scholarly journals Pengaruh Proses Fermentasi pada Daun Centella asiatica oleh Acetobacter tropicalis Terhadap Aktivitas Trombolitik

2021 ◽  
Vol 6 ◽  
pp. 1
Author(s):  
Lailatul Nuraini ◽  
Bambang Tri Purwanto ◽  
Achmad Syahrani ◽  
Riesta Primaharinastiti ◽  
Achmad Toto Poernomo

Agen trombolitik merupakan plasminogen activator yang dapat memecah fibrin menjadi fibrin degradation product (FDP) dan dapat digunakan pada terapi penyakit kardiovaskular. Agen trombolitik dapat diperoleh dari mikroorganisme seperti Acetobacter tropicalis InaCC B374 dan dari tanaman seperti Centella asiatica. Kedua sumber agen trombolitik tersebut dapat dilakukan kombinasi melalui proses fermentasi untuk meningkatkan efek terapetiknya. Proses fermentasi sendiri dipengaruhi oleh beberapa faktor termasuk media fermentasi dan waktu fermentasi. Penelitian ini bertujuan untuk mengetahui pengaruh proses fermentasi terhadap peningkatan aktivitas trombolitik dari hasil fermentasi Centella asiatica oleh Acetobacter tropicalis InaCC B374 pada berbagai variasi waktu fermentasi. Preparasi dilakukan dengan memfermentasi Centella asiatica selama 24, 48, dan 72 jam pada suhu 30°±1°C dengan kecepatan pengocokan 100 rpm kemudian ditentukan aktivitas trombolitiknya dengan metode clot lysis yang dilakukan inkubasi pada suhu 37°±1°C selama 60 menit. Hasil pengujian aktivitas trombolitik menunjukkan bahwa terjadi peningkatan aktivitas trombolitik setelah dilakukan proses fermentasi selama 24, 48 dan 72 jam dan aktivitas trombolitik maksimum tercapai pada hasil fermentasi 72 jam. Centella asiatica yang difermentasi selama 72 jam menunjukkan nilai indeks trombolitik yang paling besar (82,03) jika dibandingkan dengan infusa Centella asiatica tanpa fermentasi (37,39) dan Acetobacter tropicalis InaCC B374 (37,68). Disimpulkan bahwa proses fermentasi Centella asiatica oleh Acetobacter tropicalis InaCC B374 secara signifikan dapat meningkatkan aktivitas trombolitik keduanya

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1341-1348 ◽  
Author(s):  
CM Lawler ◽  
EG Bovill ◽  
DC Stump ◽  
DJ Collen ◽  
KG Mann ◽  
...  

Abstract The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the “Thrombolysis in Myocardial Infarction II” (TIMI II) trial. Cross- linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1–42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15–42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1–42 in the fibrinopeptide beta 15–42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15–42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1–42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin- specific tag ELISA may provide more accurate information when correlated with clinical endpoints.


Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1341-1348 ◽  
Author(s):  
CM Lawler ◽  
EG Bovill ◽  
DC Stump ◽  
DJ Collen ◽  
KG Mann ◽  
...  

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the “Thrombolysis in Myocardial Infarction II” (TIMI II) trial. Cross- linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1–42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15–42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1–42 in the fibrinopeptide beta 15–42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15–42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1–42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin- specific tag ELISA may provide more accurate information when correlated with clinical endpoints.


1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.


1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.


1988 ◽  
Vol 60 (02) ◽  
pp. 328-333 ◽  
Author(s):  
N J de Fouw ◽  
Y F de Jong ◽  
F Haverkate ◽  
R M Bertina

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.


1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.


1987 ◽  
Vol 58 (04) ◽  
pp. 1085-1087 ◽  
Author(s):  
P J Gaffney ◽  
A D Curtis

SummaryAn international collaborative study involving ten laboratories located in eight different countries was undertaken in order to replace the current International Standard (I.S.) for tissue plasminogen activator (t-PA). Two lyophilised candidate preparations of high purity were assessed in comparison with the current I.S. for t-PA using only a clot lysis assay. One preparation (coded 861670) was purified from a cultured melanoma cell supernatant and was about 98% single chain t-PA while the other preparation (coded 861624) was derived from Chinese hamster ovary (CHO) cells following DNA recombinant procedures and was 75% single chain t-PA.Both candidate preparations of t-PA compared in quite a satisfactory manner with the current I.S. from the viewpoint of the biometrics of parallel line bioassays and both preparations were quite stable for long periods at low temperatures and stable from up to 1 month at temperatures of 20° and 38° C. Both fultil the criteria to serve as a satisfactory Znd International Standard for t-PA. The Fibrinolysis Subcommittee of the International Committee for Thrombosis and Haemostasis recommended the melanoma source t-PA (861670) as the next I.S. in order to maintain continuity with the 1st I.S. which was also a melanomatype preparation. The data from the ten laboratories indicated that each ampoule of the new proposed standard contains 850 international units of t-PA activity by the clot lysis assay. It is planned to present the results of this study to the Expert Committee on Biological Standardization of the World Health Organization at its next meeting and to request that the preparation of t-PA, coded 861670, be established as the 2ndlnternational Standard for t-PA.


1979 ◽  
Vol 41 (04) ◽  
pp. 745-755 ◽  
Author(s):  
Dušan Keber ◽  
Mojca Stegnar ◽  
Irena Keber ◽  
Bojan Accetto

SummaryFibrinolysis was studied in 10 alpinists during regular physical activity of different intensity. Blood was sampled at rest and after exposure to submaximal workload on the treadmill on three occasions: before and after 6 months physical conditioning (moderate physical activity), and after 6 weeks of an alpinistic expedition (strenuous physical activity). Measurements included submaximal working capacity, fibrinogen, euglobulin clot lysis time (ELT), whole plasma clot lysis time, and estimations derived from ELT - percent increase in fibrinolytic activity after exercise (RFS), and absolute increase in fibrinolytic activity after exercise (PAR).Regular moderate activity increased the resting level of ELT, but strenuous activity decreased is. After each treadmill testing, a marked increase in fibrinolytic activity was observed. RFS was unaltered at all three testings. PAR increased after moderate activity, but decreased after strenuous activity.The results indicate that regular physical activity can lead from enhanced to decreased resting activity of plasminogen activator in blood. It is presumed that increased release of activator during prolonged stress causes partial depletion of endothelial stores with the consequence of decreased activator activity in the blood.


1992 ◽  
Vol 67 (06) ◽  
pp. 686-691 ◽  
Author(s):  
Hua Rong Lu ◽  
Herman K Gold ◽  
Zaomin Wu ◽  
Tsunehiro Yasuda ◽  
Patrick Pauwels ◽  
...  

SummaryThe effects of G4120, a cyclic Arg-Gly-Asp (RGD) containing peptide which inhibits fibrinogen binding to the platelet receptor GPIIb/IIIa, on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were investigated in a combined arterial and venous thrombosis model in heparinized dogs. The arterial thrombus model consisted of a 3 cm everted (inside-out) carotid arterial segment inserted into a transsected femoral artery which occludes within 30 min with platelet-rich material and which is resistant to recanalization with 0.5 mg/kg rt-PA. The venous thrombus was a 125I-fibrin labeled whole blood clot produced in the contralateral femoral vein.In 5 dogs given an intravenous bolus of 0.05 mg/kg G4120 followed by a continuous infusion of 0.05 mg/kg per hour for 3 h (group I), arterial occlusion persisted throughout a 4 h observation period and was still present at 24 h in all dogs; the extent of venous clot lysis after 120 min was 27 ± 7%. In 5 dogs given the same infusion of G4120 in combination with 0.5 mg/kg rt-PA over 60 min, recanalization of the arterial graft occurred in all dogs, within 13 ± 2 min and persisted throughout the observation period of 4 h (p = 0.01 versus G4120 or rt-PA alone); at 24 h, however, all grafts were occluded. Venous clot lysis in this group was 75 ± 8% (p = 0.002 versus G4120 alone andp NS versus rt-PA alone). Pathologic analysis revealed platelet-rich or mixed thrombus with platelet-rich and erythrocyte-rich zones. The last 6 dogs were given a reduced dose of G4120 consisting either of a 0.05 mg/kg bolus followed by an infusion of 0.05 mg/kg over 1 h in 3 dogs (group III) or of a single 0.05 mg/kg bolus in 3 dogs (group IV), both given in combination with 0.5 mg/kg rt-PA infused over 60 min. These protocols produced recanalization within 15 ± 2 and 34 ± 8 min, respectively, which was maintained throughout the 4 h observation period. Venous lysis in these groups was 63 ± 4 and 97 ± 1% respectively. Bleeding times prolonged from 1 to 2 min to >30 min with G4120, but returned towards baseline within 2 h after the end of the infusion. Platelet aggregation with ADP was completely inhibited with G4120 but partially recovered within 1 h after the end of the infusion. No fibrinogen breakdown was observed in association with the rt-PA infusion.Thus, G4120, a synthetic GPIIb/IIIa receptor antagonist, enhances and accelerates lysis of platelet-rich arterial thrombosis with rt-PA and prevents reocclusion during and within 3 h after the infusion. It may be useful for the conjunctive use with thrombolytic agents in patients with arterial thromboembolic disease.


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