Functional determination of soluble fibrin polymers (SFP) in plasma.
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Arginine↗
Biomedical subjects
T W Stief
Publications and source records attributed to T W Stief.
Oxidized fibrin stimulates the activation of pro-urokinase and is the preferential substrate of human plasmin.
Activated phagocytes participate in physiological thrombolysis producing non-radical excited oxidants and the important proteases elastase and urokinase. The interaction of oxidized fibrin with the proteases of the fibrinolytic system is therefore physiologically relevant. Here it is shown that human pro-urokinase is activated three- to four-fold faster in the presence of an oxidized solid fibrin matrix. In contrast, oxidized fibrin did not favour the fibrinolytic activity of urokinase or t-PA. Measurement of urokinase antigen showed that urokinase bound slightly to strongly oxidized denatured fibrin, whereas pro-urokinase did not. Plasmin degraded oxidized fibrin more rapidly than non-oxidized fibrin. Thus, singlet molecular oxygen (1O2) converts fibrin to a form that stimulates the activation of plasminogen (bound to oxidized fibrin) by pro-urokinase and that of pro-urokinase by plasmin. The oxidative modification of fibrin by 1O2 is specific. In contrast to oxygen radicals (H2O2 in high concentration) 1O2 does not directly destroy protein chains but favours subsequent fibrinolysis. Thus 1O2 prepares fibrin for its specific degradation.
Enzyme Activation↗
The susceptibility of plasma to activation of fibrinolysis.
The susceptibility of blood to fibrinolytic activation is an important parameter to predict the possible clinical outcome of thrombolysis in routine hospital practice. This paper describes a rapid method which is based on the integrated plasmin activity of a sample and which therefore does not require standardization of different fibrinolytic factors, such as plasminogen activator inhibitor type 1. Plasma is incubated with either streptokinase, urokinase (u-PA), tissue plasminogen activator (t-PA) or plasminogen streptokinase activator complex (PSAC) at room temperature. This is followed by addition of KCl, the chromogenic plasmin substrate S-2251 and chloramine T. As a blank, KCl is added before the enzyme. The incubation time is shortened three-fold at 37 degrees C. The highest fibrinolytic activity, with the smallest standard deviation, was obtained with streptokinase. When the lowest activity measured was defined as 100%, the highest activities were 273%, 297%, 344% and 961% for streptokinase, urokinase, t-PA and APSAC respectively. The susceptibility to urokinase correlated with that of t-PA. Patients reacted differently to the different plasminogen activators. Therefore, the method allows the appropriate agent and amount to be chosen before thrombolysis is started.
Blood Chemical Analysis↗
Nonradical oxidants of the phagocyte type induce the activation of plasmatic single chain- urokinase.
Single chain- urokinase (scu-PA) is the proenzyme of the plasminogen activator urokinase (tcu-PA). In human blood scu-PA is of great stability. Activated phagocytes generate large amounts of single chain- urokinase and of reactive oxidants (chloramines and HOCl). Since these cells participate in physiologic fibrinolysis, we were interested in the interaction between plasmatic scu-PA and chloramines. The oxidants dose dependently induce the activation of plasmatic scu-PA. Optimal activation of scu-PA occurs at about 3-5 mmol/l of chloramine-T. The findings suggest a control mechanism of scu-PA stability/activity by oxidatively modifiable plasma proteins, such as alpha-2-antiplasmin. The oxidation mechanism seems to be mediated by singlet molecular oxygen, an excited oxygen species. Basing on this scu-PA/oxidant synergism a sensitive and fast functional assay of scu-PA in human plasma is presented. Plasmatic inhibitors normally interfering with functional scu-PA measurements are inactivated by addition of chloramine-T, imitating the physiological oxidants generated by activated phagocytes. The scu-PA concentration in plasma of n = 36 healthy individuals has been determined to be 5.8 +/- 1.6 ng/ml. The lower detection limit of plasma scu-PA by the procedure described is about 1.5 ng/ml of plasma. By means of this technique scu-PA concentration during thrombolytic therapy can be measured within minutes in undiluted (direct) plasma samples, allowing adjustments of the scu-PA dosage. The present study gives further credence for a role of singlet molecular oxygen, possibly a new type of locally acting hormones (autacoid), in the regulation of the fibrinolytic pathway.
Adult↗
Factor XIII of blood coagulation inhibits the oxidative phagocyte metabolism and suppresses the immune response in vivo.
Factor XIII of blood coagulation (F XIII) belongs to the family of transglutaminases and is a major cell product of certain subsets of macrophages. The gene for F XIIIA is coupled to the immune response genes of the HLA-region on chromosome 6. F XIII dose- dependently inhibits the in vitro chemiluminescence response of human phagocytes. About 0.1 units of F XIII/ml (final) decreased the chemiluminescence response to about 50%. In addition, about 0.6 units of F XIII/ml inhibits 50% of the release of the lysosomal hydrolase N-acetyl-beta glucosaminidase in both immune complex stimulated and unstimulated monocytes. Intraperitoneal application of F XIII reduced the activity of phagocytes in a F XIII dose dependent manner. 0.25 units of F XIII reduced the chemiluminescence reaction of murine peritoneal M phi to about 50% of the activity of PBS treated animals after 2 or 24 hours of in vivo incubation. In the Fisher/Lewis rats skin transplantation model, injections of 5 units of F XIII/animal on days 1-7 or on days 10-17 increased the survival times of the transplants from the control value of 17.0 +/- 1.4 to 26.0 +/- 2.0 and 23.0 +/- 2.4 days, respectively. F XIII may represent a novel and physiological immune suppressive agent for a broad range of human diseases of autoimmune character.
Acetylglucosaminidase↗
Nonradical excited oxygen species induce selective thrombolysis in vivo.
All the thrombolytic agents currently in clinical use act as plasminogen activators. In this study evidence is presented that also oxidants of the phagocyte type are of fibrinolytic efficiency in vivo. Activated phagocytes participate in physiologic fibrinolysis. The cells generate plasminogen activators and reactive oxidants of the nitrogen-chlorine type. Experimental mimicry of this oxidative inflammatory response induces selective thrombolysis in a rabbit jugular vein model. Intravenous bolus administration of sub-millimolar blood concentrations of chloramine-T resulted in thrombolysis after about 30 min without notable systemic toxicity; the coagulation parameters activated partial thromboplastin time (aPTT), thrombin time, fibrinogen, and alpha-2-antiplasmin were not influenced. Control experiments with 2000 IU of urokinase/kg induced thrombolysis after about 90 min with systemic changes of the hemostatic system. The fibrinolysis promoting effect of the oxidants of the phagocyte type could be inhibited by quenchers of singlet molecular oxygen and was not affected at all by inhibitors of oxygen radicals. The data gives evidence that nonradical excited oxygen species (NEOS) act as powerful pro-fibrinolytic and anti-coagulant agents in vivo. It might be suggested that NEOS could represent a novel class of regulators of the fibrinolytic system. The long lived and hydrophilic chloramine derivatives can either accumulate or diffuse far from their site of generation. Therefore, on the one hand oxidants in high (local) concentrations might be considered as direct pro-fibrinolytic agents due to their powerful protein modulating efficacy. On the other hand, oxidants at low concentrations may act as indirect pro-fibrinolytic compounds, i.e. as chemoattractants to concentrate phagocytes to the site of a thrombus. In this case the oxidants would play the role of signal elements faraway from the thrombus, a self amplifying mechanism possibly mediated by oxidation of blood arachidonat/lipid metabolites.
Animals↗
Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin.
Evidence has recently been presented that activated macrophages (M phi) express both urinary (u-PA) and tissue type (t-PA) plasminogen activator. Major cell products of M phi and polymorphonuclear neutrophils (PMN) are reactive oxidants of the HOCl/chloramine type. Since PMN and M phi are involved in inflammatory and fibrinolytic processes, we were interested in the interaction of u-PA, t-PA, and plasmin with oxidants of the leukocyte type. The enzymes were treated with chloramine-T, which at pH 8.5 is a selective oxidant for methionine residues. Oxidation by chloramine-T of t-PA abolishes about 40% of both stimulation susceptibility of t-PA by fibrinogen degradation products (FDP) and affinity of t-PA to FDP. However, the plasminogenolytic and amidolytic activity of unstimulated t-PA as well as the plasminogenolytic activity of u-PA and the amidolytic activity of plasmin are not impaired. Identification of the amino acid residues in the t-PA responsible for the interaction with fibrin might be of great importance in order to understand the mechanism of the clot- selectivity of t-PA. The present study gives evidence that fibrin specificity of t-PA partly depends on chloramine oxidizable amino acids, presumably methionine residues. Hence, experimental data on the interaction between t-PA and fibrin, using oxidized and labelled t-PA should be interpreted with caution. It may be suggested that oxidants of the leukocyte type might regulate t-PA activity and selectivity for fibrin.
Chloramines↗
Plasminogen activator inhibitor in acute stroke.
Plasminogen activator inhibitor activity was determined in patients (26 men, 6 women) with acute ischemic stroke (n = 28) and transient ischemic attack (TIA) (n = 4). Age-matched patients (22 men, 10 women) with various nonvascular neurologic diseases served as controls. Plasma levels of plasminogen activator inhibitor activity were significantly higher in the stroke group (p less than 0.003). Serum triglycerides and plasminogen activator inhibitor activity correlated positively in the stroke group (p less than 0.03) and in controls (p less than 0.0001). Our data suggest a possible involvement of plasminogen activator inhibitor activity in the pathophysiology of stroke.
Acute Disease↗
Oxidation susceptibility of plasminogen activator inhibitors of human plasma.
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Chloramines↗
Single chain-urokinase binds to oxidized fibrin(ogen) derivatives.
Single chain- urokinase (scu-PA), in contrast to two chain urokinase, is a physiological plasminogen activator of fibrin selectivity. The mechanism of selective fibrinolysis of scu- PA has not been clarified up to now. This study has shown that fibrin selectivity might involve oxidative processes. Binding studies with immobilized oxidized fibrinogen degradation products (FDP) demonstrated higher affinity of scu-PA to oxidized than to unmodified FDP. The fibrin(ogen) domain responsible for this oxidant mediated increase of scu-PA affinity is localized in the D-subunit of fibrin(ogen). Thus, experimental data upon the interaction of scu-PA with fibrin(ogen) using oxidized and I- labelled fibrin(ogen) might be interpreted with caution: the oxidized product may behave in a distinct manner than the unoxidized, native, one. Activated leukocytes release large amounts of scu-PA and of oxidants of the chloramine type. The oxidants could contribute significantly to fibrinolysis and proteolysis in areas of inflammation, preparing fibrin for its specific degradation. The present data support the concept of an involvement of oxidative processes in the fibrinolytic pathway.
Fibrin Fibrinogen Degradation Products↗
Protection of urokinase from plasminogen activator inhibitors in human plasma by hydrophobic agents.
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Detergents↗
A direct approach in fibrinolysis diagnostic: mimicry of the leukocyte by attack by oxidants.
Activated leukocytes release large amounts of chloramine like oxidizing agents (see Weiss et al. Science 222, 625-628, 1983) which inactivate protease inhibitors, creating microenvironments of uncontrolled protease activity. The biological result is an increased proteo- and fibrino- lysis. Functional determination of tissue type (t-PA) or urinary type (u-PA) plasminogen activator, the key enzymes of fibrinolysis, is of clinical importance. Assay techniques have been developed but are troublesome due to predilution, acidification or separation steps in order to eliminate the PA and plasmin inhibiting effect of plasmatic inhibitors of anti-PA and of anti-plasmin type, respectively. In this study, evidence is presented that performance of fibrinolytic assays using N-chloroamines offers great advantage: plasmatic plasminogen activator activity both of urinary and of tissue type can be measured precisely within minutes in untreated (direct) plasma samples. Therefore, mimicking the oxidative inflammatory response, for the first time it gets feasible to analyze blood factors involved in the physiological course of fibrinolysis by means of a functional, sensitive, and rapid assay procedure.
Chloramines↗
Oxidized fibrin(ogen) derivatives enhance the activity of tissue type plasminogen activator.
Oxidation of fibrinogen degradation products (FDP) with chloramines, results in a five- fold increase of their property to stimulate plasminogen activation by tissue type plasminogen activator (t-PA). Binding studies with immobilized stimulators demonstrated greater affinity of t-PA to oxidized than to unmodified FDP. The fibrin (ogen) domain responsible for this oxidant mediated increase in t-PA stimulation is localized in the D- subunit of fibrin(ogen). Thus, experimental data with (oxidized) I-labelled fibrin(ogen) should be interpreted with caution: the oxidized product might behave in a distinct manner than the unoxidized, native, one. As activated leukocytes release large amounts of oxidants of the chloramine type (Weiss et al., Science 222, 625-628, 1983), oxidation of fibrin might contribute significantly to fibrinolysis and proteolysis in areas of inflammation. The data give further evidence for an involvement of physiological components of haemostasis in non haemostasis but inflammation related processes.
Chloramines↗
Determination of plasminogen activator inhibitor (PAI) capacity of human plasma in presence of oxidants: a novel principle.
A new functional assay of PAI-activity in human plasma is described. Hitherto known assays for fast acting PAI have some disadvantages: predilution and/or acidification steps of the sample to afford the required inactivation of alpha-2-antiplasmin (A2-PI). This approach in contrast implicates test performance in presence of chloramine T (CT), an oxidant that destroys plasma antiplasmin activity without impairing significantly the activity of urokinase (u-PA) or plasmin. The following reaction conditions were found optimal: 50 microliter of undiluted plasma, anticoagulated with citrate or EDTA, were first incubated with 1 IU u-PA in a Tris-buffer, pH 8.4 and then with Glu-plasminogen (0.85 mumol/l final), CT (2.5 mmol/l final), tranexamic acid (0.9 mmol/l final) for 5 min. at 37 degrees C. After addition of 0.3 mmol/l of the chromogenic plasmin substrate H-D-Nva-CHA-Lys-pNA (pNA = para nitroanilide) and of NaCl (250 mmol/l final) a linear kinetic with delta A405/t in the range of 0.2/min for normal plasma was recorded. In an endpoint version of the test the chromogenic substrate can be added together with plasminogen resulting an A/t2-kinetic. Dilution studies showed a linear calibration curve from 0 to 14 arbitrary u-PA inhibiting units (AU)/ml plasma. By means of PAI-standard plasmas PAI-capacity values of 20 healthy volunteers (10 males/10 females) (x = 26 years, sigma = 4.2) were determined. They ranged from 0.4 - 6.9 (x = 1.3, sigma = 0.9) AU/ml plasma. Plasma samples containing more than 14 AU/ml were prediluted with PAI-deficient plasma. Intra- and inter-assay coefficients of variation (CV) were determined to be 1.3 +/- 0.6 and 4.3 +/- 0.5%, respectively. The values of this assay correlate well with those obtained by acidification of the samples. However, the possibility of measuring plasma PAI (and PA) activities by means of a simple and direct approach can be considered as an important progress with regard to routine hospital practice. The presented oxidative inactivation of A2-PI mimics the leukocyte attack phase, suggesting that activated leukocytes create a microenvironment of uncontrolled plasmin activity.
Adult↗
Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor.
Oxidative inactivation of alpha-1-proteinase inhibitor (A1-P1) and plasminogen activator inhibitor-1 (PAI-1), both members of the serine protease inhibitor (serpin) superfamily, using mild oxidation conditions has been already demonstrated. The oxidation mechanism has been shown to involve conversion of methionine to methionine sulfoxide in the reactive center of the inhibitors. In this study evidence is presented that alpha-2-antiplasmin (A2-PI) and antithrombin III (AT III) can also be inactivated by means of oxidation. For total inactivation of 50 pM A1-PI about 10 nM chloramine T (CT) and for the same molar concentration of A2-PI and AT III about 250 nM CT were found necessary. C1-inhibitor (C1-INH) showed some resistance to oxidation that could be overcome only by increasing CT to an amount (greater than 2000 nM) already beginning to inactivate the corresponding C1-esterase. As target enzymes for A2-PI, AT III, and A1-PI plasmin, thrombin and elastase, respectively, were used. Their activity was not impaired by the oxidation conditions applied. As there is no methionine in the reactive center of AT III an additional mechanism for oxidative inactivation of serpins has to be taken into consideration. Oxidation seems to be a general mechanism for altering the balances between serine proteases and their inhibitors in favour of the protease.
Antithrombin III↗
Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants.
Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.
Animals↗
A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma.
Human plasma contains an inhibitor of activated protein C (APC) which is termed according to its function protein C inhibitor (PCI). High purification of functionally active PCI with a yield of 18% is achieved by an improved procedure consisting of 4 steps: precipitation by rivanol, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE Sephacel and chromatography on dextran sulfate Sepharose. This purification results in the isolation of a homogeneous PCI which migrates in immunoelectrophoresis with the beta-globulins of human plasma and in SDS PAGE as one single band at Mr = 57,000 both under reducing and nonreducing conditions. The specific activity of the highly purified PCI was determined to be 226 units/mg, 1 unit being equivalent to the activity of 1 ml fresh human citrated plasma. PCI forms complexes with 1:1 stoichiometry (Ki: 1.4 x 10(-8) M) resulting in a loss of the amidolytic activity of APC as measured on Tos-Glu-Pro-Arg-pNA (S 2366). The inhibition rate of APC by PCI (k: 7.5 x 10(5) M-1 min-1) is significantly increased in the presence of 5 i.u./ml heparin (kH: 2.2 x 10(7) M-1 min-1). PCI also blocks the amidolytic activities of urokinase plasminogen activator (u-PA), thrombin and factor Xa on their chromogenic substrates in a heparin-dependent manner. According to the Ki-values measured for these reactions PCI is a noncompetitive inhibitor of these proteases. The Ki-values calculated do not differ significantly from those obtained for the inhibition of APC by PCI. Immunodepleted PCI-deficient plasma still contains an inhibitory activity against APC which, however, only slowly inactivates the amidolytic activity of APC and in a time and concentration-dependent manner. Addition of heparin has no influence on the inhibition rate. This finding suggests the existence of a second, heparin-independent PCI present in human plasma.
Blood Proteins↗
A simple method for producing degradation products of fibrinogen by an insoluble derivative of plasmin.
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Chromatography, Ion Exchange↗