Thrombin-induced Clots Research Articles

DNA and histones impair the mechanical stability and lytic susceptibility of fibrin formed by staphylocoagulase.

Staphylocoagulase (SCG) is a virulence factor of Staphylococcus aureus, one of the most lethal pathogens of our times. The complex of SCG with prothrombin (SCG/ProT) can clot fibrinogen, and SCG/ProT-induced fibrin and plasma clots have been described to show decreased mechanical and lytic resistance, which may contribute to septic emboli from infected cardiac vegetations. At infection sites, neutrophils can release DNA and histones, as parts of neutrophil extracellular traps (NETs), which in turn favor thrombosis, inhibit fibrinolysis and strengthen clot structure. To characterize the combined effects of major NET-components (DNA, histone H1 and H3) on SCG/ProT-induced clot structure, mechanical and lytic stability. Recombinant SCG was used to clot purified fibrinogen and plasma. The kinetics of formation and lysis of fibrin and plasma clots containing H1 or core histones+/-DNA were followed by turbidimetry. Fibrin structure and mechanical stability were characterized with scanning electron microscopy, pressure-driven permeation, and oscillation rheometry. Histones and DNA favored the formation of thicker fibrin fibers and a more heterogeneous clot structure including high porosity with H1 histone, whereas low porosity with core histones and DNA. As opposed to previous observations with thrombin-induced clots, SCG/ProT-induced fibrin was not mechanically stabilized by histones. Similarly to thrombin-induced clots, the DNA-histone complexes prolonged fibrinolysis with tissue-type plasminogen activator (up to 2-fold). The anti-fibrinolytic effect of the DNA and DNA-H3 complex was observed in plasma clots too. Heparin (low molecular weight) accelerated the lysis of SCG/ProT-clots from plasma, even if DNA and histones were also present. In the interplay of NETs and fibrin formed by SCG, DNA and histones promote structural heterogeneity in the clots, and fail to stabilize them against mechanical stress. The DNA-histone complexes render the SCG-fibrin more resistant to lysis and thereby less prone to embolization.

Novel characteristics of soluble fibrin: hypercoagulability and acceleration of blood sedimentation rate mediated by its generation of erythrocyte-linked fibers

Soluble fibrin (SF) in blood consists of monomers lacking both fibrinopeptides A with a minor population in multimeric clusters. It is a substantial component of isolated fibrinogen (fg), which spontaneously self-assembles into protofibrils progressing to fibers at sub-physiologic temperatures, a process enhanced by adsorption to hydrophobic and some metal surfaces. Comparisons of SF-rich (FR) and SF-depleted (FD) fg isolates disclosed distinct molecular imprints of each via an adsorption/desorption procedure using gold surfaced silica microplates. Accelerated plasminogen activator-induced lysis and decreased stiffness (G′) of thrombin-induced FR fg clots were revealed by thomboelastography. Erythrocyte sedimentation (ESR) in afibrinogenemic plasma (Hematocrit 25–33%) was accelerated by FR fg nearly threefold that of FD fg. Stained smears disclosed frequent rouleaux formations and fibers linking stacked erythrocytes in contrast to no rouleaux by FD fg. Rouleaux formations were more pronounced at 4 °C than at ambient temperatures and at fiber-membrane contacts displayed irregular, knobby membrane contours. One of several FR fg isolates also displayed incomplete fiber networks in cell-free areas. What is more, pre-mixing FR fg with each of three monoclonal IgG anti-fg antibodies at 1.5 mol/mol fg, that inhibited fibrin polymerization, prevented rouleaux formation save occasional 2–4 erythrocyte aggregates. We conclude that spontaneously generated SF fibers bound to erythrocytes forming intercellular links culminating in rouleaux formation and ensuing ESR acceleration which in clinical settings reflects hypercoagulability. Also, the results can explain the reported fg binding to erythrocytes via ligands such as CD47, stable in vivo RBC aggregates in capillaries, and red areas of pathologic thrombi.

Thromboelastographic phenotypes of fibrinogen and its variants: Clinical and non-clinical implications

IntroductionThromboelastography (TEG), a widely used clinical point of care coagulation test, is poorly understood. To investigate its fibrin determinants we used normal and variant fibrinogen isolates. Materials and MethodsWe focused mainly on the TEG maximum signal amplitude (MA), a shear modulus and clot stiffness indicator. Isolates included normal des-αC, cord, and abnormal congenital variants with amino acid substitutions or deletions that impaired fibrin polymerization. Heterophenotypic congenital isolates were from cryoprecipitate-depleted plasma owing to their more diminished clot MA than their cryoprecipitate counterparts. By colorimetric assay, the amount of fibrinogen adsorbed by untreated TEG cups was 83.5±12.4 pM/cm2, n=18. Thrombin-induced clots were obtained at pH6.4 or 7.4, the latter containing 8mM CaCl2, and 14% afibrinogenemic plasma with and without gel-sieved platelets. Results and ConclusionsMeasured by the water droplet contact angle, >90% reduction of surface hydrophobicity by exposure of TEG cup and pin to ozone plasma decreased MA by 74%. Increasing normal fibrinogen or thrombin concentrations progressively increased MA. Platelets increased MA further ~2 fold, except for ≥10 fold for des-αC clots. Examined in the absence of platelets, MA of heterophenotypic fibrin variants averaged 21%, n=15. The results imply that essential MA determinants include hydrophobic fibrinogen/fibrin adsorption and each polymerization contact site, with substantial enhancement by platelets. Also, cryoprecipitate-harvested soluble fibrinogen/fibrin complexes contained mostly normal molecules, while cryoprecipitate-depleted plasma contained mostly variant molecules. Moreover, significantly decreased MA by fibrinogen anomalies and/or low level thrombin generation can potentially impact clinical interpretation of MA.

Homophenotypic Aα R16H fibrinogen (Kingsport): uniquely altered polymerization associated with slower fibrinopeptide A than fibrinopeptide B release

We detail for the first time the uniquely altered fibrin polymerization of homophenotypic Aalpha R16H dysfibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed fibrinopeptide A release approximately six times slower than its fibrinopeptide B. Turbidimetric analyses revealed unimpaired fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB fibrin monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB fibrin monomer properties attenuate their des-AA fibrin counterparts.

Differences in clot preparation determine outcome of recombinant tissue plasminogen activator treatment in experimental thromboembolic stroke.

Thrombin-induced clots used in experimental thromboembolic stroke differ from clots forming spontaneously under clinical conditions. We investigated whether this difference influences the efficacy and outcome of thrombolytic treatment. In rats, the middle cerebral artery was occluded by intracarotid injection of fibrin-rich clots, prepared either according to established methods by adding thrombin to freshly drawn arterial blood or by spontaneous coagulation. The mechanical properties of clots were determined in vitro by elasticity and plasticity tests. One hour after embolism, thrombolysis was started by intra-arterial application of recombinant tissue plasminogen activator (rtPA) (10 mg/kg). Treatment efficacy was monitored by MR measurements of blood perfusion, apparent diffusion coefficient (ADC), T2 relaxation time and blood-brain barrier permeability, and by pictorial measurements of ATP and pH. Thrombin-induced clots were classified as elastic, and spontaneously forming clots were classified as plastic. Middle cerebral artery embolism with thrombin-induced or spontaneously forming clots led to similar reduction of perfusion and ADC, but rtPA treatment efficacy differed greatly. In the spontaneously forming clot group, blood perfusion returned to or above control within 2 hours, ADC and ATP normalized, tissue pH exhibited alkalosis, and T2 and blood-brain barrier permeability did not change. In the thrombin-induced clot group, in contrast, blood reperfusion was delayed, ADC and ATP remained reduced, tissue pH was acidic, and edema developed, as reflected by increased T2 and blood-brain barrier permeability. rtPA-induced thrombolysis promotes rapid reperfusion and tissue recovery in animals embolized with spontaneously forming clots but not in those embolized with thrombin-induced clots. This difference is explained by the different mechanical and possibly molecular consequences of clot preparation and must be considered for the interpretation of thrombolysis experiments.

Batroxobin-induced clots exhibit delayed and reduced platelet contractile force in some patients with clotting factor deficiencies

Thrombin causes platelet activation via multiple pathways, and deficient thrombin generation reduces platelet contractile force (PCF) during clot retraction. We hypothesized that PCF in blood samples from clotting factor-deficient patients would be diminished due to delayed or deficient thrombin generation. Blood samples from patients with fibrinogen, and factor V, VII, VIII, IX, X, XI and XIII deficiencies were compared to samples from normal controls. PCF in patient blood clotted with thrombin (1 NIH UmL(-1)) was compared to PCF in clots formed with batroxobin (0.25 micro g mL(-1)). PCF in the former should be normal, but PCF in the latter is dependent on thrombin generation within the sample and might be deficient. In factor VII-(n = 2, P < 0.05), factor VIII-(n = 6, P < 0.005) and factor XI-(n = 2, P < 0.05) deficient platelet-rich plasmas, PCF in batroxobin-induced clots was significantly lower than in thrombin-induced clots. In factor IX deficiency (n = 2), one patient had a dramatic reduction in PCF while a second patient had increased PCF. PCF was insignificantly (P = 0.346) reduced in two patients with factor X deficiency, and was normal in one patient with factor V deficiency. The factor X result is consistent with work in model systems, which indicates that as little as 1-3% factor X activity is sufficient to restore thrombin generation to normal. The factor V result probably indicates that the deficiency is incomplete. PCF in thrombin-induced clots was normal in all of these patients. Low fibrinogen and factor XIII deficiency reduced PCF in both thrombin- and batroxobin-induced clots. These results indicate that PCF is reduced, probably due to delayed thrombin generation, in some factor-deficient platelet-rich plasma samples.

Proinflammatory Cytokines Are Not Released in the Circulation Following Acute Pulmonary Thromboembolism in Pigs

Histological examination of acute lung injury associated with sepsis often revealed thromboembolic lesions in the pulmonary microcirculation. Several inflammatory mediators such as platelet activating factor, thromboxane, and endothelins have also been implicated in the pathogenesis of acute pulmonary thromboembolism (APTE). In the present study we examined the roles of three proinflammatory cytokines, tumor necrosis factor- f (TNF- f ), interleukin 1 g (IL-1 g ), and IL-8, in the early phase of APTE. APTE was induced in 13 anesthetized piglets (22 - 4 kg) by injecting thrombin-induced blood clots directly into the left lower lobar pulmonary artery. Five animals that received only warm sterile saline served as controls. Arterial plasma samples were collected regularly over 8 h so that cytokine levels could be measured later by enzyme-linked immunosorbent assay (ELISA). Administration of clots doubled the mean pulmonary arterial pressure (from 13 - 5 to 26 - 7 mm Hg) and caused significant decrease in arterial oxygen tension (PaO 2 from 390 - 85 to 256 - 89 mm Hg while the FiO 2 was maintained at 1.0). Mean arterial blood pressure and cardiac output remained comparable throughout the experiments after initial fluid resuscitation. Plasma levels of TNF- f , IL-1 g , and IL-8 were not significantly increased in the APTE group when compared with their baseline values or the control group. Our results thus show that APTE is associated with pulmonary hypertension and deterioration of gas exchange but not with the systemic release of TNF- f , IL-1 g , or IL-8. We conclude that these cytokines have minimal impact on the systemic circulation during APTE.

Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis.

In this study, we investigated tissue-type plasminogen activator (tPA)-induced lysis of glutamic acid (glu)-plasminogen-containing or lysine (lys)-plasminogen-containing thrombin-induced fibrin clots. We measured clot development and plasmin-mediated clot disintegration by thromboelastography, and used scanning electron microscopy (SEM) to document the structural changes taking place during clot formation and lysis. These events occurred in three overlapping stages, which were initiated by the addition of thrombin, resulting first in fibrin polymerization and clot network organization (Stage I). Autolytic plasmin cleavage of glu-plasminogen at lys-77 generates lys-plasminogen, exposing lysine binding sites in its kringle domains. The presence of lys-plasminogen within the thrombin-induced fibrin clot enhanced network reorganization to form thicker fibers as well as globular complexes containing fibrin and lys-plasminogen having a greater level of turbidity and a higher elastic modulus (G) than occurred with thrombin alone. Lys-plasminogen or glu-plasminogen that had been incorporated into the fibrin clot was activated to plasmin by tPA admixed with the thrombin, and led directly to clot disintegration (Stage II) concomitant with fibrin network reorganization. The onset of Stage III (clot dissolution) was signaled by a sustained secondary rise in turbidity that was due to the combined effects of lys-plasminogen presence or its conversion from glu-plasminogen, plus clot network reorganization. SEM images documented dynamic structural changes in the lysing fibrin network and showed that the secondary turbidity rise was due to extensive reorganization of severed fibrils and fibers to form wide, occasionally branched fibers. These degraded structures contributed little, if anything, to the structural integrity of the residual clot, and eventually collapsed completely during the course of progressive clot dissolution. These results provide new perspectives on the major structural events that occur in the fibrin clot matrix during fibrinolysis.

The Influence of Fibrinogen and Fibrin on Thrombin Generation - Evidence for Feedback Activation of the Clotting System by Clot Bound Thrombin

In plasma the bulk of thrombin generation takes place after a clot has formed. We therefore investigated in what way the clot influences thrombin generation in plasma. The forming clot withdraws thrombin from free solution. Consequently less thrombin activity is found and less thrombin-inhibitor complexes are formed. The thrombin that is adsorbed to the clot reduces the lag time before thrombin generation in intrinsically or extrinsically triggered platelet poor plasma as well as in platelet rich plasma. We investigated the mechanism of this activation. Clots were obtained by recalcification of plasma or by the addition of thrombin-like enzymes (Reptilase, Agihal) from snake venoms. They were thoroughly washed until the washing fluid was devoid of any detectable clotting enzyme activity. In platelet poor plasma (PPP), thrombin-induced clots shorten the factor Va-dependent lag-time of thrombin generation in the extrinsic system as well as the factor VIIIa-dependent thrombin generation in the intrinsic system. Factor V or factor VII preparations that in itself hardly influence thrombin generation patterns acquire the capacity to shorten these lag-times when incubated with clot. The last washing fluid of the clot is inactive. Snake venom induced clots are not active either. Clots that are incubated in heparinised plasma for 1 h or more are as active as clots from normal plasma are. A role of factor Xa can not be excluded but must be minor because a clot made by addition of thrombin to plasma from which the factors II, VII, IX and X have been removed is as active as a clot from normal plasma is.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc Alters Fibrin Ultrastructure

Turbidimetric studies indicate that Zn(II) accelerates fibrin gelation [decreases clotting time (CT)] and increases maximal fibrin clot turbidity. For any given level of fibrinogen (0.2-2.6 mg/ml), the relative fibrin turbidity of thrombin-induced clots increases with Zn(II) in a concentration dependent manner. Zinc-associated turbidity increases are also observed in the presence of 2 mM (Ca(II). With citrate, similar turbidity increases are observed, though at higher cation levels. Thus, turbidimetry indicates that the gel formed with Zn(II) is coarser, or has thicker fibre strands. SEM micrographs confirm that fibre thickness ranges from 260 A to 2600 A, when Zn(II) levels range from 0-50 uM. With citrate, TEM micrographs reveal a more than 20 X fold increase in fibre diameter (100 A- greater than 2000 A) with higher Zn(II) (less than 1 mM) levels. Based on a fibrin monomer cross-section of approximately 60 A, the electron micrographs indicate that depending on the Zn(II) levels, fibrin strands are composed of between 2 to 40 monomeric fibrin molecules. Thus, at physiologically relevant levels, Zn(II) can drastically modulate fibrin ultrastructure.

The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper)

A. S. Kamiguti, M. C. C. Sousa e Silva, P. Morena and L. Nahas. The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper). Toxicon 23, 383 – 391, 1985 — An inhibitory effect of Bothrops castelnaudi venom was observed on the following systems: prothrombin time, activated partial thromboplastin time, thrombin time, thromboplastin generation time, activation of factor X by Russell's viper venom and Russell's viper venom activated factor X (factor X a). This effect did not require previous incubation and was prevented by the addition of Bothrops-antivenom. The prolonged activated partial thromboplastin time was not shortened by increased phospholipid concentration (0.5 – 10 mg/ml), suggesting that the inhibitory effect is not due to an anti-phospholipid activity. No significant fibrinogenolytic activity was detected upon incubation of human fibrinogen with the venom, since physiological levels of thrombin-clottable material were still present. Compared to Bothrops jararaca venom, the proteolytic activity on casein and on azocoll was very low. Thrombin-induced clots of human plasma and fibrinogen were not lysed by the venom within 24 hr. The results indicate that the anticoagulant effect of Bothrops castelnaudi venom is exerted at least at two levels of the blood coagulation mechanism: (1) before prothrombin activation, by inhibiting factor X-activation and factor X a activity; (2) by direct action on thrombin.

Evidence of fibrinogen degradation in rat anaphylaxis.

In active anaphylactic shock of rats pretreated with Bordetella pertussis vaccine, both plasma thrombin clotting time and the amount of antigenically active fibrinogen degradation products in the serum were increased. The formation of clottable fibrinogen fragments was shown by SDS polyacrylamide gel electrophoresis of thrombin-induced clots. When plasma of rats pretreated with 125I rat fibrinogen and then subjected to anaphylaxis was submitted to SDS polyacrylamide gel electrophoresis, fibrinogen-split products were also detected. Fibrinogen degradation results from the proteolytic effect of an activated fibrinolytic enzyme.

Nonenzymatic polymerization of fibrinogen by protamine sulfate An electron microscope study

The fine structure of polymers resulting from the action of thrombin and protamine sulfate on three specimens of purified human fibrinogen was studied with the electron microscope. The basic pattern of both polymers, “protamine sulfate-fibrin” and “thrombin-fibrin”, was very similar. The average periodicity of thrombin-fibrin was 239 Å in shadowed and 228 Å in negatively-stained preparations. The average periodicity of protamine sulfate-fibrin was 225 Å in shadowed and 223 Å in stained preparations. The network of protamine sulfate-fibrin formed from one specimen of fibrinogen (Cutter, Calif.) was extensive and exhibited branching typical of that observed in thrombin-induced clots from all specimens of fibrinogen. The networks of protamine sulfate-fibrin formed from two other specimens of fibrinogen (Kabi, Sweden and Jamieson, Washington, D.C.) were limited and were composed of short, broad strands with bluntly tapered ends. It is postulated that protamine sulfate brings about nonenzymatic polymerization of fibrinogen by neutralizing the negative charge on fibrinopeptides.

Studies on the heterogeneity of fibrinolytic activity in artificially induced intravascular clots.

SUMMARY This study was designed to investigate the effectiveness of enhancing local fibrinolytic actvity in intravascular clots preventing side reactions resulting from fibrinolytic activation in the circulating blood. For this purpose, the following procedures were performed. Artificial intravenous clots were induced in femoral and external jugular veins of dogs by three different methods. To dogs at various time intervals after clot formation, EACA or trans·AMCHA was intr a venously injected following with simultaneous human serum, and then various units of streptokinase or urokinase were intravenously administrated. Fibrinolytic activity of these clots after treatment was assayed according to the fibr in plate method, at the same time fibrinolytic activity of a thrombosed vein was assayed. The results of these experiments were as follows: 1) Artificially induced intravenous clots showed no fibrinolytic activity except thrombin-induced clots. 2) Activa tor activity of one hour to six day old clots treated with EACA or trans-AM CHA, was significantly enhanced by the simultaneous injection of strep­ tokinase or urokinase. 3) Activator activity of intima of a thrombosed vein was also enhanced with streptokinase or urokinase. 4) The administration of EACA or trans-AMCHA did not sacrifice the fibr­ inolytic activation in an intravenous clot with streptokinase or urokinase. 5) A fibrinolytic mechanism in an intravascular clot showed different charac­ terist ics from that of the circulation.

Formation and Properties of Fibrin Clots Resulting from Staphylocoagulase (SC) Action

SummarySome properties of the fibrin clots formed under the influence of staphylocoagulase (SC) and thrombin were compared.1. Mechanical properties of SC-induced clots were found to be different from thrombin-induced clots. Maximal amplitude in TEG was significantly smaller in the case of SC.2. In the system containing all other necessary components for fibrin stabilization, SC in contrasts to thrombin did not produce stabilized clots. It was shown that this was due to the lack of FSF activation by SC. SC induced fibrin was easily stabilized by the addition of FSF activated by thrombin. No difference in sialic acid content has been found when SC and thrombin induced fibrins were compared.3. Neither clotting nor esterase activity of SC were inhibited by heparin and hirudin.4. Fibrinogen degradation products (FDP) inhibit clotting activity of SC. Both early and late FDP seem to affect this activity in a similar degree, whereas early FDP show much more pronounced effect in the case of thrombin induced clotting.5. The SC-clots are much more resistant to plasmin action, although SC itself does not inhibit plasmin caseinolytic activity.6. The significance of these findings is discussed.