Modulating Plasmin Activity using Multivalent Benzamidine Inhibitors

SummaryThe activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul275, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg275- Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s−1) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s−1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s−1). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s−1 respectively). In contrast, the vs for nPlg and rPlg-Ala740 by single chain rt-PA- G1u275, two-chain rt-PA-G1u275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala740), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

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Plasminogen Depletion during Streptokinase Treatment or Two-Chain Urokinase Incubation Correlates with Decreased Clot Lysability Ex Vivo and In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649714 ◽

1993 ◽

Vol 70 (06) ◽

pp. 0998-1004 ◽

Cited By ~ 13

Author(s):

Páll T Önundarson ◽

H Magnús Haraldsson ◽

Lena Bergmann ◽

Charles W Francis ◽

Victor J Marder

Keyword(s):

Myocardial Infarction ◽

Ex Vivo ◽

Time Dependent ◽

State Variables ◽

Thrombolytic Treatment ◽

Direct Proportion ◽

Plasmin Activity ◽

Plasma Exposure ◽

The Relationship

SummaryThe relationship between lytic state variables and ex vivo clot lysability was investigated in blood drawn from patients during streptokinase administration for acute myocardial infarction. A lytic state was already evident after 5 min of treatment and after 20 min the plasminogen concentration had decreased to 24%, antiplasmin to 7% and fibrinogen 0.2 g/1. Lysis of radiolabeled retracted clots in the patient plasmas decreased from 37 ± 8% after 5 min to 21 ± 8% at 10 min and was significantly lower (8 ± 9%, p <0.005) in samples drawn at 20, 40 and 80 min. Clot lysability correlated positively with the plasminogen concentration (r = 0.78, p = 0.003), but not with plasmin activity. Suspension of radiolabeled clots in normal plasma pre-exposed to 250 U/ml two-chain urokinase for varying time to induce an in vitro lytic state was also associated with decreasing clot lysability in direct proportion with the duration of prior plasma exposure to urokinase. The decreased lysability correlated with the time-dependent reduction in plasminogen concentration (r = 0.88, p <0.0005). Thus, clot lysability decreases in conjunction with the development of the lytic state and the associated plasminogen depletion. The lytic state may therefore limit reperfusion during thrombolytic treatment.

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Determination of Plasminogen in Human Plasma by the Lysis Time Method

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655622 ◽

1966 ◽

Vol 16 (01/02) ◽

pp. 001-017 ◽

Cited By ~ 8

Author(s):

W Berg ◽

K Korsan-Bengtsen ◽

J Ygge

Keyword(s):

Human Plasma ◽

Present Method ◽

Blood Donors ◽

Fibrinogen Concentration ◽

Plasmin Activity ◽

Time System ◽

Lysis Time ◽

High Dilutions ◽

Single Determination

SummaryA one-stage lysis time system containing fibrinogen, streptokinase, thrombin, and a known, small amount of plasminogen was used to determine plasminogen in plasma.The known amount of plasminogen was added to the system in order to keep the lysis times relatively short when a highly diluted plasma was tested. High dilutions of plasma were used to reduce the influence of the plasma inhibitors.The calculation of the plasminogen concentration was made on the basis of the correlation: “plasminogen = fibrinogen/lysis time” which was valid in the system. The method allowed determination of plasminogen in plasma with varying fibrinogen concentrations, as the fibrinogen concentration in plasma was considered in the calculation.The presence of “spontaneous” plasmin activity in the plasma did not influence the plasminogen determination. Estimated by this method, the plasminogen content in plasma from 32 blood donors aged 25-45 years was 13.1 ±2.4 casein u/ml. The error of a single determination was 0.3 casein u/ml. The plasminogen content in plasma, determined with the present method, is about 3-4 times higher than the content found when a caseinolytic method is used.

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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Effects of Intermittent Pneumatic Calf Compression On Postoperative Thrombin and Plasmin Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661639 ◽

1986 ◽

Vol 56 (02) ◽

pp. 198-201 ◽

Cited By ~ 25

Author(s):

Jeffrey Weitz ◽

Jost Michelsen ◽

Kenneth Gold ◽

John Owen ◽

Duncan Carpenter

Keyword(s):

Enzyme Activity ◽

Fibrinopeptide A ◽

Plasmin Activity ◽

Neurosurgical Patients ◽

The Mean ◽

Calf Compression

SummaryA previous study of neurosurgical patients demonstrated an imbalance between thrombin and plasmin action following surgery. The present study was designed to determine the effect of intermittent pneumatic calf compression on postoperative enzyme activity. Fibrinopeptide A (FPA) and Bβ 1-42 levels, reflecting thrombin and plasmin action respectively, were measured daily in patients undergoing elective craniotomy. Two of 9 patients not receiving calf compression developed positive fibrinogen leg scans, while none of 5 patients receiving prophylaxis had positive scans. Calf compression was associated with a markedly altered pattern of changes in the fibrinopeptide values following surgery. Without compression, there was perturbation of the balance between thrombin and plasmin action on the day after surgery as reflected by an increase in the FPA/Bβ 1-42 ratio. In contrast, in those receiving prophylaxis there was no change in this ratio on the first postoperative day. Calf compression both blunted the mean postoperative increase in the FPA level (1.8 nM vs 4.7 nM; p <.05) and augmented the mean Bβ 1-42 value (3.0 nM vs 0.2 nM; p <.05) so that the mean increase in the FPA/ Bβ 1-42 ratio was only 0.1 with calf compression as compared to 2.2 without it (p <.05). Systemic modulation of both the coagulation and fibrinolytic pathways thus occurred in association with calf compression.

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