Catalysis of the generation of thrombin-antithrombin complex by insoluble anticoagulant polystyrene derivatives

The inhibition of thrombin by antithrombin III is known to be accelerated by heparin through the formation of complexes between the muccopolysaccharide and both proteins. In the preceding papers, we reported that polystyrene derivatives absorb thrombin and its inhibitor with a higher affinity for the protease than for the antiprotease. These complexes are responsible for the catalysis of the generation of thrombin-antithrombin complex which was observed either with purified proteins or in plasma. The protease-antiprotease complex has an affinity for the polymer surface which is higher than that of antithrombin but lower than that of thrombin. Therefore, the thrombinantithrombin complex generated on the insoluble material is desorbed by thrombin and a catalytic anticoagulant effect can be observed with these polymers.     

Polyether polyurethanes: biostable or not?

Certain polyether polyurethanes have been shown to be biostable in long-term implant studies. Others retain good bulk properties, but have been shown to develop cracks on their tissue contacting surfaces. Two cracking mechanisms have been identified, in vivo stress cracking and metal ion oxidation. Stress cracking is the result of an interaction between the in vivo mammalian environment and residual stress (strain) in the implanted polymer. Mild autooxidation can be initiated by stress cracking. More extensive autooxidation can be initiated and propagated by corrosion of metallic device components, especially the corrosion products of cobalt. Both mechanisms are controllable, thus, do not necessarily preclude the use of polyether polyurethanes in implantable devices.     

Biological characterization of a novel biodegradable antimicrobial polymer synthesized with fluoroquinolones.

Biomaterial-related infections continue to represent a significant challenge to the medical community. Several approaches have been utilized to incorporate antimicrobial agents at the surface of implant devices in attempts to delay or eliminate the formation of biofilms. To date, most of these strategies have focused on drug conjugation or diffusion-limited systems for the delivery of such pharmaceutical agents. More recently, work has been presented on the feasibility of incorporating drugs into the backbone of polymers as a main-chain monomer. When sequenced into the backbone of the polymer with other monomers that are hydrolytically sensitive to enzyme-catalyzed breakdown, it is thought that drugs may be able to be selectively released. Specifically, degradable polyurethanes have been synthesized with fluoroquinolone antibiotics and have shown an ability to kill bacteria when released following degradation of the polymer chains by the macrophage-derived enzyme cholesterol esterase. However, specificity of the cleavage sites in the polymer was difficult to control. Since cholesterol esterase has specificity for hydrophobic moieties, it is desirable to alter the formulation of the polyurethanes to incorporate long hydrophobic monomers immediately adjacent to the ciprofloxacin molecule. Hence, the current study focuses on evaluating the enzyme-catalyzed degradation of a degradable polyurethane synthesized with 1,12 diisocyanatododecane as a substitute for 1,6 diisocyanatohexane, which was used in previous work. Validation of specific ciprofloxacin release and the generation of antimicrobial are shown. A preliminary cell study to assess the cytotoxicity of this biodegradable antibiotic polymer shows that the material has no observable effects on cell proliferation or cell membrane structure.     

Adsorption of plasma proteins onto anticoagulant polystyrene derivatives: a fluorescence study

Quenching of fluorescence was used to monitor adsorption of thrombin (T), antithrombin (AT) and their inactive complex (T-AT) onto three anticoagulant biomaterials made of polystyrene beads bearing the functional groups of heparin. An adsorption capacity of 0.12 mumol of T per mg of polymer allowed the formation of a monolayer of protein at the polymer surface. An affinity constant of 3 x 10(7) l.mol-1 between thrombin and polymer was estimated, whatever the polymer used. The affinity of T-AT was similar although weaker. Desorption of proteins from the polymeric interface by means of polycations (polybrene and polylysine) showed that the inactive complex T-AT is more quantitatively and easily released than thrombin.     

Donor acceptor interactions in polymeric systems, 6. Effects of the molecular weight of carbazolyl containing polyesters and the length of spacer between donor groups on the complex formation with 2,4,7-trinitrofluorenone

EDA complex formation between 2,4,7-trinitrofluorenone (TNF) and polyesters bearing pendant 9-carbazolyl groups was studied in ethylene dichloride. The formation constant (K) decreases with increasing length of the main chain repeating units separating pendant donor groups and with decreasing degree of polycondensation. In all cases, the values of K in polymeric systems are larger than those for the monomer model system by factors less than three. Thermodynamic parameters for complex formation were also determined. The general trend of the polymer effect is to increase ? ΔH, while the entropy term is not much different from the monomer model system. The origin of polymer effects is interpreted as due not only to neighbouring group effects but also to remote group effects, and simultaneous participation of more than one carbazolyl group in complexing an acceptor molecule is considered. The results are compared with the EDA complex formation by ethylenic polymers and polyurethanes with pendant carbazolyl groups.     

Donor acceptor interactions in polymeric systems, 7. Structure matching effect in interpolymer EDA complex formation

Interpolymer EDA complex formation between polyesters bearing pendant carbazolyl groups and polyurethanes bearing pendant trinitrofluorenonyl groups was investigated in dioxane at 25°C. Complex formation depends on the degree of polycondensation (DP) as well as on structure matching of polymeric D and polymeric A. With increasing DP, the solubility of the D polymer-A polymer mixture trends to decrease. Even when the polymer complexes do not precipitate, the Ketelaar's plots do not always fall on a straight line. These phenomena were interpreted as due to higher order complex formation and/or dependence of the formation constant on the fraction of saturation. It was found that polymeric donor acceptor pairs in which the number of atoms in a repeating main chain unit are identical for D and A polymers form a stronger complex than other combinations. It was concluded that structure matching of an interacting pair is a favourable condition to form tight complexes as in the case of antigenantibody interactions.     

General haemostatic agents--fact or fiction?

Haemophilia is the most serious bleeding model that nature has provided us with, indicating the importance of factor FVIII and FIX in haemostasis. According to current knowledge, haemostasis is initiated by the formation of a complex between tissue factor (TF), exposed as a result of a vessel wall injury, and activated FVII (FVIIa) that is normally present in circulating blood. The TF-FVIIa complexes convert FX into FXa on the TF-bearing cell. FXa then activates prothrombin into thrombin. This limited amount of thrombin activates FVIII, FV, FXI and platelets. Thrombin-activated platelets change shape, resulting in exposure of negatively-charged phospholipids, which form the perfect template for full thrombin generation involving FVIII and FIX. In patients with haemophilia FVIII or FIX is missing. These individuals generate only initial limited amounts of thrombin as its generation is dependent on the presence of FVIII and FIX. Full thrombin generation is necessary for complete activation of FXIII and thrombin activatable fibrinolytic inhibitor to occur. Furthermore, full thrombin generation is important for the fibrin structure of the haemostatic plug. In the case of impaired thrombin generation, fibrin plugs will be loose and highly permeable. Such fibrin plugs are easily dissolved by normal fibrinolytic activity and thus prevent full and maintained haemostasis from occurring. The addition of rFVIIa to FVIII- or FIX-deficient plasma has been shown to increase thrombin generation in a cell-based in vitro model. Furthermore, extra rFVIIa was found to normalise fibrin clot permeability in vitro and to tighten the fibrin structure as studied by three-dimensional confocal microscopy. These findings indicate that administration of rFVIIa is capable of compensating for the lack of FVIII and FIX. Accordingly, the administration of exogenous rFVIIa has been found to stop bleedings in haemophilia patients and, provided it is given in doses high enough, to allow major surgery to be performed in severe haemophiliacs with inhibitors. As rFVIIa enhances thrombin generation on already activated platelets, it has been suggested that rFVIIa may also help to improve haemostasis in other situations involving impaired thrombin generation, such as platelet disorders (thrombocytopenia and functional platelet defects). Preliminary clinical data appear to support this. Patients with profuse bleeding due to extensive surgery or trauma often develop a complex coagulation pattern which includes reduced plasma levels of fibrinogen, FVIII and FV, and decreased platelet counts. These patients may well have an impaired capacity to generate thrombin. Consequently, they may benefit from one or two doses of rFVIIa in order to assist in the generation of a thrombin peak sufficient to form a firm, stable fibrin haemostatic plug and thereby reduce bleeding. This would facilitate any mechanical repair necessary for full haemostasis. Preliminary results in a few patients may support such an effect for rFVIIa. As thrombin has such a crucial role in providing haemostasis, any agent that enhances the thrombin generation in situations with an impaired thrombin formation may be characterised as a 'general haemostatic agent'. Let us look forward to more 'facts' through the 'evidence-based route'.     

Heparin cofactor II in atherosclerotic lesions from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII–thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against atherosclerosis in mice was determined to be the inhibition of thrombin. Despite this evidence, the presence of HCII in human atherosclerotic tissue has not been reported. In this study, using samples of coronary arteries obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, we explore the local relationship between HCII and (pro)thrombin in atherosclerosis. We found that HCII and (pro)thrombin are co-localized in the lipid-rich necrotic core of atheromas. A significant positive correlation between each protein and the severity of the atherosclerotic lesion was present. These results suggest that HCII is in a position to inhibit thrombin in atherosclerotic lesions where thrombin can exert a proatherogenic inflammatory response. However, these results should be tempered by the additional findings from this, and other studies, that indicate the presence of other plasma proteins (antithrombin, albumin, and α₁-protease inhibitor) in the same localized region of the atheroma.     

MASP-1 of the complement system promotes clotting via prothrombin activation

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.     

A novel hemostatic delivery device for thrombin: biodegradable poly(D,L-lactide-co-glycolide) 50:50 microspheres

Topical thrombins are locally active hemostatic agents that can be used to minimize blood loss during any surgery. The aim of this study was to design and investigate a thrombin-containing biodegradable hemostyptic device with an optimized drug release profile to promote local blood clot formation. It is effective with ongoing systemic antithrombotic therapy and can be used in all types of bone-related surgery, for example, in dental surgery. Thrombin-loaded poly(D,L-lactide-co-glycolide) microspheres were synthesized by means of complex (w/o/w) emulsion evaporation method. The resulting enzyme activity of the serine-protease thrombin was verified by the specific chromogenic substrate S-2238. The thrombin release profile depended on four factors: (1) thrombin dosage, (2) polymer concentration in the o-phase, (3) phase quotient w1:0 in the primary emulsion, and (4) the addition of pore-introducing agents. A collagenous sponge containing thrombin-loaded microspheres by means of lyophilization was developed. The impact of several production factors of the (w1/o/w2) solvent evaporation method to optimize thrombin encapsulation, morphology of the spheres, and desired drug release profile have been investigated. The in vitro thrombin release was dependent on the polymer-to-oil phase ratio, the polymer concentration, and the type of solvent and polymer. The porosity of the spheres and release rate of the active agent were enhanced by increasing the inner aqueous w1 phase. With this study, a new biodegradable hemostyptic device could be verified and established for a potentially safe and locally controlled thrombin release to manage postsurgical hemorrhage in patients undergoing anticoagulant therapy.     

Novel Poly(urethane-aminoamides): an in vitro study of the interaction with heparin

In order to obtain heparin-binding polyurethanes, tertiary amino-groups have been introduced in the polymer backbone by attributing a key-role to the chain extender, i.e. substituting butanediol, commonly used in polyurethane synthesis, with a tailor-made diamino-diamide-diol. In this work a poly(ether-urethane-aminoamide) (PEU/PIME/al) was obtained with poly(oxytetramethylene) glycol 2000, 1,6-hexamethylene-diisocyanate and the new chain extender, in the molar ratio 1 :2 : 1. The heparin binding capacity of PEU/PIME/al was evaluated with ¹²⁵I labelled heparin, using for comparison the analogous polymer obtained with a diamide-diol (i.e. the poly(ether-urethane-amide) PEU/PIBLO/al), and two commercially available biomedical polyurethanes (Pellethane 2363 and Corethane). pH and ionic strength dependence of the heparin uptake were investigated by treating all the polyurethanes with solutions of ¹²⁵I heparin into buffers from pH 4 to 9 or NaCl molarity from 0.0 to 1.0. The stability of the interaction with bound heparin was investigated by sequential washing treatments (PBS, 1 N NaOH, 2% SDS solution), then analysing the residual radioactivity on the materials. Results indicated that the heparin binding of PEU/PIME/al is significantly higher and more stable than that of the other polyurethanes, with a time-dependent kinetic. The interaction with heparin appears to be prevalently ionic, with the contribution of other electrostatic and hydrophobic interactions. Activated partial thromboplastin time (APTT), performed on human plasma with polyurethane-coated, heparinized test tubes, indicated that bound heparin maintains its biological activity after the adsorption.     

Polyurethanes bearing pendant amino acids: fibrinogen adsorption and coagulant properties.

Segmented polyurethanes based on 4,4'-diphenylmethane diisocyanate and polypropylene oxide and chain extended with a sulfonated diamine were derivatized by reaction of sulfonate groups in the polymer with amino acids. The chemical composition of the derivatized polymers was determined by elemental analysis. Tensile stress-strain measurements indicated a slight increase in modulus and elongation with incorporation of amino acids. Water uptake at room temperature showed little change following derivatization, but at 70 degrees C increased significantly. Water contact angles were not influenced by the presence of amino acids, but electron spectroscopy for chemical analysis data showed an increase in hard segment content in the near-surface layers so that bulk and surface compositions were more nearly the same in the amino-acid-containing materials. Fibrinogen adsorption from plasma, shown previously to be high on the sulfonated polyurethanes, was reduced by derivatization, due probably to the decrease in free sulfonate content. Thrombin times of plasma in contact with these materials were essentially the same for the derivatized and underivatized materials.     

On the mechanism of enhanced thromboresistance of polymeric materials in the presence of heparin

Polymeric materials with covalently immobilized heparin were shown to display enhanced thromboresistance in in vitro and in vivo experiments. This property of heparin-containing polymers is due to the specific effect of immobilized heparin for every step of interaction of a polymer with blood. The presence of heparin substantially changes the character of adsorbed proteins on a polymer surface and the number of adhered platelets. Thromboresistance enhancement is largely carried out by the interaction of immobilized heparin with plasma proteins which is accompanied by the decrease in total blood coagulant activity, by the decrease in fibrinogen, prothrombin and thrombin concentrations, and by the supression of fibrinstabilizing factor activity. The free heparin content in blood is not changed. It was found that immobilized heparin forms complexes with fibrinogen, thrombin and plasmin that produce lytic action on unstabilized fibrin.     

Effect of covalent serpin-heparinoid complexes on plasma thrombin generation on fetal distal lung epithelium

Extravascular coagulation within the lung airspace is a hallmark of respiratory distress syndrome (RDS) in premature infants. We previously showed that covalent antithrombin-heparin complex (ATH) is superior to noncovalent antithrombin (AT) + heparin (H) mixtures at inhibiting plasma thrombin generation on rat fetal distal lung epithelium (FDLE) in vitro. However, heparin cofactor II (HC) has been shown to selectively inhibit thrombin, which may be advantageous if other enzyme activities are present in the airspace. We compared the abilities of ATH, covalent HC-heparin complex (HCH), and covalent HC-dermatan sulfate (HCD) to inhibit thrombin generation on FDLE in plasmas from either adults or newborns. In the presence of ATH, peak free thrombin generation in adult plasma on the cell surface was reduced by 92% compared with controls (no anticoagulant). However, whereas HCH reduced peak free thrombin generation in adult plasma by 81%, HCD was only able to reduce activity by 33%. All covalent complexes caused a greater decrease in thrombin activity compared with that with the corresponding noncovalent serpin + heparinoid mixtures. Experiments in plasma from newborns resulted in peak free thrombin that was less than or equal to that in adult plasma when covalent conjugates were added. Relative peak free thrombin was proportional to rate of prothrombin consumption and amount of thrombin-inhibitor complexes formed. In vivo, experiments in newborn rats showed that a greater percentage of intratracheally instilled ATH and HCH could be recovered in lung lavage fluid compared withwith that for HCD. In summary, ATH, HCH, and HCD are inhibitors of thrombin generation on FDLE superior to the corresponding noncovalent mixtures, with ATH and HCH being more potent than HCD. Covalent conjugates of AT or HC with H may be preferred in treatment of extravascular coagulation.     

Autooxidative degradation of implanted polyether polyurethane devices

While certain polyether polyurethanes have been shown to be biostable, undesirable interactions between polymer, the body environment and device can occur. For example, the corrosion products of metallic parts can cause relatively rapid autooxidation of polyether soft segments. This phenomenon was first demonstrated in our laboratories by immersing polyurethane test specimens in metal ion solutions of different oxidation potentials. Subsequently, the mechanism was reasonably duplicated by immersing Pellethane 2363-80A insulated cardiac pacing leads in 3%, 37 degrees C hydrogen peroxide. In vivo studies after 1 year show that corrosion products from pure Co and Fe produce the most rapid degradation of the polyurethane.     

The use of silicone/polyurethane graft polymers as a means of eliminating surface cracking of polyurethane prostheses

The long-term biodegradation of various polyurethanes with and without surface modifications was evaluated by implanting small porous filamentous patches of these materials subcutaneously in the backs of dogs for one month. Data were compared to those obtained with spun polyurethane vascular grafts of similar materials implanted in the aorto-iliac position in dogs. The extremely high surface area of approximately 7 m2/cm3 of these porous filamentous patches provided numerous sites for surface cracking and the very fine filaments (10 microns in diameter) provided an easily identifiable structure to study the cracking phenomenon. Results from numerous one month implants clearly demonstrated that the subcutaneous implant model effectively reproduced the biodegradation behavior observed in vascular graft implants. The degradation was most pronounced in the softer Shore 80A polyurethanes and less pronounced in the harder 55D and 75D polyurethanes. The degradation could not simply be stopped by stress annealing the polyurethane and the degradation did not require the presence of metallic ions. Antioxidants, surface adsorbed albumin, poly(2-hydroxyethyl-methacrylate) grafting, silicone copolymerization, tetrafluoroethylene plasma discharge and the addition of urea linkages to the polymer were also shown to be ineffective in stopping the biodegradation process. In contrast, covalent bonding or grafting of silicone polymer to the surface of the urethane successfully inhibited the biodegradation process.     

Novel poly(urethane-aminoamides): an in vitro study of the interaction with heparin

In order to obtain heparin-binding polyurethanes, tertiary amino-groups have been introduced in the polymer backbone by attributing a key-role to the chain extender, i.e. substituting butanediol, commonly used in polyurethane synthesis, with a tailor-made diamino-diamide-diol. In this work a poly(ether-urethane-aminoamide) (PEU/PIME/al) was obtained with poly(oxytetramethylene) glycol 2000, 1,6-hexamethylene-diisocyanate and the new chain extender, in the molar ratio 1:2:1. The heparin binding capacity of PEU/PIME/al was evaluated with 125I labelled heparin, using for comparison the analogous polymer obtained with a diamide-diol (i.e. the poly(ether-urethane-amide) PEU/PIBLO/al), and two commercially available biomedical polyurethanes (Pellethane 2363 and Corethane). pH and ionic strength dependence of the heparin uptake were investigated by treating all the polyurethanes with solutions of 125I heparin into buffers from pH 4 to 9 or NaCl molarity from 0.0 to 1.0. The stability of the interaction with bound heparin was investigated by sequential washing treatments (PBS, 1 N NaOH, 2% SDS solution), then analysing the residual radioactivity on the materials. Results indicated that the heparin binding of PEU/PIME/al is significantly higher and more stable than that of the other polyurethanes, with a time-dependent kinetic. The interaction with heparin appears to be prevalently ionic, with the contribution of other electrostatic and hydrophobic interactions. Activated partial thromboplastin time (APTT), performed on human plasma with polyurethane-coated, heparinized test tubes, indicated that bound heparin maintains its biological activity after the adsorption.     

Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI)

Thrombin-activatable procarboxypeptidase B (proCPB or thrombin-activatable fibrinolysis inhibitor or TAFI) is a plasma procarboxypeptidase that is activated by the thrombin–thrombomodulin complex on the vascular endothelial surface. The activated CPB removes the newly exposed carboxyl terminal lysines in the partially digested fibrin clot, diminishes tissue plasminogen activator and plasminogen binding, and protects the clot from premature lysis. We have recently shown that CPB is catalytically more efficient than plasma CPN, the major plasma anaphylatoxin inhibitor, in inhibiting bradykinin, activated complement C3a, C5a, and thrombin-cleaved osteopontin in vitro. Using a thrombin mutant (E229K) that has minimal procoagulant properties but retains the ability to activate protein C and proCPB in vivo, we showed that infusion of E229K thrombin into wild-type mice reduced bradykinin-induced hypotension but it had no effect in proCPB-deficient mice, indicating that the beneficial effect of E229K thrombin is mediated through its activation of proCPB and not protein C. Similarly proCPB-deficient mice displayed enhanced pulmonary inflammation in a C5a-induced alveolitis model and E229K thrombin ameliorated the magnitude of alveolitis in wild-type but not proCPB-deficient mice. ProCPB-deficient mice also displayed enhanced arthritis in an inflammatory arthritis model. Thus, our in vitro and in vivo data support the thesis that thrombin-activatable CPB has broad anti-inflammatory properties. By specific cleavage of the carboxyl terminal arginines from C3a, C5a, bradykinin and thrombin-cleaved osteopontin, it inactivates these active inflammatory mediators. Along with the activation of protein C, the activation of proCPB by the endothelial thrombin–thrombomodulin complex represents a homeostatic feedback mechanism in regulating thrombin's pro-inflammatory functions in vivo.     

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin–thrombin complex to minimize the biotinylation of the residues involved in thrombin–boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²⁵I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin’s potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.     

Lipopolysaccharide-instructed, cryopreserved, human monocytes sequentially convert plasma fibrinogen to fibrin and lyse the fibrin formed

Cryopreserved human monocytes have been examined for their procoagulant and profibrinolytic capacities on lipopolysaccharide (LPS) stimulation, when given the possibility to act upon fibrinogen in heparinized plasma. As shown by the appearance of fibrinopeptide A, indicating thrombin action on the fibrinogen molecule, and by the appearance of D-Dimer, indicating the action of plasmin on fibrin, it is apparent that LPS-instructed monocytes give rise to fibrin which is subsequently lysed. Thus, delineation (fibrin formation) appears to be followed by fibrin removal (restitution).