A method for determining plasmin by the rate of fibrin gel lysis

A simple and sensitive method has been developed to assess the fibrinolytic activity of plasmin from the change in the column height of fibrin gel. Two conditions were used: 1) 37 degrees C and 16 h incubation at plasmin concentrations of 0.5-50 micrograms/ml and 2) 25 degrees C and 1-2.5 h incubation at plasmin concentrations of 50-1000 micrograms/ml. The method permits to observe the kinetics of fibrinolysis at plasmin concentrations higher that 10 micrograms/ml. The results have shown that the method is applicable for quantitation of plasminogen in human plasma. The method is precise and well reproducible.     

The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis

The effect of heparin on the proteolytic and fibrinolytic activities of plasmin and plasminogen was studied. Heparin at a concentration of 6.3.10(-6) M did not change the caseinolytic activity of plasmin and plasminogen stimulated by streptokinase but suppressed their fibrinolytic activity. At concentrations from 2.10(-8) to 0.5.10(-6) M heparin increased, whereas at 1.10(-6)-4.10(-6) M reduced the time of desAAfibrin clot half-lysis by plasmin. Within the concentration range of 2.10(-8) to 4.10(-6) M heparin did not change the time of the clot half-lysis by glu-plasminogen and slightly decreased the time of fibrin clot half-lysis by lys-plasminogen in the presence of the tissue activator. It was supposed that heparin inhibits the fibrinolytic effect of plasmin by way of formation of complexes with plasmin and reduction of plasmin specificity to the solid phase substrate, i. e., polymeric fibrin.     

Investigating the two regimes of fibrin clot lysis: an experimental and computational approach

It has been observed in vitro that complete clot lysis is generally preceded by a slow phase of lysis during which the degradation seems to be inefficient. However, this slow regime was merely noticed, but not yet quantitatively discussed. In our experiments, we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average, the slow regime lasts longer than the fast one, meaning that during most of the process, the lysis is ineffective. We proposed a computational model in which the properties of the binding of the proteins change during the lysis: first, the biochemical reactions take place at the surface of the fibrin fibers, then in the bulk, resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally.     

Activated thrombin-activatable fibrinolysis inhibitor reduces the ability of high molecular weight fibrin degradation products to protect plasmin from antiplasmin

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B-like plasma enzyme that can slow clot lysis by removing lysine residues exposed on fibrin as it is cleaved by plasmin. Previously, it was shown that fibrin treated with TAFIa is less able to promote plasminogen activation by tissue-type plasminogen activator. In this study, the effect of TAFIa modification of a fibrin surface on the rate of plasmin inhibition by antiplasmin was studied using high molecular weight fibrin degradation products (HMw-FDPs) as a soluble model for intact plasmin-modified fibrin. To quantify the inhibition, a novel end point assay was employed where plasmin, antiplasmin, and cofactors were mixed in the presence of a chromogenic substrate and the end point in the substrate hydrolysis reaction was used to measure the second order rate constant of inhibition. When HMw-FDPs were titrated in the presence of plasmin and antiplasmin, the rate constant for inhibition decreased by 16-fold at saturation (9.6 x 10(6) m(-1) s(-1) to 0.59 x 10(6) m(-1) s(-1)). When HMw-FDPs were pretreated with TAFIa, nearly two-thirds of the protective effect was lost. When 730 nm HMw-FDPs were treated for 20 min with TAFIa, the rate constant for plasmin inhibition was increased 3-fold from 1.9 x 10(6) m(-1) s(-1) to 6.2 x 10(6) m(-1) s(-1). Therefore, a novel mechanism was identified whereby TAFIa can modulate plasmin levels by increasing the susceptibility of plasmin to inhibition by antiplasmin.     

Effect of fragments E and D on the plasmin hydrolysis of the fibrin clot

It was found that fragments E (Mr = 45 000), DH (Mr = 95 000) and DL (Mr = 82 000) decrease the rate of plasmin hydrolysis of fibrin that is not cross-linked with factor XIII; the most effective inhibitor is fragment DL. The Kd values for the interactions of fragments E, DH and DL with plasmin are equal to 0.15, 0.4 and 0.04 microM, respectively.     

A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time

Background  

Determination of clot lysis times on whole blood, diluted whole blood, plasma or plasma fraction has been used for many years to assess the overall activity of the fibrinolytic system. We designed a completely computerised semi-automatic 8-channel device for measurement and determination of fibrin clot lysis. The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minute (range: 5 to 9999). We have used this new device for Euglobulin Clot Lysis Time (ECLT) determination, which is the most common test used in laboratories to estimate plasma fibrinolytic capacity.     

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the K4 and K5 plasmin heavy chain kringles in the fibrin clot structure destruction]

A comparative analysis of the rates of polymeric fibrin structure destruction by plasmin (Pm) and its proteolytic derivatives such as Val354-plasmin (c-Pm), Val442-plasmin (m-Pm) and Lys530-plasmin (mu-Pm) has been undertaken. It was shown, that Pm, c-Pm, m-Pm and mu-Pm at equal proteolytic activity, have dissolved fibrin clots with relative rates 40.3:38.0:4.6:1.0 correspondingly. The Pm, m-Pm and mu-Pm relative rates were changed by epsilon-aminocaproic acid to 4.6:1.5:1.0 correspondingly. In this case fibrin clot destruction time was increased for Pm and m-Pm and was not changed for mu-Pm. The rates of fibrinogen hydrolysis were nearly equal for these forms of enzyme. It was suggested, that the specific interactions between plasmin K4 and K5 kringles and solid phase fibrin substrate determine the polymer fibrin structure destruction rate.     

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.     

A colorimetric assay for measuring the lysis of a plasma clot.

The present study describes a simple, quantitative assay for measuring the lysis of a plasma clot. The principle of the assay is based on the release of Coomassie brilliant blue R-250 dye from the clot. Thirty microliters of freshly prepared Coomassie brilliant blue R-250 (1 mg/ml) was added to 200 microliters of diluted human plasma (1:5). After mixing, 100 microliters of thrombin (2.5 NIH units/ml) were added to mediate a plasma clot. One milliliter of streptokinase (0.1 mg/ml) was used as a plasminogen activator to initiate clot lysis. During the course of lysis, 100 microliters of soluble material were transferred to microtiter wells and the absorbance at 540 nm was determined as a measure of clot lysis. This assay was used to measure clot lysis in 18 human plasma samples. The colorimetric method (X) developed in this report correlated well with that determined using a conventional 125I-fibrinogen method (Y): Y = 0.83X + 7.98 (r = 0.91).     

A re-examination of the cleavage of fibrinogen and fibrin by plasmin.

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000 to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from its gamma' subunit of 38,000 molecular weight; however, the beta' subunit of 42,000 molecular weight and the alpha' subunit of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.     

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.     

A reconstituted dilute blood clot lysis assay for the medium throughput screening of thrombolytic compounds.

Antithrombotic and clotting factors have long been targets for drug discovery, necessitating the development of blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted blood clot lysis assay which eliminates the need for on-site donors. The assay uses whole blood stored at 4 degrees C obtained from a local blood bank, diluted 1:10 in phosphate buffer. This blood was supplemented with 125I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed clots was measured. The whole blood used in this assay, which had been stored at 4 degrees C for several days, no longer formed solid or retracting clots. Thus, platelets 5-7 days ex vivo (165 x 10(6) platelets) were added to the whole blood in the presence of thrombin (0.80 IU/ml) to form clots. Solid clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site blood donors.     

A fast-acting,modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy

To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.     

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Cardiovascular effects of epinephrine during rewarming from hypothermia in an intact animal model.

Rewarming from accidental hypothermia is often complicated by "rewarming shock," characterized by low cardiac output (CO) and a sudden fall in peripheral arterial pressure. In this study, we tested whether epinephrine (Epi) is able to prevent rewarming shock when given intravenously during rewarming from experimental hypothermia in doses tested to elevate CO and induce vasodilation, or lack of vasodilation, during normothermia. A rat model designed for circulatory studies during experimental hypothermia and rewarming was used. A total of six groups of animals were used: normothermic groups 1, 2, and 3 for dose-finding studies, and hypothermic groups 4, 5, and 6. At 20 and 24 degrees C during rewarming, group 4 (low-dose Epi) and group 5 (high-dose Epi) received bolus injections of 0.1 and 1.0 microg Epi, respectively. At 28 degrees C, Epi infusion was started in groups 4 and 5 with 0.125 and 1.25 microg/min, respectively. Group 6 served as saline control. After rewarming, both CO and stroke volume were restored in group 4, in contrast to groups 5 and 6, in which both CO and stroke volume remained significantly reduced (30%). Total peripheral resistance was significantly higher in group 5 during rewarming from 24 to 34 degrees C, compared with groups 4 and 6. This study shows that, in contrast to normothermic conditions, Epi infused during hypothermia induces vasoconstriction rather than vasodilation combined with lack of CO elevation. The apparent dissociation between myocardial and vascular responses to Epi at low temperatures may be related to hypothermia-induced myocardial failure and changes in temperature-dependent adrenoreceptor affinity.     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.     

A quantitative histological study of adrenal development during the perinatal period in intact and hypophysectomized pigs

Adrenal growth was studied between D70 of gestation (D0: day of mating) and D6 after birth in 33 fetuses and 11 new-born pigs of the Large White breed. Volume, height and mean surface, were estimated by zone, as well as cell number, cell size and nucleocytoplasmic ratio. The volume increased exponentially. In the cortex it was 4.0 mm3, 7.2 mm3, 10.5 mm3, 33.6 mm3, 55.3 mm3 at D70, D94, D106, D113 and D1-D6, respectively, after birth. In the medulla, the volume was 4.0 mm3, 6.3 mm3, 6.9 mm3, 9.6 mm3 and 20.6 mm3 at the same stages. This evolution was due to predominant longitudinal stretching between D94 and D106, then thickening, as shown by the relative variations of the height and the surface. The growth of the cortex corresponded to hyperplasia associated with hypertrophy after D110. In the medulla, hyperplasia predominated until D110; this was followed by hypertrophic phase. Twelve fetuses hypophysectomized at D44 or at D55 were recovered at D94, D106 and D112 at least 50 days after surgery. Hypophysectomy did not affect medullar development (volume and number of cells) but inhibited very significantly the growth of the cortical zone, particularly that of the fasciculata.