Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0?kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′ tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.     

Adherence of Candida species to fibrin clots in vitro

The adherence of six Candida species to fibrin clots was studied using a simple, in vitro technique. Yeast suspensions were incubated with fibrin clots and the number of adherent organisms quantified as follows: after washing, the clots were subjected to vortex mixing and the number of CPU's which subsequently grew on Sabourauds medium were counted. Adhesion was directly proportional to the concentration of Candida species in the suspension (r=0.99 p<0.001). C. albicans and C. tropicalis exhibited marked adherence whereas C. krusei, C. gulliermondi and C. glabrata adhered less readily. C. parapsilosis was intermediate in its ability to adhere.     

Interaction of the fibrinogen-binding tetrapeptide Gly-Pro-Arg-Pro with fine clots and oligomers of alpha-fibrin; comparisons with alpha beta-fibrin

The tetrapeptide Gly-Pro-Arg-Pro(GPRP) was introduced by diffusion into fine unligated clots formed from human fibrinogen at pH 8.5 and ionic strength 0.45 by batroxobin (αβ-fibrin) and by thrombin (α-fibrin). The α-fibrin clots were essentially liquefied at GPRP concentrations above 1 mM and αβ-fibrin clots above 15 mM, and the degree of polymerization of the resulting oligomers decreased progressively with increasing GPRP concentration as shown by γ-γ ligation with factor XIIIa and subsequent gel electrophoresis. Much smaller concentrations of GPRP, when introduced into unligated clots by diffusion, were sufficient to modify their mechanical properties profoundly. The shear modulus of elasticity G₂₅ measured 25 s after imposition of stress fell, for example, by a factor of 0.4 at 0.1 mM GPRP in α-fibrin and at 1.1 mM in αβ-fibrin. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. This behavior, and the corresponding decrease in steady flow viscosity, may be interpreted in terms of competition of GPRP with A sites on the E domains of fibrin monomers for bidning to “a” sites on the D domains, resulting in a moderate increase with increasing GPRP concentration of the average proportion of severed network strands and an enormous increase in the rate at which all strands dissociate and reassociate. Reassociation of severed strands in new configurations is a necessary corollary since the differential modulus or compliance remains constant during creep and creep recovery. The greater susceptibility of α-fibrin clots to interaction with GPRP is attributed to stabilization of contacts between monomer units by Bb associations in αβ-fibrin. Ligated clots, with or without GPRP, exhibited essentially no time-dependent creep and no irrecoverable deformation, corresponding to an absence of any severance of network strands.     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.     

Ligation of fibrinogen by factor XIIIa with dithiothreitol: mechanical properties of ligated fibrinogen gels

Human fibrinogen (concentration 8.4 mg/mL) was ligated (cross-linked) with factor XIIIa and dithiothreitol (DTT) at pH 8.5, ionic strength 0.45. With 7.5 μg/mL of factor XIIIa alone, there was almost no γ-γ ligation, but with 2 mM DTT added, oligomers appeared, and γ-γ and Aα-Aα ligation was nearly complete after 3 days. At 38 μg/mL of factor XIIIa, some γ-γ and Aα-Aα ligation occurred even without DTT. For fibrinogen concentrations of 4.0 and 8.4 mg/mL, 38 μ/mL factor XIIIa, 2.0 mM DTT, clot-like gels formed and the shear modulus of elasticity increased slowly over several days to a constant value. The final modulus was similar in magnitude to those of ligated clots of α-fibrin (clotted by thrombin) and α-fibrin (clotted by batroxobin) under the same conditions. However, the opacity was somewhat higher; whereas in fine fibrin clots there is minimal lateral association of the protofibrils, in fibrinogen gels at the same pH and ionic strength the protofibrils (which are presumably single chains of fibrinogen monomers joined end to end at their D domains) are evidently associated in bundles (although not to the degree seen in coarse fibrin clots). Creep and creep recovery measurements showed almost perfect elastic behavior, with essentially no creep under stress and complete recovery after removal of stress. The modulus was scarcely affected by introduction of lithium bromide by diffusion to a concentration of 0.6M, which in unligated fibrin clots causes substantial softening. Whereas in fine fibrin clots (both αβ-fibrin and α-fibrin) factor XIIIa causes only γ-γ ligation, addition of 2 mM DTT produced some α-α ligation in these also.     

体外循环血栓非侵入在线电阻抗成像方法

目的 在体外循环系统中,血栓的在线检测和可视化具有重要意义。本文提出了基于电阻抗成像（EIT）的体外循环血栓非侵入在线检测方法。方法 首先通过联合仿真研究了传感器尺寸对成像效果的影响。其次,根据仿真结果设计了直径为20 mm的16铜质电极EIT传感器,搭建了循环流动实验平台,并设计了静态和循环流动实验。使用尺寸为3～6 mm的猪血块代替血栓,将血块置于新鲜猪血样本中,采用Tikhonov正则化算法进行成像。将3 mm和5 mm的血块分别置于循环系统中,重建血块在传感器截面的大小和位置图像,并与高速相机拍摄结果进行对比。结果 仿真结果显示当目标物与传感器面积比（AR）不小于0.01时,传感器直径为20 mm和30 mm对应的图像相关系数（IC）均大于0.06,成像效果较好。静态成像结果显示,相对尺寸覆盖率误差（RCR）小于等于0.1。循环流动实验显示,血块经过传感器时,检测到归一化后的相对电导率变化值分别为80和200,结果显示该方法能够检测到循环系统中的血块。结论 该方法具有实时性和非侵入的优点,有望应用于体外血栓的检测。     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

Protofibrin clots induced by calcium and zinc

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin-activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration-dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)- or Zn(II)-induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation-sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.     

Fibrin clot retraction by human skin fibroblasts: effects of ADP and thrombin.

Thrombin stimulated human skin fibroblasts to retract fibrin clots. When Bothrops marajoensis thrombinlike enzyme was substituted for thrombin, no retraction occurred. Fibroblasts were found to contain 12 nmole of ATP and 3.6 nmole of ADP/mg of protein, a value closely resembling that of nonmetabolic adenine nucleotides in platelets. Thrombin caused neither release of adenine nucleotides from the suspension of fibroblasts harvested enzymatically nor did addition of ADP stimulate fibroblasts to retract fibrin clots.     

Label-free high frame rate imaging of circulating blood clots using a dual modal ultrasound and photoacoustic system

Deep vein thrombosis (DVT) is a disorder when a blood clot (thrombus) is formed in one of the deep veins. These clots detach from the original sites and circulate in the blood stream at high velocities. Diagnosing these blood clots at an early stage is necessary to decide the treatment strategy. For label-free, in vivo, and real-time detection, high framerate photoacoustic imaging can be used. In this work, a dual modal clinical ultrasound and photoacoustic (PA) system is used for the high framerate PA imaging of circulating blood clots in blood at linear velocities up to 107 cm/sec. Blood clot had 1.4 times higher signal-to-noise ratio (SNR) in the static mode and 1.3 times higher SNR compared to blood PA signal in the flow experiments. This work demonstrates that fast-moving circulating blood clots are easy to recognize against the background PA signal and may aid in early diagnosis.     

Protein inhibitors of fibrin stabilizing factor FXIII

The ability of cysteine proteinase inhibitors (CPIs) isolated from tubers of potato (Solanum tuberosum) to suppress transpeptidase activity of fibrin stabilizing factor (FXIIIa) through the direct effect on the essential SH group of the enzyme active site has been studied. The formation of fibrin clots soluble in 5 M urea and 2% acetic acid as well as spectrophotometric turbidity analysis of the stabilization and resistance of fibrin clots formed in the presence of FXIIIa and CPIs from potato tubers to plasmin, and electrophoresis of reduced fibrin samples indicate the decrease or absence of covalent crosslinking of fibrin chains. In addition, CPIs added to the substrate proved to decelerate fibrinogen polymerization almost twice relative to control. It is concluded that natural CPIs can both take part in the regulation of FXIIIa transpeptidase activity in vitro and modify the substrate.     

Rheological studies of creep and creep recovery of unligated fibrin clots: comparison of clots prepared with thrombin and ancrod

Mechanical creep and creep recovery in small shearing deformations have been studied in unligated clots formed with both thrombin and ancrod. In thrombin clots, both A binding sites (which interact with “a” sites to link monomer units within a protofibril) and B sites (which interact with “b” sites to form links between protofibrils) are exposed to enable formation of linkages; in ancrod clots, only the A sites are exposed. Fine clots (with minimal lateral aggregation of protofibrils), coarse clots (with substantial aggregation of fibril bundles), and clots of intermediate coarseness were compared. Fine thrombin clots showed less creep at short times but more creep at long times than coarse or intermediate clots and had more irrecoverable deformation relative to the initial elastic deformation. Ancrod clots had greater irrecoverable deformation than the corresponding thrombin clots, both fine and coarse. The permanent deformation in fine ancrod clots was enormous, corresponding almost to fluid character; the rate of permanent deformation was larger than that in fine thrombin clots by more than two orders of magnitude. For all types of clots, differential measurements of compliance (or its reciprocal, elastic modulus), as well as the applicability of the Boltzmann superposition principle to calculation of creep recovery, showed that the overall density of structure remained constant throughout the mechanical history; i.e., if structural elements were breaking, they were reforming at the same rate in different configurations. The possibility that the weakness of ancrod clots is attributable to partial degradation of α-chains rather than absence of Bb linkages was eliminated by comparisons of clots made with thrombin, ancrod, and ancrod plus thrombin; the last two showed identical partial degradation of α-chains (by gel electrophoresis), but the first and third had essentially identical initial elastic moduli and creep behavior. Two alternative mechanisms for irrecoverable deformation in fine clots are discussed, involving rupture of protofibrils and slippage of twisted segments, respectively.     

A novel source of fibrinolytic activity: <Emphasis Type="Italic">Bionectria</Emphasis> sp., an unconventional enzyme-producing fungus isolated from Las Yungas rainforest (Tucumán,Argentina)

Fibrinolytic enzyme production was evaluated in fungal specimens isolated from the sub-tropical Las Yungas Pedemontana forest (Tucumán, Argentina). Proteolytic and fibrinolytic activities were evaluated in freeze-thaw crude extracts from 230 fungal isolates on 1% w/v skimmed-milk or 0.25% w/v fibrin-agar plates, respectively. Proteolytic activity was positive in 62% of the isolates, whilst only three of them were able to produce extracellular fibrinolytic enzymes on solid nutritive medium. Fibrinolytic-positive extracts were able to degrade fibrin clots in a direct plasminogen-independent way. Selected isolates were identified by sequencing the 26S rDNA D1/D2 domain. Isolates LY 4.1 and LY 4.4 showed a 99.9% similarity with Bionectria ochroleuca, while LY 4.2 showed a 99.9% identity with Cladosporium cladosporioides. Under submerged culture conditions, LY 4.1 and LY 4.4 were able to excrete fibrinolytic enzymes, reaching a maximum at 120 h of cultivation of 100.2 and 107.9 U/ml in plasmin-equivalent units, respectively. Fibrinolytic enzyme production could be scaled-up to fermenter scale reaching similar values. Fibrin zymography showed that fibrinolytic activity was associated with ~173-, 153- and 80-kDa protein fractions. Extracellular fibrinolytic enzymes from Bionectria species may be potentially related to pathogenesis mechanisms, as already demonstrated for serine-proteases from the nematicidal anamorph Clonostachys rosea. This work reveals the potential of Bionectria strains as an unconventional and unexplored production alternative to already known thrombolytic agents. The value of Las Yungas forests as a reservoir of fungal species with promising biotechnological value could be also highlighted.     

Peroxide-Induced Damage to Plasminogen Molecules

Doklady Biochemistry and Biophysics - Plasminogen is a zymogenic form of plasmin, an enzyme that plays a fundamental role in the dissolution of fibrin clots as well as in many other physiological...     

The marine bacterium Alteromonas piscicida--a producer of enzymes with thrombolytic action

The ability of marine bacteria A. piscicida to produce exoproteases that were able to lyse human blood clots has been studied. Optimal conditions for biosynthesis of these enzymes have been found. The enzyme has been partially purified. In concentration of 1 mg/ml it has activity corresponding to that of 500 micrograms/l plasmine and 100 micrograms/ml trypsine. The enzyme activity was completely inhibited after incubation in human blood plasma.     

Identification of factor XI deficiency in Holstein cattle in Turkey

Background  

Factor XI (FXI) is a plasma protein that participates in the formation of blood clots. Factor XI deficiency is autosomal recessive hereditary disorder that may be associated with excess bleeding in Holstein cattle.     

Chimeric derivative of fibrolase, a fibrinolytic enzyme from southern copperhead venom, possesses inhibitory activity on platelet aggregation

Fibrolase, a metalloproteinase isolated from the venom of Agkistrodon contortrix contortrix (southern copperhead snake), is a direct acting fibrinolytic enzyme that has been used to digest occlusive blood clots in animal models. The snake venom enzyme directly degrades fibrin associated with platelet rich blood clots and does not rely on plasminogen activation. Rethrombosis is a serious complication that is experienced in a significant percentage of patients treated with thrombolytic agents to remove occlusive vascular thrombi. The involvement of platelets in the initiation of rethrombosis is well known. Arg-Gly-Asp-(RGD)-containing agents have been shown to inhibit rethrombosis following thrombus dissolution by plasminogen activators. In an effort to create a more effective fibrinolytic enzyme and to target the enzyme to platelet-rich thrombi, thereby decreasing the potential for rethrombosis, a chimeric derivative of fibrolase has been produced. This report describes the construction and biochemical characterization of the chimeric enzyme and an evaluation of its in vitro activities. The chimera was formed by covalently incorporating an RGD-like peptide into fibrolase. The site of peptide attachment was determined to be a single lysine residue remote from the enzymes active site. Covalent modification of fibrolase with the RGD-like peptide did not inhibit either fibrinolytic activity of the enzyme nor platelet aggregation inhibitory activity of the peptide. The chimera not only retained the same level of enzymatic activity as native fibrolase, but also acquired the ability to inhibit platelet aggregation by binding to the fibrinogen receptor (integrin alphaIIbbeta3) on platelets.     

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.