Thrombin-induced Fibrinogen Clotting Research Articles

Antithrombotic Effect of Chikusetsusaponin IVa Isolated fromIlex paraguariensis(Maté)

The triterpene chikusetsusaponin IVa was isolated from the fruit of Ilex paraguariensis. Using biochemical and pharmacological methods, we demonstrated that chikusetsusaponin IVa (1) prolongs the recalcification time, prothrombin time, activated partial thromboplastin time, and thrombin time of normal human plasma in a dose-dependent manner, (2) inhibits the amidolytic activity of thrombin and factor Xa upon synthetic substrates S2238 and S2222, (3) inhibits thrombin-induced fibrinogen clotting (50% inhibition concentration, 199.4 ± 9.1 μM), and (4) inhibits thrombin- and collagen-induced platelet aggregation. The results also indicate that chikusetsusaponin IVa preferentially inhibits thrombin in a competitive manner (K(i)=219.6 μM). Furthermore, when administered intravenously to rats, chikusetsusaponin IVa inhibited thrombus formation in a stasis model of venous thrombosis, although it did not induce a significant bleeding effect. Chikusetsusaponin IVa also prolonged the ex vivo activated partial thromboplastin time. Altogether, these data suggest that chikusetsusaponin IVa exerts antithrombotic effects, including minor hemorrhagic events. This appears to be important for the development of new therapeutic agents.

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus ( Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

A thrombin inhibitor from the gut of Boophilus microplus ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin-Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.

Protease Nexin-1 Interacts With Thrombomodulin and Modulates Its Anticoagulant Effect

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1-overexpressing human aortic endothelial cells and PN-1-underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.

Ecotin modulates thrombin activity through exosite-2 interactions

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human α-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin–α-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC 50 = 3.2 μM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC 50 = 4.5 μM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-α-thrombin (EC 50 = 3.5 μM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.

Suramin interaction with human α-thrombin: inhibitory effects and binding studies

Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human α-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC 50 = 40 μM), and the thrombin-induced fibrinogen clotting (IC 50 = 20 μM). The latter is completely reversed by albumin (30 mg mL −1) suggesting that, at therapeutic concentrations, suramin is unable to affect α-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing V max without changing the K m for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in α-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in α-thrombin tertiary structure, without affecting the secondary structure content. Interaction with α-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to α-thrombin inhibition at extra-vascular sites.

Lonofibrase, a novel α-fibrinogenase from Lonomia obliqua caterpillars

Envenomation caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by a profound hemorrhagic disorder. In the present study, we describe the characterization of a fibrin(ogen)olytic factor (lonofibrase) isolated from a venomous secretion of the caterpillars. The crude extract showed a dose-dependent inhibitory effect in the rate of thrombin-induced fibrinogen clotting and produced fragmentation of fibrinogen. Isolation of the fibrin(ogen)olytic enzyme was achieved by combining ion exchange chromatography followed by gel filtration in a fast protein liquid chromatography (FPLC) system. A single 35-kDa band was identified and the isolated enzyme named lonofibrase. Lonofibrase rapidly degrades Aα and Bβ chains of fibrinogen, also being able to cleave fibrin in a distinct way from that observed with plasmin. The presence of lonofibrase with both fibrinogenolytic and fibrinolytic activities in L. obliqua secretion is coherent with the severe hemorrhagic clinical profile resulting from envenomation caused by these insects.

SdrG, a Fibrinogen-binding Bacterial Adhesin of the Microbial Surface Components Recognizing Adhesive Matrix Molecules Subfamily from Staphylococcus epidermidis, Targets the Thrombin Cleavage Site in the Bβ Chain

Staphylococcus epidermidis is an important opportunistic pathogen and is a major cause of foreign body infections. We have characterized the ligand binding activity of SdrG, a fibrinogen-binding microbial surface component recognizing adhesive matrix molecules from S. epidermidis. Western ligand blot analysis showed that a recombinant form of the N-terminal A region of SdrG bound to the native Bbeta chain of fibrinogen (Fg) and to a recombinant form of the Bbeta chain expressed in Escherichia coli. By analyzing recombinant truncates and synthetic peptide mimetics of the Fg Bbeta chain, the binding site for SdrG was localized to residues 6-20 of this polypeptide. Recombinant SdrG bound to a synthetic 25-amino acid peptide (beta1-25) representing the N terminus of the Fg Bbeta chain with a KD of 1.4 x 10(-7) m as determined by fluorescence polarization experiments. This was similar to the apparent K(D) (0.9 x 10(-7) m) calculated from an enzyme-linked immunosorbent assay where SdrG bound immobilized Fg in a concentration-dependent manner. SdrG could recognize fibrinopeptide B (residues 1-14), but with a substantially lower affinity than that observed for SdrG binding to synthetic peptides beta1-25 and beta6-20. However, SdrG does not bind to thrombin-digested Fg. Thus, SdrG appears to target the thrombin cleavage site in the Fg Bbeta chain. In fact, SdrG was found to inhibit thrombin-induced fibrinogen clotting by interfering with fibrinopeptide B release.

Purification and cDNA Cloning of Salmorin that Inhibits Fibrinogen Clotting

A new protein, salmorin, that inhibits fibrinogen clotting was purified to homogeneity from the snake venom of Agkistrodon halys brevicaudus. Salmorin was characterized to be a heterodimeric protein composed of 15 kDa and 14 kDa subunits. Purified salmorin inhibited not only thrombin-induced fibrinogen clotting but also factor Xa-induced prothrombin activation. Experimental evidence indicated that the hetero-dimeric protein does not bind to the thrombin catalytic site but binds to thrombin and prothrombin exosites. Analyses of the cDNA sequences encoding the two separate polypeptide chains revealed that the mature subunits are composed of 131 and 122 amino acids, respectively. In the isolated cDNAs, N-termini of both chains are preceded by hydrophobic signal peptides of 23 residues. The deduced amino acid sequence exhibited considerable identity with other snake venom C-type lectin-like proteins derived from various snake venoms.

Monoclonal antibodies against human thrombomodulin whose epitope is located in epidermal growth factor-like domains

Thrombomodulin (TM) on endothelial cells is a glycoprotein that functions as a cofactor for thrombin-catalyzed activation of protein C. The structural requirement for thrombin binding and cofactor activity were investigated using monoclonal antibodies (moAbs) against TM and site-directed mutagenesis of recombinant human soluble TM (rsTM). Results showed that moAb 2A2 inhibited thrombin binding to rsTM and also abolished its functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation, moAb 1F2 did not affect its activity as an anticoagulant, but inhibited its cofactor activity, and moAb 10A3 did not inhibit either activity. Epitope analysis was carried out by site directed mutagenesis of rsTM expressed in CHO cells. Some proteins with mutations within the second disulfide loop of the fourth EGF-like domain showed reduced affinity for moAb 1F2, but retained cofactor activity. These results suggest that the epitope of moAb 1F2 includes the second disulfide loop of the fourth EGF-like domain, which is close to a region required for cofactor activity. Mutant proteins of the third disulfide loop of the fifth EGF-like domain showed loss of interaction with moAb 2A2. Thus the epitope of moAb 2A2 may include the third disulfide loop of the fifth EGF-like domain. Furthermore, replacement of Asn-439 by Gln decreased the cofactor activity and anticoagulant activity, and resulted in low affinity for either moAb 1F2 or 2A2, suggesting that Asn-439, which is located in the second disulfide loop of the sixth EGF-like domain, is critical for determining the functional conformation of the EGF-like domains 4–6.

Antithrombotic Effects of Recombinant Human Soluble Thrombomodulin in a Rat Model of Vascular Shunt Thrombosis

Thrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTM alpha, 0.15 mg/kg rsTM beta, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTM alpha or 0.4 mg/kg rsTM beta resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of the Possible Recognition Sites for Thrombomodulin, Procoagulant, and Anticoagulant Proteins around the Active Center of α-Thrombin1

alpha-Thrombin has several characteristic module structures around its active center. Previously we showed that a synthetic peptide, TWTANVGKGQPS, corresponding to the residues Thr147 to Ser158 of the B-chain of human thrombin, a possible interaction site of thrombomodulin near the active center of thrombin, specifically blocked the interactions between thrombin and thrombomodulin, fibrinogen, Factor V, or platelets [Suzuki, K. & Nishioka, J. (1991) J. Biol. Chem. 266, 18498-18501]. To elucidate further the role of the other module structures, we studied the effects of several synthetic peptides; FRKSPQELL, LLYPPWDKNF, RIGKHSRTRYER, LEKIYIHP, RYNWREN, DSTRIRI, EGDSGGP, and SWGEGCDRDGK, respectively corresponding to the residues Phe19 to Leu27, Leu45 to Phe54, Arg62 to Arg73, Leu81 to Pro88, Arg89 to Asn95, Asp175 to Ile181, Glu202 to Pro208, and Ser226 to Lys236 of the B-chain of human thrombin, which are located around the active center, as well as TWTANVGKGQPS, on the interaction between thrombin and thrombomodulin, protein C, fibrinogen, Factor V, antithrombin III, or hirudin. Thrombin-thrombomodulin interaction was inhibited significantly by RYNWREN as well as TWTANVGKGQPS, and partially by LLYPPWDKNF. The inhibitory effects of the two former peptides were additive and thrombomodulin directly bound to them. RYNWREN and TWTANVGKGQPS also increased the Km values 3-7 times for protein C as compared with the conditions without peptide. Thrombin-induced protein C activation in the absence of thrombomodulin was specifically blocked by EGDSGGP. Thrombin-induced fibrinogen clotting was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS at lower concentrations, and by RYNWREN and DSTRIRI at higher concentrations. Thrombin-induced Factor V activation was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Unexpected Effects of Aurin Tricarboxylic Acid on Human Platelets

Aurin tricarboxylic acid (ATA) is a potent inhibitor of ristocetin-mediated platelet agglutination and of shear-induced, von Willebrand factor (vWf)-mediated platelet aggregation, probably via inhibition of vWf interaction with glycoprotein Ib (GPIb). We examined the effects of ATA (both the sodium salt and a solution of ATA in ethanol) on platelet functions in citrated plasma (PRP) and in suspensions of washed platelets in Tyrode-albumin solution (contains 2 mM Ca2+). ATA (42-211 micrograms/ml) blocked aggregation and release of granule contents induced by thrombin (0.15 U/ml in PRP; 0.03 U/ml in platelet suspension). Responses to higher concentrations of thrombin were not inhibited. ATA also prolonged thrombin-induced clotting of fibrinogen. Since ATA had no effect on fibrinogen-induced responses of chymotrypsin-treated platelets, ATA probably acts on thrombin rather than on fibrinogen. In PRP and platelet suspensions, ATA (acid form 106 micrograms/ml; sodium salt 122 micrograms/ml) had little effect on ADP-induced platelet aggregation. The sodium salt of ATA (61-122 micrograms/ml) enhanced collagen-induced aggregation and release by platelets in citrated plasma and by washed platelets; the enhancement was extensively inhibited by aspirin. With platelet suspensions, ATA significantly enhanced aggregation and release caused by low concentrations of sodium arachidonate (15-50 microM); aggregation and release caused by higher concentrations of arachidonate were somewhat inhibited by ATA. Arachidonate-induced aggregation and release were also enhanced by ATA in PRP. ATA enhanced aggregation and release induced by the calcium ionophore A23187; aspirin had little effect on the enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of rabbit thrombomodulin on thrombin inhibition by antithrombin in the presence of heparin

Thrombomodulin acts as a cofactor for protein C activation by thrombin (PC activation cofactor activity) and inhibits thrombin-induced fibrinogen clotting (direct anticoagulant activity). In addition, rabbit thrombomodulin has been shown to promote thrombin inactivation by antithrombin (AT-dependent anticoagulant activity). However, a non-acidic form (i.e non-retarded on ion-exchange chromatography) of thrombomodulin generated by limited proteolysis retained only the PC activation cofactor activity. The acidic form (retarded on ion-exchange chromatography) of thrombomodulin is now shown to prevent the rapid inactivation of thrombin by antithrombin in the presence of heparin, presumably by preventing the formation of the ternary thrombin-AT-heparin complex. This effect was not observed with non-acidic thrombomodulin. When submitted to chondroitinase digestion, thrombomodulin was converted into an essentially non-acidic form that lacked both the AT-dependent and the direct anticoagulant activities but showed a PC activation cofactor function indistinguishable from that of native thrombomodulin. This chondroitinase-digested form did not prevent the catalytic effect of heparin on the inhibition of thrombin by AT. It is concluded that the acidic domain of rabbit thrombomodulin, a chondroitin (dermatan) sulfate glycosaminoglycan, interacts with a site of the thrombin molecule that is not involved in the protein C activation cofactor function, but is essential to the cleavage of fibrinogen or binding of heparin.

Thrombin-induced alterations in endothelial permeability.

Figure 15 summarizes the current understanding of mechanisms of endothelial permeability alterations induced with thrombin. If thrombin generation exceeds the antiprotease activity, thrombin results in clotting of fibrinogen and intravascular fibrin accumulation. Pulmonary neutrophil sequestration also occurs after fibrin deposition, and this is related to the degree and duration of fibrin sequestration. Neutrophil activation appears to be an essential requirement for the mediation of the pulmonary vascular injury. Thrombin-induced intravascular coagulation results in the generation of lipid mediators (LTB4 and HETEs), which may be involved in increasing endothelial permeability. The release of thrombin in higher concentrations during lysis of fibrin (sequence; see text) FIGURE 15. Hypothesis showing mechanisms of thrombin-induced increase in endothelial permeability to proteins. Thrombin may have direct effects on endothelial permeability, or thrombin induced fibrinogen clotting, activation of neutrophils, and the release of lipid metabolites that subsequently lead to an increase in endothelial permeability. clots may induce a direct formation of interendothelial "gaps." Therefore, the vascular injury induced by neutrophil activation and the formation of endothelial "gaps" induced directly by thrombin can both increase the endothelial permeability to proteins. Thrombin is an important mediator of increased endothelial permeability to macromolecules, and may participate in the inflammatory response. In this regard, thrombin may be similar to other mediators (such as histamine and serotonin) that have been previously documented to increase macromolecule transport across the endothelium. The implications of free thrombin in increasing endothelial permeability may be greater because thrombin not only has a direct effect on endothelial permeability, but also induces clotting of fibrinogen and the subsequent generation of mediators that activate neutrophils and that in turn can induce endothelial injury.

Isolation and characterization of thrombomodulin from bovine lung

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent M r of 78 000 and 105 000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60°C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca 2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.

Characterization of a fibrinogenase from northern copperhead ( Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead ( Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 ± 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide powder azure and had an intraperitoneal LD 50 value in mice of less than 5.1 μg/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the α-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl 2.

Addendum

The thrombin-induced clotting of fibrinogen does not lead to hemostasis (Duckert et al, 1960). The fibrin molecules in the clot are connected only with hydrogen bonds, and are readily dispersed in concentrated urea solutions (Laki and Lorand 1948; Laki and Chandrasekhar, 1963).It was discovered by Laki and Lorand (1948) that the intervention of a plasma component (called: Laki-Lorand Factor = LLF, Fibrin Stabilizing Factor = FSF, Fibrinase) in the presence of Ca-ions was needed to render the clot insoluble in urea solutions. The plasma component introduced bonds between the fibrin molecules so that urea could not disperse the clot. A few years ago, Lowey et al (1961) succeeded in purifying this plasma component and showed that it was an enzyme. The purpose of this communication is to show that it is now possible to describe the mechanism by which the primary bonds are introduced between the fibrin molecules.

The mode of action of the Laki-Lorand factor in the clotting of fibrinogen

The thrombin-induced clotting of fibrinogen does not lead to hemostasis ( Duckert et al, 1960). The fibrin molecules in the clot are connected only with hydrogen bonds, and are readily dispersed in concentrated urea solutions ( Laki and Lorand 1948; Laki and Chandrasekhar, 1963). It was discovered by Laki and Lorand (1948) that the intervention of a plasma component (called: Laki-Lorand Factor = LLF, Fibrin Stabilizing Factor = FSF, Fibrinase) in the presence of Ca-ions was needed to render the clot insoluble in urea solutions. The plasma component introduced bonds between the fibrin molecules so that urea could not disperse the clot. A few years ago, Lowey et al (1961) succeeded in purifying this plasma component and showed that it was an enzyme. The purpose of this communication is to show that it is now possible to describe the mechanism by which the primary bonds are introduced between the fibrin molecules.