Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.     

Molecular weight of human fibrinogen derived from phosphorus determinations

1. Fibrin clots obtained from diluted human plasma with bovine thrombin often contain amounts of phospholipids that cannot be diminished by further plasma dilution. 2. The ;cold insoluble residue' obtained during fibrinogen preparation has a higher phosphorus content than the purified fibrinogen. 3. Evidence showed that adsorption of phospholipids or phosphorus-containing fibrinopeptides on purified fibrinogen or fibrin was unlikely. 4. O-Phosphorylserine was detected in acid hydrolysates of human fibrin. 5. On the basis of phosphorus determinations the average molecular weight of human fibrinogen cannot be less than 342000 (304000-383000) for a group of ten donors, and 265000 for two other persons, assuming 1 phosphorus atom/molecule and incomplete splitting of the phosphorus-containing fibrinopeptide. Complete splitting of the phosphopeptide would require molecular weights twice as high. 6. Fibrinolysis was a possible cause of lower phosphorus contents found in isolated fibrinogen and fibrin from a donor who showed apprehension during blood collection and in a fibrinogen preparation that had been submitted to prolonged dialysis.     

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ～0.1 and ～2 molecules/μm². Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot’s structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot’s occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow.     

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

A unique group of scabies mite pseudoproteases promotes cutaneous blood coagulation and delays plasmin-induced fibrinolysis

BackgroundScabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi.Methodology/Principal findingsA series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C—modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin.Conclusions/SignificanceThe SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

The alternatively spliced V region contributes to the differential incorporation of plasma and cellular fibronectins into fibrin clots

During blood clot formation in vivo, plasma fibronectin (pFN) is cross-linked to fibrin by coagulation factor XIIIa. Cellular FN (cFN), which localizes to connective tissue, is distinguished from pFN by the inclusion of alternatively spliced segments. To determine if these two FNs are functionally equivalent in blood clotting, the cross-linking of rat pFN and cFN to fibrin was compared in an in vitro clotting assay. Fibrinogen and FN were incubated at physiological ratios in the presence of thrombin and factor XIIIa. Cross-linking of FN to fibrin was monitored by SDS-PAGE and immunoblotting. Over 24 h, cFN was incorporated at a significantly slower rate than pFN and was not completely cross-linked to fibrin at a temperature that favors this interaction (0 degrees C). This difference was observed with purified fibrinogens from human, rat, and bovine and with rat plasma and was maintained even after incubation of pFN with rat fibroblasts for several days. Using the same assay, purified recombinant V(+)-V0 and V(+)-V+ FN dimers resembling pFN and cFN, respectively, showed a similar difference in cross-linking kinetics. These results suggest that the asymmetric distribution of the V region among pFN dimers plays a role in regulating its incorporation into blood clots. In fibrin clots, cFN was converted into a set of cross-linked intermediates distinct from those of pFN. For example, while pFN was initially cross-linked into a pFN-fibrin alpha heterodimer, this product was not a major intermediate in clots formed with cFN. This finding, in conjunction with evidence for the formation of factor XIIIa-catalyzed cFN-cFN cross-links, indicated that cFN molecules interact with each other, and with fibrin, differently from pFN. Together, these results show an important functional distinction between pFN and cFN.     

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO⁻, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO⁻ (25–300 µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000 µM stressor. Concentrations of ONOO⁻ affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO⁻ (threshold conc. 100 µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO⁻ (threshold conc. 100 µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO⁻ concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO⁻ were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO⁻ (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO⁻, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO⁻ concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.     

The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.     

Fibrinolytic and thrombolytic properties of thiol-dependent serine proteinase from Thermoactinomyces vulgaris in vitro

Fibrinolytic and thrombolytic properties of the subtilisin-like thiol-dependent serine proteinase were studied. At concentrations from 50 to 4000 micrograms/ml the enzyme causes lysis of fibrin plates and activates plasminogen. At concentrations above 100 micrograms/ml it shows a pronounced thrombolytic effect on the clots formed in vitro from both plasma and human and rat blood. Plasma inhibitors partly inactivate the thiol-dependent serine proteinase. The enzyme hydrolyses also fibrinogen, thrombin, plasmin and plasminogen.     

Coarse-grained molecular dynamics simulations of fibrin polymerization: effects of thrombin concentration on fibrin clot structure

Studies suggest that patients with deep vein thrombosis and diabetes often have hypercoagulable blood plasma, leading to a higher risk of thromboembolism formation through the rupture of blood clots, which may lead to stroke and death. Despite many advances in the field of blood clot formation and thrombosis, the influence of mechanical properties of fibrin in the formation of thromboembolisms in platelet-poor plasma is poorly understood. In this paper, we combine the concepts of reactive molecular dynamics and coarse-grained molecular modeling to predict the complex network formation of fibrin clots and the branching of fibrin monomers. The 340-kDa fibrinogen molecule was converted into a coarse-grained molecule with nine beads, and using our customized reactive potentials, we simulated the formation and polymerization process of a fibrin clot. The results show that higher concentrations of thrombin result in higher branch-point formation in the fibrin clot structure. Our results also highlight many interesting properties, such as the formation of thicker or thinner fibers depending on the thrombin concentration. To the best of our knowledge, this is the first successful molecular polymerization study of fibrin clots to focus on thrombin concentration.     

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn²⁺ in this interaction because Zn²⁺ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn²⁺ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn²⁺-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn²⁺-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn²⁺ is present. These results reveal the mechanism by which Zn²⁺ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.     

Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome): Association with Thrombin Generation and Eosinophilia

Objectives

Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome), we investigated whether fibrin clot properties are unfavorably altered in EGPA.

Methods

Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male), aged 48 (range, 21–80) years. The control group comprised 34 age- and sex- matched volunteers.

Results

Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, K_s, 7.30±0.93 vs 10.14±1.07 10⁻⁹ cm²), faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s), thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07), higher maximum levels of D-dimer released from clots (DD_max 4.10±0.46 vs 3.54±0.35 mg/L), and prolonged clot lysis time (t_50%; 9.50±1.45 vs 7.56±0.87 min); all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that K_s was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t_50%.

Conclusion

This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease.     

Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria

Aeromonas sobria infection often advances to sepsis, in which interaction of bacterial components with plasma proteins possibly causes various disorders. This bacterium releases a serine protease (ASP), a putative virulence factor, and binds to fibrinogen. To study the ASP effect on fibrinogen, we incubated fibrinogen or plasma with ASP and investigated their clotting elicited by thrombin, which converts fibrinogen to a fibrin clot. Enzymatically active ASP retarded plasma clotting in a dose-dependent manner starting at an ASP concentration of 10 nM. ASP also retarded fibrinogen clotting at 3 nM and above, which appeared to correspond to ASP cleavage of fibrinogen at the A alpha-chain. Consistent with containing serine protease activity for an ASP-specific substrate, the culture supernatant of an ASP gene-introduced strain retarded plasma and fibrinogen clotting more than that of the wild-type strain. The culture supernatant of an ASP gene-disrupted strain that releases negligible serine protease activity for the ASP-specific substrate did not affect plasma clotting. These results indicate that ASP is the main fibrinogenolytic protease released from A. sobria. Impaired plasma clottability induction through fibrinogen degradation is a new virulence activity of ASP and may contribute to hemorrhagic tendencies in sepsis caused by infection with this bacterium.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Disseminated intravascular coagulation following intravenous Corynebacterium parvum injection in the rat

Summary Corynebacterium parvum (C. parvum) was administered by a single IV injection (14 mg/kg) to rats, and platelet counts, plasma fibrinogen concentrations and thrombin clotting times were monitored for up to 7 weeks. During this time histological and ultrastructural studies were also conducted. Thrombocytopoenia, hypofibrinogenaemia, and prolongation of the thrombin clotting time rapidly followed C. parvum injection and were accompanied by the appearance of platelet clumps and fibrin within blood vessels in a variety of tissues. This initial episode of disseminated intravascular coagulation (DIC) subsided 12–24 h after injection, but a more prolonged second episode of DIC occurred 1–3 days after injection. The results suggest that caution should be observed when systemic immunotherapy with C. parvum is proposed.     

Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.     

Mechanisms involved in fibrin formation

That the role of thrombin in the conversion of fibrinogen to fibrin is essentially enzymatic, is established not only by the minute amounts of thrombin which are effective but also by the complete independence of fibrin yields and thrombin concentrations over a very wide range of thrombin dilutions and clotting times. The thrombin-fibrinogen reaction, in the phase beyond the "latent period" at least, seems fundamentally "first order." Technical requirements of the experiments leading to these conclusions include: (1) a highly purified (e.g. 97 per cent "clottable") fibrinogen, (2) absence of traces of thrombic impurities in the fibrinogen, (3) absence of fibrinolytic protease contaminant of the thrombin and the fibrinogen, and (4) sufficient stability of the thrombin even at very high dilutions. Four conditions affecting thrombin stability have been investigated. Fibrin yields are not significantly modified by numerous experimental circumstances that influence the clotting time, such as (1) temperature, (2) pH, (3) non-specific salt action due to electrical (ionic) charges, which alter the Coulomb forces involved in the fibrillar aggregation, (4) specific ion effects, whether clot-accelerating (e.g. Ca⁺⁺) or clot-inhibitory (e.g. Fe(CN)₆'), (5) occluding (adsorptive) colloids, which have a "fibrinoplastic" action, e.g. (a) acacia and probably (b) fibrinogen which has been mildly "denatured" by salt-heating, acidification, etc. The data with which several European workers have attempted to substantiate the idea of a two-stage thrombin-fibrinogen reaction with an intermediary "profibrin" (allegedly partly "denatured") have been reanalyzed with controls which lead us to very different conclusions, viz. (1) denaturation and fibrin formation are independent; (2) partial denaturation is "fibrinoplastic" (see above); and (3) conditions of strong salinity and acid pH (5.1) usually do not completely prevent the thrombin-fibrinogen reaction but merely prolong the "latent" phase and lessen the time required for completion of essentially the same reaction (fibrin polymerization) when more favorable clotting conditions are restored. Thus, our experiments advance the modern concepts concerning the coagulation mechanisms along lines that, for the most part, agree with those of the Harvard physical chemists, and we oppose the European views concerning a two-stage reaction, "profibrin," and "the denaturase theory" of clotting.