Quantitation of the Coagulation Inhibition in Vivo Due to Breakdown Products of Fibrin

1970 ◽  
Vol 23 (03) ◽  
pp. 477-485 ◽  
Author(s):  
P. S Mitchell ◽  
F. K Beller
Keyword(s):  
Blood Clotting ◽  
Fibrinogen Level ◽  
Degradation Products ◽  
Thrombin Time ◽  
Clotting Time ◽  
Human Fibrinogen ◽  
Quantitative Basis ◽  
Coagulation Inhibition

SummaryDegradation products of human fibrinogen were prepared by in vitro lysis of fibrin clots by urokinase activation and injected into rabbits on a quantitative basis. The dose necessary to anticoagulate the animal was equal to 2½ to 3 times the animals’ fibrinogen level. The effect on whole blood clotting time and thrombin time lasted for approximately 2 hrs. The “r” time of the TEG returned to normal after 1 hr while the “Max” value remained abnormal for more than 120 min. Degradation product E was shown to clear more rapidly than D by immunochemical techniques. The overall T ½ clearance was found to be approximately 12 hrs.

A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

1979 ◽  
Author(s):  
A.S. Bhargava ◽  
J. Heinick ◽  
Chr. Schöbel ◽  
P. Günzel
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Thrombin Time ◽  
Beagle Dogs ◽  
Clotting Time ◽  
Relative Potencies ◽  
Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

Intravascular Coagulation and Fibrinolysis by Stapliylocoagiilase. Comparison with Thrombin

1965 ◽  
Vol 13 (02) ◽  
pp. 457-469 ◽  
Author(s):  
J Jeljaszewicz ◽  
S Niewiarowski ◽  
A Poplawski ◽  
J Prokopowicz ◽  
K Worowski
Keyword(s):  
Intravenous Injection ◽  
Antithrombin Iii ◽  
Fibrinogen Level ◽  
Thrombin Time ◽  
Clotting Time ◽  
Short Time ◽  
Coagulation And Fibrinolysis ◽  
In Vitro Comparison

SummaryIntravascular clotting in rabbits after intravenous injection of 30 N.I.H. units of thrombin/kg and 5/mg/kg of staphylocoagulase were studied. Following determinations were made : clotting time, thrombin time, fibrinogen, factors V, VII, VIII, X, XI and XII, “true prothrombin”, two stage prothrombin, P-P test, prothrombin consumption, fibrinolysis in serum euglobulins, plasminogen, antithrombin III and VI, and coagulase reacting factor. Intravenous staphylocoagulase produced in rabbits precipitous drop of fibrinogen level in a very short time, which was not observed with the dose of thrombin used. Activity of staphylocoagulase in vivo was very much higher than of thrombin, as it could be expected on basis of the in vitro comparison. Together with defibrination caused by staphylocoagulase, fibrinogen breakdown products, identified as antithrombin VI, appear in plasma. There were no such changes observed when thrombin was used. Slight decrease of prothrombin and antithrombin III levels, together with a lack of significant changes in factors V, VII, VIII, X, XI and XII content, were observed after staphylocoagulase injection. Slight increase of CRF content in staphylo co agulase clotted plasma was noted. Fibrinolytic system was not activated during clotting process caused by staphylo-coagulase. Significance of the data obtained is discussed.

scholarly journals The Immunosuppressive Activity of Plasmic Degradation Products of Human Fibrinogen

1977 ◽  
Author(s):  
T.S. Edgington ◽  
L.K. Curtiss ◽  
E.F. Plow
Keyword(s):  
Cell Response ◽  
Degradation Products ◽  
Thymidine Uptake ◽  
Immunosuppressive Activity ◽  
Human Fibrinogen ◽  
Peptide Fraction ◽  
Intact Molecule ◽  
Stimulation Of

Plasmic cleavage of human fibrinogen leads to generation of immunosuppressive activity not expressed by the intact molecule, and which is demonstrable in vitro and in vivo. This activity is not associated with the high molecular weight derivatives X, Y, D and E, but is present in the small dialyzable peptide fraction obtained from plasmic digestion. The peptides inhibit in a non-toxic fashion, the stimulation of 3H-thymidine uptake and blastogenesis of lymphocytes by phytohemagglutinin (PHA) and allogeneic cells (MLC) under conditions of both macrophage dependence and macrophage independence. The peptides also suppress the plaque-forming cell response of mice to sheep red blood cells in vivo. Approximately 30 μg peptides/culture leads to a 50% inhibition of the PHA and MLC systems, and approximately 400 μg/mouse produces a 50% suppression of the plaque-forming cell response. Intact fibrinogen chains exhibit negligible activity, but plasmic digests of Aα chain are suppressive. Consistent with derivation from the Aa chain was the demonstration that the activity was generated from limited plasmic digest of fibrinogen which produced fragment X, and this activity was soluble at 80°C for 10 minutes. The release of the active peptide by limited plasmic degradation, and the activity of these peptides at physiologic concentrations suggests that this system may be of importance in vivo in association with local fibrinogenolysis or fibrinolysis at sites of thrombosis. This has been in part substantiated by the experimental initiation of fibrinogenolysis in vivo with streptokinase.

Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5129-5129
Author(s):  
Jawed Fareed ◽  
Debra Hoppensteadt ◽  
Omer Iqbal ◽  
Jeanine M. Walenga ◽  
Bruce E Lewis
Keyword(s):  
Protein Interactions ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Anticoagulant Effect ◽  
Thrombin Time ◽  
Clotting Time ◽  
Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

THE EFFECT OF THE VENOM OF THE ORIENTAL HORNET ON COAGULATION FACTORS

1987 ◽  
Author(s):  
A Kornberg ◽  
S Kaufman ◽  
L Silber ◽  
J Ishay
Keyword(s):  
Human Plasma ◽  
Degradation Products ◽  
Anticoagulant Activity ◽  
Coagulation Factors ◽  
Plasma Fibrinogen ◽  
Thrombin Time ◽  
Clotting Factors ◽  
Viii And Ix

The extract from the venom sac of Vespa orientalis (VSE) inactivates exogenous and endogenous thromboplastin (Joshua and Ishay, Toxicon, 13:11-20,1975). The prolongation of both prothrombin time (PT) and recalcification time suggests inactivation of other factors. The aim of the present study is to investigate the effect of VSE on clotting factors. A lyophilized VSE with protein concentration of 5 mg/ml was used. Studies were performed in vitro with human plasma and in vivo in cats. Routine methods were employed for the assay of PT, activated tissue thromboplastin (APTT), thrombin time (TT), fibrinogen degradation products (FDP), fibrinogen and factors V,VII,VIII,IX,X. Human plasma was incubated with various concentrations of VSE (0,1,5,10,50,100 μg/ml) for 60 min and for various incubation times (0,5,15,30,+ 60,90,120 min) with 50 μg/ml VSE (n=8). 1 μg/ml VSE prolonged PT from 13.5 to 16 sec (p<0.05) and APTT from 62 to 180 sec. PT was maximal (17.7 sec) with 10 μg/ml and APTT (442 sec) with 50 μg/ml VSE. Factors V,VII,X decreased gradually from 94-105% to 11%,11% and 29% with 100 μg/ml VSE and VIII and IX to 1% even with 1 μg/ml VSE. After 5 min with constant concentration of VSE (50 μg/ml) PT was 14.9 sec (normal 13 sec) and APTT 165 sec (normal 54 sec). Both were maximal (17.5 and 298 sec) after 60 min. Factors VII and X decreased to 13% and 32% and VIII and IX to >1% after 60 min of incubation. Injection of 5 mg/kg VSE to cats (n=6-8) resulted in prolongation of PT from 9.4 to 11.2 sec and of APTT from 19.5 to 63 sec after 5 min. Both were maximal after 90 min (12.3 and 127 sec). Factors V,VII and X decreased from 100% to 7.6%, 13% and 37% and VIII and IX to 1% after 10 min. In all experiments TT and plasma fibrinogen were not affected and FDP were normal. Heating of VSE for 5 min at 80°C abolished completely the anticoagulant activity but dialysis for 24 hr at 4°C had no effect on it. The activity was eluted on Sephadex-25 both in void and post void volumes. The results show that VSE has a potent anticoagulant activity against various factors. Factors VIII and IX are markedly decreased. The effect on V, VII and X is moderate. Plasma fibrinogen is not affected. The nature and clinical significance of the anticoagulant activity merit further investigation.

scholarly journals Effect of Angiography on Blood Coagulation

1977 ◽  
Author(s):  
W. H. Krause ◽  
A. Lang
Keyword(s):  
Contrast Media ◽  
Prothrombin Time ◽  
Antithrombin Iii ◽  
Degradation Products ◽  
Anticoagulant Activity ◽  
Thromboembolic Complication ◽  
Thrombin Time ◽  
Media Concentration

It is known, that angiographic contrast media have an anticoagulant activity in vitro. The purpose of the present study was, to investigate this effect in vivo. The catheter was introduced percutaneously according to Seldinger into the femoral artery. The prothrombin time, activated thromboplastin time (APTT), thrombin time, reptilase time, fibrinogen, plasminogen, antithrombin III, platelets, fibrin/fibrinogen degradation products (FDP), haematocrit and contrast media concentration were studied in a series of 50 patients before and following abdominal aorto-arteriographic procedures up to 6 hours. Thrombin time and reptilase time were prolonged significantly 30 and 60 minutes after angiography. There was a correlation between clotting tiies and contrast media concentrations. Prothrombin time, APTT, and platelet counts remained unchanged. Fibrinogen, plasminogen,and antithrombin III levels showed a significant reduction after 30 minutes. FDP concentrations were increased significantly up to 6 hours, there was no correlation between contrast media concentrations and split products. The results were corrected for contrast media dilution according to the haematocrit. No thromboembolic complication was observed. The results suggest that angiographic procedure may initiate an intravascular coagulation with an activation of the fibrinolytic system. In addition the contrast media showed an inhibition of fibrin polymerization in vivo.

Lupus-Like Anticoagulant In A Patient With Severe Factor VIII Deficiency

1981 ◽  
Author(s):  
R S Weinger ◽  
J L Moake ◽  
D Deykin ◽  
A Lopez ◽  
J D Olson
Keyword(s):  
Fibrinogen Level ◽  
Specific Inhibitor ◽  
Test System ◽  
Rabbit Brain ◽  
Clotting Time ◽  
Normal Plasma ◽  
Tissue Thromboplastin ◽  
Dilute Suspension

A severe hemophiliac (<1% FVIII-AHG) was transfused with FVIII concentrate and had orthopedic surgery without complication. His in vivo FVIII-AHG survival was normal (T½= 8hrs), as was his fibrinogen level, prothrombin (PT) and thrombin times. His prolonged (>100 sec.) activated partial thromboplastin time was incompletely corrected (40 sec.; normal<36 sec.) by the in vitro addition of an equal volume of pooled normal plasma, and did not become further prolonged after ½ and 2 hr. incubations at 37°C. A specific inhibitor to FVIII-AHG was not present. However, a thromboplastin inhibition test using patient plasma and progressively higher dilutions of rabbit brain thromboplastin (whole phospholipoprotein membranes) in a PT test system was positive. When either normal gel-separated platelets (GSP) or a dilute suspension of inosithin was used as a source of phospholipid, and clotting then initiated by the simultaneous addition of calcium and Russell’s viper venom (to activate FX directly), patient plasma clotting times were similar to the clotting times of normal plasma and other severe FVIII-AHG deficient plasmas. In contrast, when both normal GSP and dilutions of tissue thromboplastin reagent were added to plasma, and clotting then initiated by recalcification, patient plasma clotting time was prolonged in comparison to the clotting times of normal plasma and other FVIII-AHG deficient plasmas. This “lupus-like” anticoagulant had no effect on in vivo hemostasis, even in our patient with severe hemophilia A. The anticoagulant differs from a lupus anticoagulant recently well characterized (Thiagarajan et al., J.Clin. Invest. 66;397,1980) in that it interacts better with phospholipoprotein membranes than with free phospholipids, and the in vitro defect is not corrected by the addition of normal platelets.

The Action of Brinase in Vitro and in Vivo

1975 ◽  
Author(s):  
P. J. Gaffney ◽  
K. Lord ◽  
R. D. Thornes
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Size Range ◽  
Degradation Products ◽  
Molecular Size ◽  
Human Fibrinogen ◽  
Rate Limiting Step ◽  
Rate Limiting

Brinase (an extract of Aspergillus Oryzae) was shown to rapidly digest human fibrinogen in vitro to aggregable degradation products with a molecular size range of 310,000 to 230,000 the latter fragments being more slowly digested to core fragments, Dbr and Ebr. The fibrinogen polypeptide chain susceptibility to Brinase attack was in the order Aα, γ, Bβ, Lysis of the Bβ chain seems to be the rate limiting step in the conversion of the high molecular weight fragments (MW 310,000–230,000) to the core fragments Dbr and Ebr. The conservation of NH2 terminal Tyrosine during fibrinogen digestion and the very transient existence of D dimer fragments during totally crosslinked fibrin lysis suggest that the carboxy end of the γ chain is prone to Brinase attack. The crosslinked α chains of fibrin, while resistant to plasmin, are vigorously digested by Brinase. The plasma of cancer patients being treated with Brinase contained degraded fibrinogen (lacking intact Aα chains) and their aggregates. These aggregates contained some crosslinked γ chains (γ-γ dimers) suggesting that Brinase in vivo exorcises both a lytic and coagulant effect. Thrombin mediated clots in all the plasmas examined contained no crosslinked α chains. Positive plasma ethanol gelation tests can be explained by the presence of the aggregable high molecular weight fragments observed during the in vitro lysis of fibrinogen by Brinase.

scholarly journals Four-Factor Prothrombin Complex Concentrate (4F-PCC) Effectively Reverses Rivaroxaban Induced Bleeding in a Rabbit Model of Acute Bleeding

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4220-4220
Author(s):  
Eva Herzog ◽  
Jochen Mueller-Cohrs ◽  
Franz Kaspereit ◽  
Wilfried Krege ◽  
Peter Niebl ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Blood Clotting ◽  
Prothrombin Complex Concentrate ◽  
Rabbit Model ◽  
Off Label Use ◽  
Clotting Time ◽  
Acute Bleeding ◽  
Off Label

Abstract Rivaroxaban is an oral, selective direct factor Xa inhibitor approved for several indications in patients at risk of thrombotic events. One limitation of this agent is the lack of data pertaining to its reversal in situations where urgent response is critical (e.g. acute bleeding events or emergency surgery). This study therefore evaluated the effectiveness of a four-factor prothrombin complex concentrate (4F-PCC; Beriplex® P/N, Kcentra®, CSL Behring), for the reversal of rivaroxaban-associated bleeding in an in vivo rabbit model of acute bleeding. In addition, the study evaluated the correlation between in vitro coagulation parameters and haemostasis in vivo in order to gain more information on the predictivity of in vitro markers for effective bleeding reversal. Administration of single intravenous doses of rivaroxaban (150–450 μg/kg) resulted in increased and prolonged bleeding following standardised kidney injury compared to a vehicle treated control group as determined by measurements of total blood loss and time required to achieve full hemostasis. Subsequent treatment with 4F-PCC (25−100 IU/kg) resulted in a dose-dependent reversal of rivaroxaban-associated bleeding signals. Of the in vitro coagulation markers tested, thrombin generation (TGA) and whole blood clotting time (WBCT) correlated well with in vivo measures of PCC-mediated effects. Thrombin generation was highly reagent-dependent, with the assay initiated using tissue factor reagent being most sensitive to the anticoagulant effects induced by Rivaroxaban, while the phospholipid-only reagent being the most predictive of 4F-PCC mediated effective haemostasis in vivo. In summary, in a rabbit model of acute bleeding, 4F-PCC was able to effectively reduce bleeding to control levels following rivaroxaban 150 μg/kg and 300 μg/kg administrations. The 4F-PCC mediated bleeding reversal correlated best with the in vitro endpoints thrombin generation and whole blood clotting time. Disclosures Herzog: CSL Behring GmbH: Employment. Off Label Use: The use of Beriplex P/N for reversal of Rivaroxaban anticoagulation represents off-label use. Mueller-Cohrs:CSL Behring GmbH: Employment. Kaspereit:CSL Behring GmbH: Employment. Krege:CSL Behring GmbH: Employment. Niebl:CSL Behring GmbH: Employment. Doerr:CSL Behring GmbH: Employment. Schulte:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment.

scholarly journals Inhibition of Platelet Aggregation by Brinolase Degradation Products of Fibrinogen and Serum Albumin

1977 ◽  
Author(s):  
W. H. E. Roschlau
Keyword(s):  
Platelet Aggregation ◽  
Serum Albumin ◽  
Degradation Products ◽  
Platelet Rich Plasma ◽  
Biological Effects ◽  
Response Curves ◽  
Human Fibrinogen ◽  
Inhibition Of Platelet Aggregation

Brinolase (fibrinolytic enzyme from Aspergillus oryzae) was observed to possess significant platelet aggregation inhibitory properties during and after thrombolytic therapeutic use. These platelet effects were found in vitro to be caused in part by intermediate products of fibrinogen digestion, namely low-molecular-weight peptides of approx. MW 2500. Human fibrinogen peptides were isolated, purified, and shown to have high inhibitory activity in platelet-rich plasma. Quantitative comparisons of attainable platelet inhibition in vitro and observed responses in vivo during administration of equivalent enzyme doses, however, suggested that total available fibrinogen, even if it were entirely converted to degradation products (which it is not), would be insufficient to account for observed platelet effects of brinolase therapy.Human serum albumin is also readily degraded by brinolase. Albumin degradation products were prepared in vitro by optimal incubation with the enzyme. Dose-response curves of inhibition of platelet aggregation were obtained with lyophilized peptides in platelet-rich plasma in vitro, and significant inhibition of platelet aggregation was observed in vivo following infusion of albumin degradation products into rabbits. The enzyme doses and amounts of substrates employed in all experiments were equivalent to the conditions of therapeutic fibrinolysis.Thus, albumin degradation products are considered to contribute a significant, if not the major, portion of platelet-active intermediates during clinical brinolase therapy. Albumin cleavage, which is unique to brinolase amongst clinical fibrinolytic enzymes, was shown to have biological effects of its own, but it may also serve to protect coagulation proteins from enzymatic destruction through competition for the enzyme during systemic brinolase therapy.

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