Modification of a tyrosine residue in the carboxyterminal portion of human alpha2-antiplasmin.
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Amino Acids↗
Biomedical subjects
B Wiman
Publications and source records attributed to B Wiman.
Relationship between preoperative status of the fibrinolytic system and occurrence of deep vein thrombosis after major abdominal surgery.
This study comprised 50 patients subjected to major abdominal surgery, of which 13 developed deep vein thrombosis (DVT) according to the 125I-fibrinogen test. Plasma was sampled preoperatively, for the specific analysis of tissue plasminogen activator (t-PA) before and during venous occlusion. The recently described fast t-PA inhibitor and plasmin alpha 2-antiplasmin complex (PAP) were also measured. The result of the laboratory analyses were correlated to the development of DVT. From the data obtained it is concluded that the evaluation of t-PA release during venous occlusion is a poor predictive factor for the occurrence of DVT after major abdominal surgery. The level of the t-PA inhibitor appears to be raised in these patients, but the values obtained in this material were not related to the development of postoperative DVT. Patients with elevated PAP levels, as shown previously, have a lesser tendency to develop postoperative DVT.
Adult↗
Prediction of deep vein thrombosis after extensive abdominal operations by the quotient between plasmin-alpha 2-antiplasmin complex and fibrinogen concentration in plasma.
In 97 patients over 49 years of age who underwent extensive abdominal operations, post-operative deep vein thrombosis was prospectively diagnosed by the 125I-fibrinogen test. Blood was sampled preoperatively for analysis of the plasma levels of PAP (by a radioimmunoassay) and fibrinogen. The development of postoperative deep vein thrombosis was correlated to the quotient between the preoperative plasma concentration of PAP and fibrinogen. The quotient was significantly lower in the 30 patients who had thrombosis develop postoperatively as compared with the patients who did not. A simple linear discrimination analysis showed that, in the 31 patients with a quotient of more than 0.48, only two patients (6 per cent) had deep vein thrombosis. The results emphasize the importance of the preoperative fibrinolytic status in the development of postoperative deep vein thrombosis. The results of the study indicate that the quotient between PAP and fibrinogen concentration in plasma might be used for the prediction of patients with a high risk for having deep vein thrombosis develop after undergoing extensive abdominal operations.
Adult↗
Plasma fibrinolytic activity in patients undergoing major abdominal surgery.
The fibrinolytic system was studied in 30 patients undergoing elective cholecystectomy and in 30 with more advanced elective abdominal surgery. Blood was sampled before, during and after operation for determination in plasma of the tissue plasminogen activator (t-PA), the recently described fast t-PA inhibitor, and plasmin alpha 2-antiplasmin complex (PAP). In addition the t-PA activity during venous occlusion was determined preoperatively. Most of the patients showed raised t-PA levels during surgery, but the interindividual variation was wide and was not correlated to fibrinolytic capacity measured preoperatively as enhancement of t-PA activity during venous occlusion. The levels of the t-PA inhibitor rose during and immediately after surgery, and were higher in patients without increased t-PA activity during surgery. The patients with more advanced disease had higher levels of the inhibitor than the cholecystectomy patients. The data suggest that the t-PA inhibitor may influence the fibrinolytic response to surgical trauma and may explain the previously reported shutdown in fibrinolysis in the early postoperative period. PAP, used as reflecting the overall fibrinolytic activity, was increased in plasma after the first postoperative days.
Adult↗
The role of the fibrinolytic system in deep vein thrombosis.
Some aspects of the function of the fibrinolytic system have been investigated in 37 patients with a recent incident of symptomatic and confirmed deep vein thrombosis and compared with findings in 20 healthy persons. New specific methods to measure tissue plasminogen activator (t-PA) activity and antigen before and after venous occlusion and the recently discovered fast inhibitor to t-PA were employed. Thirteen of the patients with deep vein thrombosis (35%) had t-PA activity less than 0.5 IU/ml after venous occlusion, whereas the lowest activity found among the healthy individuals was 0.56 IU/ml. The t-PA inhibitor level in the total patient group was 3.8 +/- 3.7 U/ml (range 0 to 15.0 U/ml; median 2.9 U/ml) as compared with 0.7 +/- 0.7 U/ml in the healthy (median 0.5, range 0 to 2.4 U/ml). In the 13 patients with low t-PA activity in postocclusion plasma samples the inhibitor level was 6.0 +/- 4.4 U/ml. Furthermore, in this group of patients a significantly lesser release of t-PA antigen (3.7 +/- 2.8 micrograms/L) was found as compared with that in the healthy individuals (9.5 +/- 6.0 micrograms/L). Thus, two months after their first incident of symptomatic deep vein thrombosis many of the patients (35%) were found to have decreased fibrinolytic activity. This is the result of highly increased plasma levels of a novel fast inhibitor toward t-PA in combination with a poor ability to release t-PA. Possibly the decreased fibrinolytic activity did play a role in the pathogenesis of deep vein thrombosis in these patients.
Adult↗
A novel fast inhibitor to tissue plasminogen activator in plasma, which may be of great pathophysiological significance.
A novel, fast inhibitor to tissue plasminogen activator (t-PA) has been demonstrated in plasma samples. The inhibitor can be titrated in plasma. Upon addition of t-PA to plasma, an inactive complex is formed with an estimated rate constant of about 10(7)M-1s-1. The molecular weight of the complex between t-PA and the inhibitor has been determined as about 120,000. Thus, a molecular weight of about 55,000 would be expected for the inhibitor moiety in the complex. However, by gel filtration of plasma, rich in the inhibitor, an apparent molecular weight of about 200,000 was found. About 100,000 fold purification of the complex from plasma has been achieved by employing immuno adsorption chromatography (antibodies against porcine t-PA and monoclonal antibodies against human t-PA), ion-exchange chromatography and gel-filtration. Low concentrations of the new t-PA inhibitor were normally found in healthy individuals (0.7 +/- 0.7 U/ml). However, many patients with thrombosis or coronary heart disease had increased levels (3.8 +/- 3.7 U/ml and 2.8 +/- 2.2 U/ml, respectively). In normal pregnancy the concentration of the fast t-PA inhibitor increases linearly from week 10 to the time about term (5.1 +/- 0.9 U/ml). At present the origin as well as the physiological role of this novel t-PA inhibitor remains unclear. The increased levels observed in many patients with thrombotic diseases or in conditions frequently associated with thrombotic complications anticipates a role in the development of thrombosis. However, more work is needed to prove this hypothesis.
Chromatography, Gel↗
The fast inhibitor of tissue plasminogen activator in plasma during pregnancy.
The novel fast inhibitor to tissue plasminogen activator in plasma has been determined in 20 healthy non-pregnant women, 48 apparently healthy pregnant women and 136 women with risk pregnancies (preeclampsia, suspected fetal growth retardation, thrombosis or previous history of thrombosis, diabetes and others). In healthy fertile non-pregnant women, the inhibitor concentration was found to be 0.4 +/- 0.7 U/ml. In pregnant women the concentration stayed at this level until week 10, but then an almost linear increase was found, reaching about 6.5 U/ml at week 40. In plasma samples from the patients with risk pregnancies many with deviating concentrations were found. Thus, in the last trimester a range of 0-24.0 U/ml was found in these patients, as compared to 4.0-6.4 U/ml in healthy pregnant women. The pathophysiological impact of this finding is at present unclear.
Female↗
Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor.
A new specific and sensitive method for determination of tissue plasminogen activator (t-PA) in plasma samples has been used to demonstrate the presence of a fast inhibitor to t-PA in plasma. By adding [35S]Met internally labeled t-PA (Mr approximately 70,000) to plasma, we were able to demonstrate the rapid formation of a stable complex with an apparent molecular weight of about 115,000 as estimated by gel filtration. The complex was partially purified by immunoadsorbtion chromatography on insolubilized antibodies against porcine t-PA, and a molecular weight of about 120,000 was found by dodecyl sulfate-polyacrylamide gel electrophoresis. From the apparent molecular weight of the complex (120,000) and the molecular weight of t-PA (70,000), a molecular weight of about 50,000 would be expected for the inhibitor. However, gel filtration of inhibitor-rich plasma resulted in the appearance of a symmetrical peak of t-PA inhibitory activity with an apparent molecular weight of about 205,000. The reason for this discrepancy is not known, but several different models are possible.
Chromatography, Gel↗
Plasminogen content in diabetic wounds.
Plasminogen concentrations in secretions from distal wounds of 10 diabetic and 5 non-diabetic patients were determined spectrophotometrically. The plasminogen content in the secretions from the wounds was very low, only about 2% of the plasminogen content in plasma. There was no significant difference between diabetic and non-diabetic wounds concerning plasminogen content.
Aged↗
Fibrinolytic activity in plasma and deep vein thrombosis after major abdominal surgery.
In a prospective study of the frequency of deep vein thrombosis (DVT) in 45 patients subjected to major abdominal surgery, 17 patients showed signs of DVT as assessed by the 125I-fibrinogen test. In 15 of the patients the DVT was diagnosed during the first four postoperative days. Blood samples were taken pre- and postoperatively and analysed for fibrinogen, prothrombin complex, APTT, platelet-count, plasminogen, alpha 2-antiplasmin, fibrin(ogen) degradation products, and plasmin-alpha 2-antiplasmin complex (PAP). The latter was used in order to reflect the fibrinolytic activity. Preoperatively, and postoperatively on day 3, the levels of PAP were significantly higher in patients without postoperative DVT. The data suggests that patients subjected to major abdominal surgery, who have enhanced fibrinolytic activity preoperatively, have a lesser tendency to develop postoperative DVT. Patients with postoperative DVT may have decreased fibrinolytic ability. From the data of the other parameters it is concluded that patients with DVT can have increased levels of FDP at the time of development of thrombosis.
Abdomen↗
Structural and circular-dichroism studies on the interaction between human C1-esterase inhibitor and C1s.
The reaction between complement factor C1s and C1-esterase inhibitor has been investigated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and c.d. studies. It is confirmed that a very stable stoichiometric 1:1 complex with a molecular weight of about 180000 is formed, involving the light chain of C1s. On the sodium dodecyl sulphate/polyacrylamide gels a small peptide with a molecular weight of about 5000 can be seen, which may be released from the C-terminal portion of the inhibitor moiety in a manner analogous to that occurring in other similar proteinase-inhibitor reactions. By N-terminal amino acid analysis, a newly formed threonine residue is found in the complex, suggesting that the inhibitor peptide chain is cleaved in the complex between C1s and C1-esterase inhibitor. The stabilizing bond may therefore be an ester bond. C.d. studies of the native C1-esterase inhibitor indicated the presence of about 38% alpha-helix, about 24% beta-structure and about 38% unordered structure. By gradual cleavage of the disulphide bridges under non-denaturating conditions, gradual changes in the c.d. spectra occurred, suggesting loss of ordered secondary structures. The c.d. spectra of the complex between C1s and C1-esterase inhibitor indicate that tryptophan residues are affected by the complex-formation.
Amino Acids↗
A new simple method for determination of C1-esterase inhibitor activity in plasma.
A convenient method for the determination of C1-esterase inhibitor activity in plasma samples is described. The method is based on addition of purified C1s to plasma and measuring excess C1s with a new chromogenic tripeptide-p-nitroanilide substrate with a recording spectrophotometer. Addition of C1s to C1-esterase inhibitor-depleted plasma did not result in any appreciable inactivation of the enzyme for three hours. The concentration of C1-esterase inhibitor in 19 healthy individuals was estimated as 1.63 +/- 0.27 (SD) mumol/l. The correlation with C1-esterase inhibitor antigen in these individuals and 19 patients with varying concentrations of C1-esterase inhibitor was excellent. The correlation with an antikallikrein assay was found to be poor.
Complement Activating Enzymes↗
Plasminogen activator release during venous stasis and exercise as determined by a new specific assay.
A method for the specific determination of tissue plasminogen activator in plasma samples has been developed. The method is based on a recently described parabolic rate assay for tissue plasminogen activator (Rånby and Wallén, 1980) measuring plasmin activity by a chromogenic tripeptide-paranitroanilide substrate. The potential interference by plasmin inhibitors in plasma is overcome by acidification of the plasma samples to pH 4 for 15 min prior to the analysis. The baseline activity (after 10 min of rest) in 22 healthy individuals was determined to be 0.05 +/- 0.03 (SD) IU/ml (0.2 +/- 0.1 ng/ml). After venous occlusion for 10 min at 100 mm Hg the activator concentrations had increased to 1.2 +/- 1.2 (SD) IU/ml (5.2 +/- 5.2 ng/ml). Furthermore, prior to venous occlusion almost no plasmin-alpha 2-antiplasmin complex was found in the samples, whereas a pronounced increase in this complex was noted in 90% of the subjects after venous occlusion. During exercise, a gradual work-load dependent increase of tissue plasminogen activator concentration was observed. At maximal work-load the concentration was 2.3 +/- 1.9 (SD) IU/ml (9.9 +/- 8.1 ng/ml active enzyme), which after 10 min rest decreased to 26 +/- 12 (SD) % of the values at maximal work-load. In these samples only small amounts of plasmin-alpha 2-antiplasmin complex were detected, although the activator content was of the same order of magnitude as after venous occlusion.
Adult↗
Kinetics of the reaction between human C1-esterase inhibitor and C1r or C1s.
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Complement Activating Enzymes↗
Determination of plasmin-alpha 2-antiplasmin complex in plasma samples by means of a radioimmunoassay.
Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with rather high specificity towards the complex. These antibodies do not react with plasminogen or plasmin in complex with other inhibitors than alpha 2-antiplasmin. However, they react fully with native alpha 2-antiplasmin, but a 200-fold higher concentration, as compared to plasmin-alpha 2-antiplasmin complex, is needed to obtain a similar displacement curve in a double-antibody radioimmunoassay. The results indicate a conformational change in the vicinity of the reactive site in alpha 2-antiplasmin, as a result of complex formation with plasmin. A method for determination of plasmin-alpha 2-antiplasmin complex in plasma has been elaborated using the described radioimmunoassay. About 1.5 mg plasmin-alpha 2-antiplasmin complex/l can be detected, which equals the condition when about 1% of the alpha 2-antiplasmin in plasma is in complex with plasmin. In normal individuals plasmin-alpha 2-antiplasmin complex could be detected only rarely. However, patient with acute processes, as evidenced by high fibrinogen levels, surgical patients postoperatively or patients with malignancy have often detectable levels.
Antifibrinolytic Agents↗
Partial primary structure of human alpha 2-antiplasmin-homology with other plasma protease inhibitors.
Human alpha 2-antiplasmin was digested with trypsin and with chymotrypsin and about 70 percent of the amino acids were sequenced and aligned in peptides ranging from 2 to 33 residues. Here we report five sequences of 21 to 33 residues. When these were compared with the primary structures of antithrombin III, alpha 1-antitrypsin and ovalbumin, which belong to the same protein superfamily (Hunt and Dayhoff [1980] Biochem Biophys Res Commun 95: 864-871), three peptides showed clear homologies with these proteins, indicating that alpha 2-antiplasmin also belongs to that superfamily. In addition, alpha 2-antiplasmin appeared to contain at least one internal homology.
Amino Acid Sequence↗
Studies on a form of alpha 2-antiplasmin in plasma which does not interact with the lysine-binding sites in plasminogen.
A method for the specific determination of a form of alpha 2-antiplasmin which does not interact with the lysine-binding sites in plasminogen (NPB-AP) has been elaborated. Basically, the method is an electroimmnoassay utilizing an intermediate gel, which adsorbs the plasminogen-binding form of alpha 2-antiplasmin (PB-AP). The plasma concentration of NPB-AP in healthy individuals was determined as 0.40 mumol/1 +/- 0.14 (SD), constituting 35% +/- 11 (SD) of the total plasma alpha 2-antiplasmin concentration. During extensive plasmapheresis of two pregnant severely D-immunized women the NPB-AP form decreased significantly, while the PB-AP form increased, thus maintaining the total alpha 2-antiplasmin at an almost constant level. The increased biosynthesis rate of alpha 2-antiplasmin during the extensive plasmapheresis is thus accounted for by PB-AP indicating this form to be the one primarily synthesized and that the non-binding form is formed in plasma from PB-AP secondarily.
Binding Sites↗
Circular dichroism studies on alpha 2-antiplasmin and its interactions with plasmin and plasminogen.
Spectropolarimetric studies of alpha 2-antiplasmin in the far ultraviolet region indicates a content of 16% alpha-helix, 18% beta-structure and 66% random coil. Two of its three disulphide bridges are reduced under non-denaturing conditions without major changes in conformation of functionality. Cleavage of the third disulphide bridge requires denaturing agents and is accompanied by complete loss of activity. The interaction of alpha 2-antiplasmin with plasminogen or fragments derived from plasminogen by elastase digestion has been studied by circular dichroism analysis in the near ultravoilet region. The results indicate that the fragment of plasminogen constituting the three NH2-terminal triple-loop structures contains at least two lysine-binding sites: one with high affinity and one with low affinity. One of these sites, probably the high-affinity site, is involved in the interaction with alpha 2-antiplasmin. This site seems also to be exposed in the intact plasminogen molecule. The formation of the stable complex between plasmin (EC 3.4.21.7) and alpha 2-antiplasmin is also accompanied by conformational changes.
Binding Sites↗