rFC-based assay is less prone to false positives than the LAL test because rFC lacks glucan-sensitive factor G. Regardless of the vendor, these nine investigations all showed that readily accessible rFC assays detected endotoxins as well as or better than LAL. A wide range of research has demonstrated the effectiveness of rFC. The breadth of these studies also revealed good properties for the pharmaceutical use of rFC, such as high precision, exceptional dependability, and excellent performance across various applications [5].

8. Conclusion

The horseshoe crab’s blue blood has an advanced defense mechanism that is particularly sensitive to infections and other factors. Amebocytes detect a trace amount of LPS molecules by hemolymph coagulation reaction. Protease inhibitors, antimicrobial peptides, proteins, and agglutinins are abundant in the plasma and hemocytes. Recently, antimicrobial peptides have been considered potential candidates for therapeutic anti-infectives due to their fast bactericidal effect. Numerous antimicrobial peptides are now in clinical trials for the treatment of a variety of bacterial infections. Additionally, specific antimicrobial peptides have a broad spectrum of activity, which is beneficial in some therapeutic regions. Antimicrobial peptides obtained from horseshoe crab could be a promising sector for therapeutic and antimicrobial agents, which can play a significant role in the pharmaceutical and biomedical industries.


LAL:Limulus amebocyte lysate
FDA:Food and Drug Administration
CFH:Cell-free hemolymph
LICI:Limulus intracellular coagulation inhibitors
GPCR:G-protein-coupled receptor
CRP:C-reactive protein
AMP:Antimicrobial peptide
DPV:Differential pulse voltammetry
rFC:Recombinant factor C.

Conflicts of Interest

The authors declare that they have no conflicts of interest.