Functional correlation between kallikrein and factor XII activated in human plasma

Plasma kallikrein and FXIIa were assayed in acetone-treated human citrated plasma (CPLa) with the chromogenic peptide Bz-Ile-Glu-Gly-Arg-pNA (S-2222) as substrate. In end point assays with short incubation periods (1–10 min.) nearly all kallikrein present could be blocked by a low concentration of soybean trypsin inhibitor (STI). In 30 min. assays the main part of the kallikrein was recovered in a functional state not inhibited by STI, and at the same time the level of FXIIa (as amidase activity blocked by corn inhibitor, C.I.) was reduced to about 2/3 of the initial value. The formation of an association between FXIIa and kallikrein is suggested. In fractions from gel filtration of CPLa kallikrein was assayed as S-2302 amidase, high molecular weight kininogen (HK) was measured in rocket immunoassays, and HK and FXII were studied in PAGE immunoblot experiments. Kallikrein appeared as one peak together with HK (gel mol. wt. 300 KD), about 40% of HK was free (220 KD), and no FXII was observed in the kallikrein or HK peaks, but in two areas corresponding to 78–79 KD and 39–42 KD. When experiments, however, were carried out with plasma acetone-activated and gel filtered in the presence of benzamidine (5 mM), part of the amidase activity present in kallikrein peak fractions was blocked by C.I. This observation supports the above suggestion of an association between FXIIa and kallikrein.     

Radioisotopic measurement of plasma kallikrein

A rapid and sensitive assay for plasma kallikrein has been developed using the radiolabeled tripeptide substrate N-benzoyl-L-prolyl-L-phenylalanyl-L-arginyl-(¹⁴C)-anilide. This assay can measure kallikrein in plasma at concentrations ranging from 25–1000 ng/ml. The assay does not require inactivation of kallikrein inhibitors.     

Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma

Effects of tissue-type plasminogen activator (t-PA), urokinase(u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1,1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma .     

A new assay for high molecular weight kininogen in human plasma using a chromogenic substrate

High molecular weight kininogen (HK), the cofactor of contact-activated plasma proteolysis, is currently assayed by coagulant or immunological methods. The former is limited by the need for rare, congenitally-deficient plasma and a high coefficient of variation (CV), and the latter, by failure to distinguish nonfunctional protein. The surface activation of factor XI requires HK as its cofactor to transport its zymogen form to a negatively-charged surface where it is converted to its enzymatic form by factor XIIa. Based on this principle, we developed an assay for HK using the chromogenic substrate 相似文献   

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.     

Species specificity in the acceleration of tissue-type plasminogen activator-mediated activation of plasminogens, by fibrinogen cyanogen bromide fragments

Activation of human plasminogen by human tissue-type plasminogen activator (t-PA) is accelerated in the presence of cyanogen bromide digests of human fibrin(ogen).

In the present study a possible species specificity of this phenomenon was investigated. All combinations of the plasminogens, fibrin monomers and cyanogen bromide digests of the fibrinogens of man, pig, rat, cat and monkey (Macaca mulatta), and three t-PA species (man, rat and pig) were studied.

No species differences were noted with the fibrin monomers i.e. the activation rate of all five plasminogens increased more than 20-fold in the presence of all five fibrin monomer species, irrespective if man, rat or pig t-PA was used.

However, we found that species specificities come to expression when cyanogen bromide digests of the corresponding fibrinogens were used as accelerators.

Our results indicate that the plasminogen species and not the source of t-PA or fibrinogen dictates if accelerated activation occurs in the presence of a fibrinogen CNBr digest. The plasminogens can be roughly divided in two groups:

- One group, comprising human, monkey and cat plasminogen, which are activated at a higher rate by all three t-PA species in the presence of fibrinogen digest independent on the fibrinogen species from which the digest was prepared.

- An other group, comprising pig and rat plasminogen, which is not or only marginally faster activated by the three t-PA species, irrespective of the fibrinogen species from which the CNBr digest was prepared.     

Insight into the profibrinolytic activity of dermatan sulfate: effects on the activation of plasminogen mediated by tissue and urinary plasminogen activators

INTRODUCTION: Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II to enhance antithrombin action. It has also been suggested that DS has a profibrinolytic effect, although the exact molecular mechanism is as yet unknown. MATERIALS AND METHODS: An in vitro amidolytic method was used to study the effect of high and low molecular weight-DS on the activation of Glu and Lys-plasminogen by tissue and urinary plasminogen activators (t-PA and u-PA). RESULTS: Both high and low molecular weight-DS exhibited a stimulating effect on the activation of plasminogen by PAs. Interestingly, high molecular weight-DS stimulated Glu and Lys-plasminogen activation by t-PA and u-PA in a way and to an extent similar to that in which fibrin(ogen) degradation products (PDF) increased the t-PA assay. Meanwhile low molecular weight-DS had a lower effect. No DS had any effect on plasmin or u-PA amidolytic activity. The facilitation of the conversion of Glu-plasminogen to plasmin in the presence of DS was confirmed by SDS-PAGE; high molecular weight-DS effect was greater than low molecular weight-DS in accordance with the chromogenic assays. Moreover, the combination of PDF and high and low molecular weight-DS, respectively, did not further stimulate t-PA activation of either Glu or Lys-plasminogen suggesting that both substances may compete for the same binding sites. CONCLUSIONS: Through in vitro assays we demonstrated that high and low molecular weight-DS enhance plasminogen activation by u-PA and t-PA, suggesting that the profibrinolytic activity of DS might be via potentiation of plasminogen conversion to plasmin.     

Activation of plasminogen by tissue activator is increased specifically in the presence of certain soluble fibrin(ogen) fragments

Tissue activator-mediated plasminogen activation is potentiated both by fibrin and by some soluble fibrin(ogen) fragments. The potentiating effect of the different fragments decreases in the order fibrin monomer greater than D-dimer greater than Y greater than D EGTA greater than Dcate greater than X. Fibrinogen and the fragments Ecate, E EGTA and E fibrin have almost no effect. The existence of a fibrin polymer is apparently not a prerequisite for this potentiating effect. The plasminogen activation by various urokinase preparations is not potentiated by fibrin and fibrin(ogen) fragments.     

Influence of SK-potentiator and fibrinogen degradation products on the activation of human plasminogen by streptokinase

When fibrinogen was degraded by plasmin, very early degradation products (FgDP-5′) enhanced the activity of SK-activator (SK-plasminogen or SK-plasmin) to the largest extent, and further degradation, such as extentsive degradation of β-chain (FgDP-20′), resulted in less enhancement of SK-activator activity than fibrinogen itself which also enhanced it. Fibrin enhanced the activation rate of plasminogen by SK to the largest extent, more than SK-potentiator. The action of thrombin on SK-potentiator was as effective as fibrin in the enhancement, thus removal of fibrinopeptides from fibrinogen or SK-potentiator is important in the activator activity of the trimolecular complex of SK-plasminogen-potentiator. SDS-PAGE indicated that native plasminogen was converted to plasmin faster in the presence of SK and SK-potentiator than SK alone.     

Synergistic antithrombotic effect of a combination of NO donor and plasma kallikrein inhibitor

The aim of the present study was to investigate the effect of a NO donor (GSNO) and a plasma kallikrein inhibitor (PKSI-527) alone and in combination on global haemostatic status. A new in vitro test was employed which allows the measurement of both platelet function and spontaneous thrombolysis. Sixteen healthy young and 18 elderly volunteers were enrolled in this study. When GSNO (1 mM) or PKSI-527 (20 microM) was added to native human blood, platelet reactivity was significantly inhibited in both age groups. The combination of GSNO and PKSI-527 had additive inhibitory effect on platelets. Addition of either GSNO or PKSI-527 to blood samples did not significantly affect spontaneous thrombolysis, while added together, spontaneous thrombolysis was significantly enhanced. The thrombolysis enhancing effect was more prominent in elderly subjects. Our present findings suggest that the combination of NO donor and plasma kallikrein inhibitor may have clinical antithrombotic potential.     

Methylprednisolone induces activation of the contact system in a dose-dependent manner. An in vitro study

The effect of methylprednisolone sodium succinate (MP) on the contact system of plasma was studied in human citrated pool plasma. Contact activation was demonstrated by the presence of plasma kallikrein (KK) activity and activated Hageman factor (FXIIa) and/or KK in complex with Cl inhibitor (Clinh), detected by chromogenic peptide substrates or radio immunoassays, using monoclonal antibodies directed to neodeterminants exposed on complexed Clinh, respectively.

When plasma and different doses of MP were incubated for a period of 24 hours, the highest dose of MP (10 mg/ml) gave rapid and marked increases in KK activities and concentrations of Clinh complexes. MP at 5 mg/ml plasma also induced activation of the contact system, although this activation was less pronounced. Even the lower dose of MP (1 mg/ml), which is equivalent to doses used in humans, increased plasma concentrations of KK-Clinh complexes.

In conclusion, this study shows that MP in a dose-dependent way activates the contact system of plasma.     

Inhibition of plasminogen activation by polymerized ampicillin

The polymerized β-lactam antibiotic ampicillin inhibits the proteolytic activity of human plasmin upon ¹²⁵I-labeled fibrin clots. The inhibition is dose-dependent, with half-maximal inhibition occurring at 1.25 mM of the polymerized antibiotic. Polymerized ampicillin also inhibits binding of plasmin to fibrin, and 38% inhibition of binding occurs at 10 mM of the antibiotic. Furthermore, polymerized ampicillin inhibits the activation of plasminogen by either urokinase-like plasminogen activator (uPA) or tissue type-plasminogen activator (tPA). At 7.5 mM of polymerized ampicillin, the uPA-mediated plasminogen activation is suppressed by 94%, and half-maximal inhibition is obtained at 0.66 mM. The direct activity of uPA on the chromogenic substrate L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride (S-2444) is unaffected by polymerized ampicillin levels of up to 10 mM. The inhibitory effects of the polymerized antibiotic on the activation of plasminogen by both uPA and tPA is totally abolished in presence of fibrin. These interactions may serve as a novel model for ligands that enhance the clot-specificity of thrombolytic agents.     

Differences in effects of fibrin(ogen) fragments on the activation of 1-glu-plasminogen and 442-val-plasminogen by tissue-type plasminogen activator

The activation rate of plasminogen by tissue-type plasminogen activator can be increased by fibrin(ogen) fragments. There is a remarkable difference in the effect of these fragments on the stimulation of 1-glu-plasminogen activation and 442-val-plasminogen (mini-plasminogen) activation. Fibrin monomer as well as plasmic fragments Y, D EGTA and D-dimer have a stimulating effect on both 1-glu-plasminogen and 442-val-plasminogen activation, whereas cyanogen bromide fragment FCB2 stimulates only the activation of 1-glu-plasminogen. Results indicate that two types of sites may be operational in fibrin and fibrin(ogen) fragments Y, D EGTA and D-dimer. One type of site (FCB2-related) interacts probably with plasminogen and may be dependent on the kringle 1-4 region; the other type of site probably interacts either with plasminogen in a non-kringle 1-4 region-dependent manner or with tissue-type plasminogen activator.     

The fibrinolytic action of the synthetic agent S-1623 in a purified system of fibrin and plasminogen

The effects of the synthetic fibrinolytic agent α(isobutyl-4 cyclohexene-1 yl) propionic acid (S-1623) were investigated in a purified system. Bovine fibrin clots were incubated at 37°C with varying concentrations of S-1623 (0–10 mM) and a constant concentration of human plasminogen (1 μM). Two methods were used to measure lysis, e.g. visual observation of the clots and SDS-polyacrylamide gel electrophoresis. Plasmin formed during incubation was measured quantitatively by a spectrophotometric method using a chromogenic substrate. After 24 hours of incubation, a partial or total degradation of the clots was observed visually for S-1623 concentrations between 3 and 8 mM, with maximum degradation occuring at 4–5 mM. Analysis of the samples by acrylamide gel electrophoresis revealed total fibrinolysis at a concentration of 4–5 mM, partial fibrinolysis at 3 and 6–7 mM, and no fibrinolysis at other concentrations (0–2 mM and 8–10 mM). Proteolysis of plasminogen also occured, with maximum effect at 4–5 mM, and resulted in the production of fragments smaller than the intact plasmin molecule. The amidolytic activity coincided with the lytic concentrations of S-1623, with maximum activity occuring at 4–5 mM. No activity was observed in the absence of fibrin, plasminogen, or S-1623. Amidolytic activity was also generated, though to a lesser extent, when the fibrin substrate was replaced by fibrinogen. These results seem to show that the agent S-1623 is capable of generating plasmin-like activity in this system.     

The potentiating effect of platelet on plasminogen activation by tissue plasminogen activator

A new role for platelets in fibrinolysis is proposed. Platelets (euglobulin from platelet rich plasma and from human platelet extract) may potentiate plasminogen activation by tissue plasminogen activator(tPA). The potentiating activity was detected by both chromogenic substrate and fibrin plate analysis. The fibrinolysispotentiating substance in the platelets required the presence of both tPA and plasminogen, suggesting that it potentiates the activation of plasminogen by tPA. This substance was not related to fibrinogen degradation products because it was also present in platelets from two afibrinogenemic patients and did not lose its activity when separated from fibrinogen-related antigen by Sepharose 2B gel filtration. Since platelets contain both activator(s) and inhibitor(s) of plasminogen activation by tPA, a balance between activator(s) and inhibitor(s) in platelets may also be required for control of the fibrinolytic pathway.     

The resistance of fibrin-stimulated tissue plasminogen activator to inactivation by a class pai-2 inhibitor (minactivin)

Solid phase fibrin was an efficient stimulator of the tissue-type plasminogen activator (t-PA), and the plasmin produced could be detected by colorimetric assay of the soluble phase above the fibrin. However the fibrin-stimulated activity of t-PA was not inhibited by minactivin. This result was in contrast to that obtained with poly-D-lysine (PL) stimulated t-PA, where minactivin was a potent inhibitor. However, if PL was added to fibrin-bound t-PA, the enzyme once again became susceptible to minactivin inhibition. This occurred without release of t-PA from the fibrin matrix. Minactivin alone did not bind to fibrin or to the t-PA fibrin complex. It was therefore concluded that minactivin normally has no significant role in the regulation of t-PA mediated fibrinolysis, but this effect can be induced by PL.     

Evaluation of plasma urokinase-type plasminogen activator and urokinase-type plasminogen-activator receptor in patients with acute and chronic hepatitis B

Introduction

Urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) are known to be important factors in the pathogenesis of tumors and certain non-viral inflammatory diseases. However, their role in infectious virus diseases such as hepatitis B has been less well studied. This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are directly related to the inflammatory damage to liver cells caused by the hepatitis B virus. We therefore analyzed their role and clinicopathological significance in patients with acute or chronic hepatitis B.

Materials and methods

Eighty patients with acute or chronic hepatitis B, together with 30 healthy controls, were enrolled. uPA and uPAR in plasma were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits.

Results

The levels of uPA and uPAR in patients with acute or chronic hepatitis B significantly exceeded those in healthy controls (p < 0.05). Patients with severe chronic hepatitis B had significantly higher levels of uPA and uPAR than those with moderate and mild chronic disease (p < 0.05) and those with acute hepatitis B (p < 0.05). Moreover, the plasma uPA and uPAR markedly increased in the acute stage (p < 0.05) and dramatically decreased in the remission stage (p < 0.05), but in all stages levels exceeded those in healthy subjects (p < 0.05). In addition, the concentration of plasma uPAR was positively correlated with prothrombin (PT) (r = 0.605, p < 0.01) and total bilirubin (TBIL) (r = 0.649, p < 0.01).

Conclusions

It is suggested that the plasma levels of uPA and uPAR are closely related to the degree and period of inflammation in patients with acute or chronic hepatitis B, and that uPA and uPAR might be important indicators for disease progression.     

A simple and rapid method to study the association of the contact proteins of blood coagulation

Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The “mobility shift” assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.