ARICHemostasis Study - I. Development of a Blood Collection and Processing System Suitable for Multicenter Hemostatic Studies

1989 ◽  
Vol 61 (01) ◽  
pp. 015-019 ◽  
Author(s):  
A C Papp ◽  
H Hatzakis ◽  
A Bracey ◽  
K K Wu
Keyword(s):  
Processing System ◽  
Antiplatelet Agents ◽  
Epidemiologic Studies ◽  
Tissue Type ◽  
Factor Vii ◽  
Blood Collection ◽  
Atherosclerosis Risk In Communities ◽  
Type Plasminogen Activator ◽  
Hemostatic Factors

SummaryIn order to carry out a multicenter study aimed at understanding the association of hemostatic factors with atherosclerotic vascular disorders for the Atherosclerosis Risk In Communities (ARIC) Study, we compared a blood collection and processing system developed in our laboratory with the state-of-the-art- procedures. The salient features of our system included the use of a new phlebotomy set for venipuncture, the use of Millipore filters for removing platelet residues in the plasma and the use of a mixture of anticoagulants and antiplatelet agents for inhibiting the in vitro activation of platelets, coagulation and fibrinolytic system. The results derived from systematic evaluations indicate that this newly developed system yields the lowest values of plasma ßTG, PF 4 and FPA when compared with the reported values. The technique also gave reliable values of representative hemostatic measurements such as fibrinogen, factor VII, factor VIII, von Willebrand factor, antithrombin-III, protein C, tissue- type plasminogen activator, and serum thromboxane B2. Further experiments revealed that the samples withstood temporary storage at −70° C and overnight “shipping” manipulations without significant changes in the hemostatic values. We conclude that the described blood collection and processing system may be a valuable asset for conducting multicenter cooperative clinical trials and epidemiologic studies involving blood collection by multiple field centers or clinics.

scholarly journals Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1213-1217
Author(s):  
W Witt ◽  
B Baldus ◽  
P Bringmann ◽  
L Cashion ◽  
P Donner ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Desmodus Rotundus ◽  
Type Plasminogen Activator ◽  
Blood Clots ◽  
Hemostatic Factors ◽  
The Difference ◽  
Vampire Bat

rDSPA alpha 1 (recombinant Desmodus salivary plasminogen activator alpha 1) is a recombinant protein corresponding to a natural plasminogen activator from the vampire bat Desmodus rotundus. The thrombolytic properties of rDSPA alpha 1 and tissue-type plasminogen activator (t-PA) were compared in a rat model of pulmonary embolism. Whole blood clots, produced in vitro and labeled with 125I-fibrinogen, were embolized into the lungs of anesthetized rats. Thrombolysis was calculated from the difference between initial clot radioactivity and that remaining in the lungs at 60 minutes. Blood was sampled for gamma counting, measurement of hemostatic factors, and plasminogen activator antigen levels. Thrombolysis at 3, 10, 30, and 100 nmol/kg intravenously (10% bolus, 90% over 60 minutes) amounted to 30% +/- 2%, 51% +/- 4%, 85% +/- 4%, 98% +/- 0% for rDSPA alpha 1 and 30% +/- 3%, 41% +/- 3%, 57% +/- 6%, 93% +/- 2% for t-PA (controls: 29% +/- 2%; mean +/- SEM, n greater than or equal to 6). t-PA at 100 nmol/kg significantly decreased fibrinogen, plasminogen, and alpha 2- antiplasmin levels by 33% +/- 7%, 38% +/- 8%, and 61% +/- 9%, whereas rDSPA alpha 1 at 100 nmol/kg only lowered alpha 2-antiplasmin significantly (by 29% +/- 6%). Compared with t-PA, rDSPA alpha 1 is the more potent and more clot selective (fibrin specific) thrombolytic agent. These results suggest that rDSPA alpha 1 may be safer and more efficacious than currently used thrombolytics.

IN VITRO EFFECTS OF RECOMBINANT TISSUE TYPE PLASMINOGEN ACTIVATOR ON FIBRINOLYTIC AND COAGULATION PARAMETERS AND ITS PREVENTION BY SPECIFIC ANTIBODY, D-PHE-PRO-ARG-CTUCl AND APROTININ

1987 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Blood Collection ◽  
Freezing And Thawing ◽  
Blood Samples ◽  
Hemostatic Factors ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

Monitoring of systemic effects during rt-PA therapy has shown of depletion of fibrinogen-antiplasmin, plasminogen and other hemostatic factors. Because in vitro activation of plasminogen may occur between blood collection and freezing and thawing before assaying we analysed the influence of 0,0.2, 2.0 and 10.0,ug rt-PA/ml citrate blood (final conc.) on hemostatic and fibrinolytic parameters and its inhibition by 3 different inhibitors. Addition of rt-PA to citrated whole blood without an inhibitor induced a concentration-dependent depletion of Fbg, Plgα2-Apl,α2-M, C1 - I, α2-Atrp, a loss of activity of FV, VIII,IX, XIII and alterations of the global coagulation assays. No effect of rt-PA was observed on F II, VII, X, XI, XII, AT III and Protein C. To prevent in vitro fibrinogenolysis 0.1, 0.5 and 1 mg/ml of a polyclonal sheep anti-rt-PA-antibody, 0.3, 1.0 and 10 Aimol/1 PPACK (D-Phe-Pro-Arg-CH2Cl), 75 and 150 KlU/ml aprotinin (final conc.) and saline as a control were added to pooled citrate blood.All samples containing rt-PA and/or inhibitors and/or saline were incubated for 45 min on ice, centrifuged, aliquotted, snap frozen and stored at ™20° C until analysis. Pretreatment of blood samples with anti-rt-PA IgG prevented interferences with all fibrinolytic and most clotting assays in plasma at a dose of 2 ,ug rt-PA/ ml. PPACK was of limited utility in clotting assays, but enabled correct analysis of fibrinolytic assays. Aprotinin was suitable only for a restricted range of both assay types. It is concluded that collection of blood samples on an appropriate antibody may be the most suitable procedure to get correct measurements of in-vivo effects of rt-PA on the hemostatic system in patients undergoing fibrinolytic therapy.

Monitoring of Hemostasis Parameters During Coronary Thrombolysis with Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 59 (02) ◽  
pp. 133-137 ◽  
Author(s):  
D C Stump ◽  
E J Topol ◽  
A B Chen ◽  
A Hopkins ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Fibrinogen Level ◽  
Tissue Type ◽  
Poor Correlation ◽  
Blood Collection ◽  
Tissue Type Plasminogen Activator ◽  
Optimal Monitoring ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryThe monitoring of changes in the blood coagulation and fibrinolytic systems during thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) may be complicated by artifacts due to in vitro activation after blood collection and to interference of other agents (e. g., heparin) in the assays. In 106 patients with early acute myocardial infarction, infused with 150 mg of rt-PA (G11044) intravenously over 5 to 8 hours, blood samples were collected into liquid citrate supplemented with the plasmin inhibitor aprotinin (200 KlU/ml plasma) or on a lyophilized mixture of acidified citrate and the synthetic t-PA inhibitor D-Phe-Pro-Arg-CH2Cl (PPACK). A good correlation between precipitable (sulphite) and functional (clotting rate) fibrinogen levels was observed in plasma collected on citrate before therapy (r = 0.76) and in samples collected after 3 hours on either aprotinin (r = 0.87) or PPACK (r = 0.82). Precipitable fibrinogen levels were approximately 10% higher than functional level, in baseline samples collected on citrate alone and approximately 20% higher in 3 hour samples collected on either PPACK or aprotinin. Fibrinogen levels measured with both assays correlated well, but were somewhat higher in samples collected on PPACK than on aprotinin. rt-PA antigen levels assayed in plasma collected in either inhibitor correlated well (r = 0.90) but were 10-20% higher in PPACK containing samples. Addition of heparin up to 9 units/ml to plasma had no effect on the functional fibrinogen assay.Even with these precautions for assay artifact, a very poor correlation (r =-0.15) was observed between the plasma rt-PA level and the residual functional fibrinogen level, both after 3 hours and towards the end of the rt-PA infusion. A decrease of the fibrinogen level at the end of the infusion to below 1 g/l was observed in 36% of the patients and to below 0.5 g/l in 11%. Optimal monitoring of hemostasis during rt-PA infusion is achieved by fibrinogen assays with a clotting rate method on samples collected on either PPACK or aprotinin. Heparin at therapeutic levels does not interfere with this assay.

scholarly journals Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1213-1217 ◽  
Author(s):  
W Witt ◽  
B Baldus ◽  
P Bringmann ◽  
L Cashion ◽  
P Donner ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Desmodus Rotundus ◽  
Type Plasminogen Activator ◽  
Blood Clots ◽  
Hemostatic Factors ◽  
The Difference ◽  
Vampire Bat

Abstract rDSPA alpha 1 (recombinant Desmodus salivary plasminogen activator alpha 1) is a recombinant protein corresponding to a natural plasminogen activator from the vampire bat Desmodus rotundus. The thrombolytic properties of rDSPA alpha 1 and tissue-type plasminogen activator (t-PA) were compared in a rat model of pulmonary embolism. Whole blood clots, produced in vitro and labeled with 125I-fibrinogen, were embolized into the lungs of anesthetized rats. Thrombolysis was calculated from the difference between initial clot radioactivity and that remaining in the lungs at 60 minutes. Blood was sampled for gamma counting, measurement of hemostatic factors, and plasminogen activator antigen levels. Thrombolysis at 3, 10, 30, and 100 nmol/kg intravenously (10% bolus, 90% over 60 minutes) amounted to 30% +/- 2%, 51% +/- 4%, 85% +/- 4%, 98% +/- 0% for rDSPA alpha 1 and 30% +/- 3%, 41% +/- 3%, 57% +/- 6%, 93% +/- 2% for t-PA (controls: 29% +/- 2%; mean +/- SEM, n greater than or equal to 6). t-PA at 100 nmol/kg significantly decreased fibrinogen, plasminogen, and alpha 2- antiplasmin levels by 33% +/- 7%, 38% +/- 8%, and 61% +/- 9%, whereas rDSPA alpha 1 at 100 nmol/kg only lowered alpha 2-antiplasmin significantly (by 29% +/- 6%). Compared with t-PA, rDSPA alpha 1 is the more potent and more clot selective (fibrin specific) thrombolytic agent. These results suggest that rDSPA alpha 1 may be safer and more efficacious than currently used thrombolytics.

ARIC Hemostasis Study -II. Organizational Plan and Feasibility Study

1990 ◽  
Vol 64 (04) ◽  
pp. 521-525 ◽  
Author(s):  
K K Wu ◽  
A C Papp ◽  
W Patsch ◽  
R Rock ◽  
J Eckfeldt ◽  
...  
Keyword(s):  
Pilot Study ◽  
Processing System ◽  
Time Lapse ◽  
Factor Vii ◽  
Blood Collection ◽  
Multicenter Studies ◽  
Platelet Factor ◽  
Significant Difference ◽  
Aric Study

SummaryIn our previous paper, we reported the development of a blood collection and processing system (BCPS) suitable for the ARIC multicenter hemostasis study. As an additional step of preparation for the ARIC study, we incorporated this BCPS into an organizational plan to increase efficiency and minimize errors. We initially designed organizational trays for blood collection tubes and aliquot tubes and developed a coordinated step-by-step plan for the orderly processing of blood samples. Once the plan was considered workable, we carried out a pilot study to test the feasibility of this integrated organizational plan. Included in the pilot study were 95 healthy subjects randomly selected from 4 ARIC field centers, whose age and gender were comparable to those projected for the ARIC population. We determined the time lapse of filling the first tube as an index of blood flow. The overall mean time-lapse was 23 s (S.D. = 5). There was no significant difference among the field centers. We also determined the entire time lapse required for completing the sample processing. The total processing time was always less than 60 min. By performing the processing of samples in pairs, all the samples from two subjects could be completely processed in 70 min. This greatly increased the efficiency of field center operation. We evaluated the potential in vitro hemostasis activation by measuring plasma Β-thromboglobulin and platelet factor 4 levels. The geometric means of both proteins were comparable to our previously reported results. Fibrinogen, factor VII, factor VIII, von Willebrand factor, antithrombin III, protein C and activated partial thromboplastin time were analyzed. All the hemostatic measurements exhibited no shift of the values. We conclude that the organizational plan together with the BCPS facilitate the incorporation of hemostasis measurements into the ARIC study. It should be valuable as a general guide for other multicenter studies measuring hemostatic factors.

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 310-315 ◽  
Author(s):  
P W Koppert ◽  
E Hoegee-de Nobel ◽  
W Nieuwenhuizen
Keyword(s):  
Enzyme Immunoassay ◽  
Degradation Products ◽  
Blood Clot ◽  
Tissue Type ◽  
Fibrin Degradation Products ◽  
Type Plasminogen Activator ◽  
Strong Positive Reaction ◽  
Sandwich Type ◽  
Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

A Specific Immunologic Assay for Functional Plasminogen Activator Inhibitor 1 in Plasma – Standardized Measurements of the Inhibitor and Related Parameters in Patients with Venous Thromboembolic Disease

1992 ◽  
Vol 68 (05) ◽  
pp. 486-494 ◽  
Author(s):  
Malou Philips ◽  
Anne-Grethe Juul ◽  
Johan Selmer ◽  
Bent Lind ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Normal Subjects ◽  
Tissue Type ◽  
Blood Collection ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Significant Difference ◽  
Activator Inhibitor ◽  
Pai 1

SummaryA new assay for functional plasminogen activator inhibitor 1 (PAI-1) in plasma was developed. The assay is based on the quantitative conversion of PAI-1 to urokinase-type plasminogen activator (u-PA)-PAI-l complex the concentration of which is then determined by an ELISA employing monoclonal anti-PAI-1 as catching antibody and monoclonal anti-u-PA as detecting antibody. The assay exhibits high sensitivity, specificity, accuracy, and precision. The level of functional PAI-1, tissue-type plasminogen activator (t-PA) activity and t-PA-PAI-1 complex was measured in normal subjects and in patients with venous thromboembolism in a silent phase. Blood collection procedures and calibration of the respective assays were rigorously standardized. It was found that the patients had a decreased fibrinolytic capacity. This could be ascribed to high plasma levels of PAI-1. The release of t-PA during venous occlusion of an arm for 10 min expressed as the increase in t-PA + t-PA-PAI-1 complex exhibited great variation and no significant difference could be demonstrated between the patients with a thrombotic tendency and the normal subjects.

Pharmacokinetics and Haemostatic Status During Consecutive Infusions of Recom binant Tissue-Type Plasminogen Activator in Patients with Acute Myocardial Infarction

1989 ◽  
Vol 61 (03) ◽  
pp. 497-501 ◽  
Author(s):  
E Seifried ◽  
P Tanswell ◽  
D Ellbrück ◽  
W Haerer ◽  
A Schmidt
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Plasminogen Activator ◽  
Peak Plasma ◽  
Peak Plasma Concentration ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Precise Control ◽  
Type Plasminogen Activator

SummaryPharmacokinetics and systemic effects of recombinant tissue type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 12 acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in 2 minutes resulting in a peak plasma concentration (mean ±SD) of 3310±950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of. 2210±470 nglml and 930±200 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380±74 ml/min, t,½α was 3.6±0.9 min and t,½β was 16±5.4 min.After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 52%, α2-antiplasmin to 25%, α2-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin D-dimer concentrations increased from 0.40 to 2.7 μg/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

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