Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.     

A cysteine-rich protein from an arthropod stabilizes clotting mesh and immobilizes bacteria at injury sites

Hemolymph coagulation in arthropods plays key roles in host defense, including sealing wounds to staunch bleeding and immobilizing invading microorganisms. We have previously reported that horseshoe crab transglutaminase (TGase) promotes cross-linking of a clotting protein (coagulin) with hemocyte-derived proteins (proxins), resulting in the formation of stable coagulin fibrils. Here we show that TGase also cross-links proxins to another hemocyte-derived protein named stablin. Stablin is a cysteine-rich protein of 131 residues. Surface plasmon resonance analysis revealed the specific interaction of stablin with proxin-1 at K(d) = 4.0 x 10(-9) m. Stablin was predominantly localized in the large granules of hemocytes and secreted by lipopolysaccharide-induced exocytosis. Interestingly, stablin bound to chitin at K(d) = 1.5 x 10(-8) m, as determined by using a quartz-crystal microbalance. Stablin also interacted with lipopolysaccharides and lipoteichoic acids and exhibited bacterial agglutinating activity against Gram-positive and -negative bacteria. Immunostaining showed that stablin is co-localized with coagulin in the clotting fibrils that effectively trap bacteria. Moreover, an anti-stablin antibody strongly inhibited the proper formation of the clotting fibrils. These data suggest that stablin promotes the formation of the clotting mesh and the immobilization of invading microbes at injury sites. In arthropods, the TGase-mediated cross-linking may play an important role in the initial stage of host defense, wound closure, and healing, as in the case of mammals.     

Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.     

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.     

Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.     

Preparation of vitamin K-dependent proteins,such as clotting factors II,VII, IX and X and clotting inhibitor protein C

A review is given of preparative methods for the isolation of the vitamin K-dependent clotting factors II, VII, IX, X and clotting inhibitor protein C, all derived from human plasma. Factor II, activated factor VII and activated protein C are also obtained from recombinant animal cells. The methods for their purification are described. The problem of difference in posttranslational modifications between plasma derived and recombinant protein is discussed with regard to therapeutic proteins.     

The review of contemporary ideas about the influence of flow rate on blood clotting

Normal blood clotting is vitally important for mammals. The diffusion-convection transfer of clotting factors plays a key role in blood clot formation. Since the shear rates of blood flow are very high (up to 7000 s⁻¹), clot formation critically depends on the flow rate. The methods of study of the flow effect on clotting are indirect and the processes are rather complex; therefore, mathematical models of this process are significant for interpretation of results and understanding of the mechanisms. The review expounds the main experimental data on the effect of flow on the blood clotting cascade, some hypotheses and mathematical models explaining different regimes of the functioning of this system. The review is focused on specific problems encountered by researchers in this field. Some of the experimental works have shown that flow decreases the size of the formed fibrin clot and that the dependence of clot formation period on the flow shear rate is a threshold function. However, there are also experimental evidence that the flow can increase production of clotting factors (factor Xa), which must be expressed in the procoagulant action of the flow. Mathematical models of different aspects of clotting give no unified predictions either. Nevertheless, the combined analysis of results of detailed modeling and experiments, in our opinion, may result in understanding of the mechanisms, which determine the behavior of clotting in a flow.     

Processing and expression of rat and human clotting factor-X-encoding cDNAs

The cDNA encoding clotting factor X, which participates in the middle stage of the blood coagulation cascade was cloned from a rat liver cDNA library. Sequencing of the rat factor-X-encoding cDNA revealed that this vitamin-K-dependent protein has a dibasic Arg-Arg sequence at the propeptide cleavage site, as occurs in other vitamin-K-dependent proteins. Although the human and rat deduced amino acid sequences are remarkably similar (76% identical), they do significantly differ in that human factor-X contains a unique Thr-Arg sequence at the propeptide cleavage site [Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985) 3591–3595], where a dibasic sequence would normally be expected. This specific site is the recognition motif for the endoprotease, furin, which is located in the Golgi apparatus. Both rat and human cDNAs expressed in Cos-1 cells resulted in secretion of a mixture of single- and two-chain forms of factor X. The two-chain forms were devoid of the propeptide and were produced at similar rates by the transfected cells. The efficient processing of human factor X, when compared to rat factor X, may indicate that an additional protease(s), which recognizes the Thr-Arg motif, may be involved in proteolytic processing of the human enzyme     

On enzymatic clotting processes. II. The colloidal instability of chymosin-treated casein micelles.

The enzymatic clotting of casein micelles dispersed in 0.01 M CaCl₂ was monitored by turbidimetry and electrophoresis. The relation between the duration of the lag phase and the enzyme concentration, (e), can be represented by t = K(e)^?γ, where K is a constant and the exponent γ is found to vary between 0.92 and 1.00. This result is interpreted in terms of a flocculation rate constant increasing with the concentration of the enzyme. It is shown that the colloidal instability of chymosin-treated casein micelles cannot be explained on the basis of the well-known theory of the stability of lyophobic colloids, but that clotting is achieved through short-range interactions. The short-range effects that most probably account for the clotting are: hydrophobic bond formation, Ca-bridgas and electrostatic interactions. Under typica'. experimental conditions (33°C; maximum rate of enzymatic product formation about 1.8 × 10^?10 mol ml^?1 s^?1) the flocculation rate constant of clotting micelles was found to be 5 × 10⁵ mlmol^?1s^?1. Various factors, which could be responsible for this low value, are discussed. In the initial stages of the clotting process the turbidity of the system passes through a shallow minimum, which is ascribed to the cleavage of a macropeptide from K-casein by the clotting enzyme. The condition for the minimum has been derived.     

Capture of Lipopolysaccharide (Endotoxin) by the Blood Clot: A Comparative Study

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.     

Turbidimetric studies of Limulus coagulin gel formation.

The turbidity during trypsin-induced coagulin gel formation was studied over a range of wavelengths. The range of wavelengths used (686-326 nm) also made it possible to investigate the dependence of turbidity on wavelength (the wavelength exponent). Using the results from that work, and structural information on coagulin and the coagulin gel from other studies, a model gel-forming system was designed that consists of species for which the turbidity can be calculated relatively simply. These species include small particles (small in all dimensions relative to the wavelength of incident light); long rods and long random coils (particles that are large in just one dimension relative to the wavelength of incident light); and reflective regions (aggregated material that is large in more than one dimension relative to the wavelength of incident light). The turbidimetric characteristics of the real coagulin gel-forming system are compared with those of the model system.     

On enzymatic clotting processes. I. Kinetics of enzyme-triggered coagulation reactions

The kinetics of enzymatic clotting reactions such as the clotting of blood or milk, is analyzed. The appearance of a lag phase in the clotting is shown to be due to the difference in reaction order of enzymatic production and flocculation. The weight-average particle weight of the product formed is found to be a quadratic function of the reaction time. The condition for the clotting time is t square root of ksV/2 = C, where t is the clotting time, ks the flocculation rate constant, V the maximum rate of enzymatic product formation and C a constant. In agreement with this result double-logarithmic plots of t versus enzyme dilution are always observed to be linear over a wide range of enzyme concentrations. No statistical evidence could be produced for the widely held belief that the clotting time should be inversely proportional to the enzyme concentration. Varying exponents of the latter in its relation to the clotting time are discussed in terms of von Smoluchowski's theory of the slow coagulation of colloidal particles.     

On enzymic clotting processes V: Rate equations for the case of arbitrary rate of production of the clotting species

The rate theory for enzyme-triggered coagulation reactions, such as the clotting of fibrin or casein, is extended to the case of an arbitrary rate of production of the clotting species. It is shown that the general expression for the growth of the weight-average molecular weight of the clotting product, -M_w, is given by -M_w = M₁{1 + k_s {∫₀^tP(t)² dt}/P(t)}, where M₁ is the “monomer” molecular weight, k_s the smoluchowskian flocculation rate constant and P(t) the total number of monomers produced by the enzyme in t. In the purely smoluchowskian case P(t) stands for the total number of monomers at the beginning of the clotting process. Numerical examples in which the rate of enzymic production is governed by complete Michaelis-Menten kinetics, are compared to cases in which this rate equals V_max- It is shown that after exhaustion of the substrate the system continues to coagulate in a purely smoluchowskian way. Turbidimetric experiments on the clotting of micelles of whole and κ-casein are presented which suggest inactivation of the enzyme by non-productive binding in the flocs formed.     

Influence of clotting time on the protein composition of serum samples based on LC-MS data

Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC-MS data is not affected by clotting times varying from 2 to 8h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC-MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than 1h. For prospective studies, we recommend to let blood clot for at least 2h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting.     

Intravascular clotting activation and bleeding in patients with hematologic malignancies

The association between thrombosis, bleeding and neoplastic disease is well recognized. There are distinctive features of the thrombotic and bleeding complications associated with specific hematologic malignancies. A number of procoagulants can initiate intravascular clotting including tissue factor, cancer procoagulant and interleukin-1. The hematologic malignancy most often associated with intravascular clotting and bleeding is acute promyelocytic leukemia. The pathogenesis of the life-threatening bleeding disorder associated with this uncommon subtype of acute myeloid leukemia (AML) is complex and involves disseminated intravascular coagulation, fibrinolysis and proteolysis. Both all-trans retinoic acid and arsenic trioxide result in relatively rapid resolution of the coagulopathy. Intravascular clotting may also be induced by hyperleukocytosis in AML and by the hyperviscosity syndrome observed in multiple myeloma and Waldenstr?m's macroglobulinemia. In the setting of hematologic malignancies, when thromboembolic complications occur, the presence of comorbid thrombophilic conditions should be excluded. Abnormal platelet production and function contribute to the development of thrombosis in patients with myeloproliferative disorders. The Budd-Chiari syndrome may be observed in patients with myeloproliferative disorders. A number of medications have thrombogenic potential, including corticosteroids, thalidomide, L-asparaginase, all-trans retinoic acid and arsenic trioxide.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

Structural analysis of N-linked sugar chains of human blood clotting factor IX

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.     

Injecting skin fibroblasts into muscles to express human clotting factor IX cDNA

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Application and validation of the lattice Boltzmann method for modelling flow-related clotting

The purpose of this paper is to present a simple clotting model, based on residence time and shear stress distribution, that can simulate the deposition over time of enzyme-activated milk in an in vitro system. Results for the model are compared with experiments exhibiting clot deposition in the region of a sharp-edged stenosis. The milk experiments have been shown to be a valuable analogue for the experimental representation of flow-induced blood clotting, particularly in the context of separation of hydrodynamic from biochemical factors. The facility to predict the flow-induced clotting of the blood analogue, in which the chemistry reduces to what is effectively a zeroth order reaction, gives confidence in this physics-based approach to simulation of the final part of the coagulation cascade. This type of study is a necessary precursor to the development of a complex, multi-factorial, biochemical model of the process of thrombosis. In addition to the clotting simulations, comparisons are reported between the computed flow patterns prior to clot deposition and flow visualisation studies. Excellent agreement of hydrodynamic parameters is reported for a Reynolds number of 100, and qualitative agreement is seen for the complex, disturbed flow occurring at a physiologically relevant Reynolds number of 550. The explicit, time-stepping lattice Boltzmann approach may have particular merit for the transitional flow at this higher Reynolds number.