Effects of Bradykinin on Coagulation and Fibrinolysis Study in Vitro and in Vivo

1965 ◽  
Vol 14 (03/04) ◽  
pp. 508-518 ◽  
Author(s):  
G. G Neri Serneri ◽  
P. L Rossi Ferrini ◽  
P Paoletti ◽  
A Panti ◽  
G D’Ayala Valva
Keyword(s):  
Thrombin Generation ◽  
Thrombin Time ◽  
Lysis Time ◽  
Time Determination ◽  
Recalcification Time ◽  
Generation Test ◽  
Coagulation And Fibrinolysis

SummaryThe effects of bradykinin on coagulation and fibrinolysis have been studied both “in vitro” and “in vivo” in man. “In vitro” bradykinin employed at different concentrations does not affect the coagulation and fibrinolysis processes in any appreciable way. Bradykinin, intraarterially injected in man in the dose of 10 y, does not modify coagulation studied both with global investigations (thrombelastogram, recalcification time) and with analytical researches (Quick’s time, activation test of intrinsic thromboplastin, thrombin generation test and thromboplastin test, thrombin time, determination of antithrombin II and III). Bradykinin instead produces an activation of fibrinolysis both in the thrombelastographic investigation and in the lysis time of euglobulins. The decrease in the activity of the proactivator and of plasminogen supports our view that bradykinin produces an activation of the fibrinolytic system by liberating tissue kinases which act on the proactivator. The authors have discussed the physiological and physiopathological significance of the observed findings.

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.

scholarly journals Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

2020 ◽  
Vol 26 ◽  
pp. 107602962096082
Author(s):  
Dalia Qneibi ◽  
Eduardo Ramacciotti ◽  
Ariane Scarlatelli Macedo ◽  
Roberto Augusto Caffaro ◽  
Leandro Barile Agati ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Thrombin Generation ◽  
Area Under The Curve ◽  
Factor Xa ◽  
In Vivo Studies ◽  
In Vitro Tests ◽  
Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

Fibrinogen Break-Down Products and Clotting Parameters

1967 ◽  
Vol 17 (03/04) ◽  
pp. 467-481 ◽  
Author(s):  
J Gormsen ◽  
B Laursen
Keyword(s):  
Fibrinolytic Activity ◽  
Thrombin Generation ◽  
Specific Activity ◽  
Disc Electrophoresis ◽  
Clot Formation ◽  
Immunological Methods ◽  
Thrombin Time ◽  
Antigenic Properties

SummaryFibrinogen and fibrin break-down products (b. p.) in vitro and in vivo are identified by immunodiffusion, Immunoelectrophoresis and acryl-disc-electrophoresis with immunodiffusion.The clotting parameters obtained in vitro by addition of b. p. to plasma system are compared with similar tests in patients with spontaneous occurence of b. p.B. p. formed after short plasmin digestion of fibrinogen prolong significantly the thrombin time, reduce the maximal thrombin activity in the thrombin generation test and inhibit the clot formation in TEG, increasing the k- and reducing the mA values. These b. p. are further digested. The plasmin resistant fractions exhibiting D or E antigenic properties are less active. The specific activity of the different fractions were not investigated.The clotting parameters in patients with spontaneous occurence of b. p. (D fraction) are very different. Defects similar, but not identical, to those obtained in vitro by addition of b. p. to plasma systems are seen. Accelerated thrombin generation, enhanced fibrin polymerization and accelerated clot formation in TEG are found in others. Similar differences in fibrinolytic activity are demonstrated. The presence of b. p. might be the only abnormality found. B. p. are suggested to develop either as the result of localized thrombo-fibrinolysis or during a more generalized increase in fibrinolytic activity.Immunological methods can demonstrate b. p. in the concentration of 0.05 to 0.10 mg%. The thrombin time is not significantly prolonged until b. p. are present in the concentration of 0.5 mg% (early b. p.) - 2 mg% (later b. p.) as estimated spectrophotometrically.

The Influence of Infusions of 1-Desamino-8-D-Arginine Vasopressin (DDAVP) In Vivo on Thrombin Generation In Vitro

1992 ◽  
Vol 68 (01) ◽  
pp. 037-039 ◽  
Author(s):  
S H Ibbotson ◽  
J A Davies ◽  
P J Grant
Keyword(s):  
Arginine Vasopressin ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Occlusive Disease ◽  
Lag Phase ◽  
Computer Assisted ◽  
Chromogenic Substrate ◽  
Generation Test

SummaryTo investigate the effect of increasing FVIII:C in-vivo on coagulation ex-vivo, DDAVP was infused over 15 min in 10 volunteers and in-vitro thrombin generation measured. FVIII:C rose from 0.42 and 0.43 IU/ml before DDAVP to 1.38, 1.73 and 1.78 IU/ml at 15, 30 and 60 min respectively (p <0.001).A computer-assisted thrombin generation test was performed in defibrinated plasma using chromogenic substrate, S2238. Time to reach 50% maximal thrombin activity (T50/s) and lag phase of thrombin generation (lag/s) were measured. Lag shortened from 75 and 60 s before to 45 s during and after infusion (p <0.001). T50 shortened from 78.5 and 76.0 to 62.5, 60.0 and 58.5 s at times 15 (p <0.01), 30 (p <0.001) and 60 (p <0.001) min. FVIII:C correlated inversely with lag and T50 (r = –0.847, p <0.001, r = –0.826, p <0.001, n = 10) respectively.These findings show that acute elevations of FVIII:C in-vivo accelerate in-vitro thrombin production. This work suggests that elevated FVIII:C levels in-vivo may be important in thrombo-occlusive disease.

A HEP ARINE-LIKE ANTICOAGULANT IN A PATIENT WITH WEGENER’S GRANULOMATOSIS

1987 ◽  
Author(s):  
L Chrobák ◽  
V Rozsíval ◽  
V Herout
Keyword(s):  
Wegener’S Granulomatosis ◽  
Wegener's Granulomatosis ◽  
Thrombin Time ◽  
Protamine Sulphate ◽  
Normal Limits ◽  
Significant Prolongation ◽  
Lysis Time ◽  
Coagulation Tests

In a 23-year-old man with Wegener’s granulomatosis and mild bleeding coagulation studies revealed a significant prolongation of the coagulation time (CT) prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), failure of TT and aPTT to correct in a 1:1 mixture with pooled normal plasma (PNP), correction of the prolonged TT with toluidine blue and correction of TT and aPTT both in vitro and in vivo^protamine sulphate (P.S.).All other coagulation tests, i.e.,bleeding time, platelet count? fibrinogen level, euglobulme lysis time were within normal limits. The patient did not receive heparine. TT after administration of 5ml of protamine sulphate i.v. to the patient became normal - 18,2 s (19,7 s).In summary a patient with Wegener s granulomatosis associated with an endogenous heparine-like anticoagulant is reported. The anticoagulant could be corrected both in vitro and in vivo by protamine sulphate.

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.

ASPIRIN ACETYLATES FIBRINOGEN AND ENHANCES FIBRINOLYSIS IN VIVO. FIBRINOLYTIC EFFECT IS INDEPENDENT OF CHANGES IN PLASMINOGEN ACTIVATOR LEVELS

1987 ◽  
Author(s):  
D Thorir ◽  
M D Bjornsson ◽  
Henry Berger
Keyword(s):  
Plasminogen Activator ◽  
Time Course ◽  
Clot Lysis ◽  
Thrombin Time ◽  
Lysis Time ◽  
Coagulation Tests ◽  
Clot Lysis Time ◽  
Increased Susceptibility

In addition to its antiplatelet effect, aspirin has been reported to have fibrinolytic and hypoprothrombinémie effects. The objective of this study was to investigate possible mechanisms underlying the enhanced fibrinolysis observed after aspirin. Five healthy subjects received 650 mg of aspirin ql2hr for five days. Blood samples were collected before aspirin (control) and immediately before (0 hr) and two hours after (2 hr) the last dose for determinations of clot lysis time, time course of thrombin-induced fibrin aggregation, tissue plasminogen activator (tPA), intrinsic pathway fibrinolytic activity (IPFA), plasminogen, fibrinogen, aspirin and salicylic acid, and the coagulation tests activated partial thromboplastin time, thrombin time and prothrombin time. Clot lysis time was shorter after aspirin, control: 9.1±12.4 min (mean±s.d.), 0 hr: 4.6±4.0 min, 2 hr: 5.7±6.2 min (p:0.04), and the fibrin aggregation curves showed increased turbidity (expressed as AUC over 10 min), control: 72.7±17.8 mnumin, 0 hr: 94.6±1.6 mnumin, 2 hr: 112.8±45.1 mnumin (p:0.02). Control values of tPA (0.11±0.04 IU/ml), IPFA (2.20±0.59 IU/ml), plasminogen (10.9±1.0 mg/dl), fibrinogen (288±37 mg/dl), and the coagulation tests were not differnet from those after aspirin. Plasma aspirin concentrations were below detection limits at 0 hr and averaged 1.63±0.97μg/ml at 2 hr. In vitro studies using fibrinogen-free plasma and added acetylated fibrinogen sfiowed an inverse relationship between the extent of acétylation and clot lysis time. Studies using 14C-acetyl-labeled aspirin and fibrinogen showed that fibrinogen is acetylated to form ε-N-acetyl-lysine on both D and E domains of the molecule (50.6±2.0 and 49.4±2.0%, respectively) and on α β and γchains of the molecule (34.4±1.6, 30.7±3.4 and 34.9±4.7%, respectively), with preferential acétylation on the E domain. On the average, 2.88±1.49 ε-N-acetyl-lysyl residues were formed on each fibrinogen molecule. These results suggest that N-acetylation of lysyl residues of fibrinogen is responsible for the increased susceptibility of fibrin clots to lysis after aspirin.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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