Modified Tests for Measuring the Thrombogenic Potential of Factor IX Concentrates.

1979 ◽  
Author(s):  
Sarah M. Middleton ◽  
Jessie T. Douglas ◽  
C.D. Forbes ◽  
C.R.M. Prentice
Keyword(s):  
Factor Xa ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Procoagulant Activity ◽  
In Vitro Tests ◽  
Factor V ◽  
Thrombogenic Potential ◽  
Sepharose 4B ◽  
Generation Test

In vitro tests for screening potential thrombogenicity of factor IX concentrates are unsatisfactory as it has been shown that the non-activated partial thromboplastin time (NAPTT) and the TGt50 (reflecting thrombin generation by the concentrate after recalcification} do not correlate with each other. We have modified the TGt50 by addition of optimum concentrations of factor V and phospholipid, and compared it with the NAPTT and factor Xa generation test using the chromogenic substrate S2222. The modified TGtSO correlates with both the NAPTT and the factor Xa generation test suggesting that these tests are measuring the same entity. Chromatography of concentrates on sepharose 4B indicates that the high MW void volume material has procoagulant activity as measured by the unmodified TGt50 whilst the retained volume he procoagulant activity as measured by the TGt50 and the NAPTT. When, however, phospholipid is added to the unmodified TG150 the activity of the high MW component is lost and only that of the retained volume is still present. The data suggests that the modified TGt50 and the NAPTT measure the same procoagulant activity in these concentrates.

In Vitro Thrombogenicity Tests of Factor IX Concentrates. II: Effects of Phospholipids and Heparin

1979 ◽  
Vol 42 (05) ◽  
pp. 1368-1377 ◽  
Author(s):  
C V Prowse ◽  
M C Boffa ◽  
C Guthrie ◽  
D S Pepper
Keyword(s):  
Total Phospholipid ◽  
Factor Xa ◽  
Relative Effect ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Low Levels ◽  
Recalcification Time ◽  
Generation Test

SummaryMeasurement of the total phospholipid (and that portion active in coagulation) in factor IX concentrates revealed no correlation with in vitro tests of potential thrombogenicity, except in the case of the recalcification time and the thrombin generation test which may detect coagulant phospholipid as well as the presence of thrombogenic enzymes. This is probably due to separation of the prothrombin complex proteins from most phospholipid during ion-exchange chromatography. Although low levels of phospholipid remain in the final product these are apparently insufficient to effect appreciable activation of factor IX concentrates despite low levels of antithrombin III.Two tests which measure the formation of thrombin and factor Xa after recalcification of concentrates were affected by the addition of exogenous phospholipid. However this is a relative effect such that differences are quantitative rather than qualitative.Heparin addition during production of factor IX concentrate was found to have only minor effects on the results of in vitro thrombogenicity tests of the final product. This was confirmed in the laboratory by incubation of unheparinised products with heparin for periods of up to 6 hr.

In Vitro Thrombogenicity Tests of Factor IX Concentrates

1979 ◽  
Vol 42 (05) ◽  
pp. 1355-1367 ◽  
Author(s):  
C V Prowse ◽  
A Chirnside ◽  
R A Elton
Keyword(s):  
Factor Viii ◽  
Partial Thromboplastin Time ◽  
Generation Time ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Factor Viii Inhibitor ◽  
Factor Ixa ◽  
Viii Inhibitor

SummaryVarious factor IX concentrates have been examined in a number of in vitro tests of thrombogenicity. The results suggest that some tests are superfluous as in concentrates with activity in any of these tests activation is revealed by a combination of the non-activated partial thromboplastin time, the thrombin (or Xa) generation time and factor VIII inhibitor bypassing activity tests. Assay of individual coagulant enzymes revealed that most concentrates contained more factor IXa than Xa. However only a small number of concentrates, chiefly those that had been purposefully activated, contained appreciable amounts of either enzyme.

Measurement of Activated Factor IX in Factor IX Concentrates: Correlation with In Vivo Thrombogenicity

1995 ◽  
Vol 73 (04) ◽  
pp. 675-679 ◽  
Author(s):  
Elaine Gray ◽  
Jill Tubbs ◽  
S Thomas ◽  
A Oates ◽  
M Boisclair ◽  
...  
Keyword(s):  
Significant Positive Correlation ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Prothrombin Complex Concentrates ◽  
Thrombotic Risk ◽  
Chromogenic Assay ◽  
The Mean ◽  
Thrombogenic Potential

SummaryCurrent in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15–1.2 U/1000 iu FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25–4. There was a significant positive correlation (r = 0.55, p <0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

Studies Of Factor VIII Inhibitor Bypassing Activity (FEIBA)

1981 ◽  
Author(s):  
T W Barrowcliffe ◽  
E Gray ◽  
G Kemball-Cook
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Ix ◽  
Procoagulant Activity ◽  
Two Stage ◽  
Human Antibodies ◽  
One Stage ◽  
Viii And Ix ◽  
The One

The activities of an activated Factor IX concentrate (FEIBA, Immuno AG) were studied by two in vitro assays: a one-stage method using VUI-deficient plasma as substrate, and a two-stage assay based on the thrombin generation test. The nature of the active principle was explored by measuring the reduction in activity when FEIBA was incubated with specific antibodies.Incubation of FEIBA with human antibodies to Factor VIII reduced its activity by about 30% in the one-stage assay, and about 50% in the two-stage assay, suggesting that FEIBA contains Factor VIII procoagulant activity. Inactivation of the Factor VIII in FEIBA was somewhat slower than that of normal Factor VIII, indicating partial protection from inhibition. Human antibodies to Factor IX inhibited the one stage activity by about 30%, and incubation with both antibodies also produced a 30% reduction in activity. The remaining procoagulant activity decayed only slowly when incubated with non-inhibitor plasma. In contrast, purified human Factor Xa lost activity rapidly on incubation in normal plasma, as did a purified fraction from a Factor IX concentrate, which had high activity in the one-stage assay.These results suggest that the in vitro activity of FEIBA is due to at least two components. One component appears to be dependent on both Factors VIII and IX and may be a complex of VIII and IXa. The other component acts later than Factors VIII and IX in the coagulation cascade but, unlike purified Factor Xa, is relatively resistant to inactivation by plasma inhibitors such as antithrombin III. FEIBA was also found to contain phospholipid, and it may be that the phospholipid protects both the Factor VIII and activated enzymes from their inhibitors.

Removal of Potentially Thrombogenic Material from a Concentrate of Factors II, IX & X by Polyethylene Glycol Precipitation

1979 ◽  
Author(s):  
P. R. Foster ◽  
M. R. Patterson ◽  
A. J. Johnson ◽  
S. M. Middleton
Keyword(s):  
Polyethylene Glycol ◽  
Hepatitis B ◽  
Partial Thromboplastin Time ◽  
Hepatitis B Surface Antigen ◽  
Factor Xa ◽  
Activated Partial Thromboplastin Time ◽  
In Vitro Tests ◽  
Factor Ii ◽  
Polyethylene Glycol Precipitation

Polyethylene glycol(PEG) precipitation has beer used for the removal of hepatitis B surface antigen (HBsAg) from a concentrate of factors II, IX & X of intermediate purity. HBsAg is precipitated into a fraction PI at 20% (w/v) PEG and pH 6.5 while factors II, IX & X remaining in the supernatant are subsequently precipitated at 30% (w/v) PEG and pH. 5.2These various fractions have been assessed for the presence of potentially thrombogenic materials by in vitro tests. In comparison with the starting material the precipitate rich In factors II, IX & X has a significantly increased non-activated partial thromboplastin time (NAPTT) and a decreased rate of factor Xa generation. By contrast the waste fraction (PI) exhibits a significantly decreased NAPTT and an increased rate of factor Xa generation suggesting that potentially thrombogenic material has been selectively precipitated. This procedure has been used to prepare a factor II, IX & X concentrate containing 100 U FIX/ml with reduced contamination by both HBsAg and thrombogenic materials.

Coagulation activation and microparticle-associated coagulant activity in cancer patients

2012 ◽  
Vol 108 (07) ◽  
pp. 160-165 ◽  
Author(s):  
Frederiek van Doormaal ◽  
René Berckmans ◽  
Nigel Mackman ◽  
David Manly ◽  
Pieter Kamphuisen ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Coagulation Activation ◽  
Activation Markers ◽  
Coagulant Activity ◽  
Generation Test

SummaryCancer increases the risk of venous thromboembolism (VTE). Here, we investigated the contribution of microparticle (MP)-dependent procoagulant activity to the prothrombotic state in these patients. In 43 cancer patients without VTE at study entry and 22 healthy volunteers, markers of in vivo and MP-dependent coagulation were measured and patients were prospectively followed for six months for the development of VTE. Procoagulant activity of MPs was measured in vitro using a tissue factor (TF)-independent phospholipid dependent test, a factor Xa-generation assay with and without anti-TF, and a fibrin generation test (FGT) with and without anti-factor VII(a). Markers of in vivo coagulation activation and total number of MPs at baseline were significantly elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/l, D-dimer 0.76 vs. 0.22 mg/l and 5.53 x 106 vs. 3.37 x 106 MPs/ml). Five patients (11.6%) developed VTE. Patients with VTE had comparable levels of coagulation activation markers and phospholipid-dependent MP pro-coagulant activity. However, median TF-mediated Xa-generation (0.82 vs. 0.21 pg/ml, p=0.016) and median VIIa-dependent FGT (13% vs. 0%, p=0.036) were higher in the VTE group compared with the non-VTE group. In this exploratory study the overall hypercoagulable state in cancer patients was not associated directly with the MP phospholipid-dependent procoagulant activity. However, in the patients who developed VTE within six months when compared to those who did not, an increased MP procoagulant activity was present already at baseline, suggesting this activity can be used to predict VTE.

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.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

1966 ◽  
Vol 15 (03/04) ◽  
pp. 349-364 ◽  
Author(s):  
A.H Özge ◽  
H.C Rowsell ◽  
H.G Downie ◽  
J.F Mustard
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Factor Ix ◽  
Clotting Factor ◽  
Blood Ph ◽  
Order Of Magnitude ◽  
Dose Dependent ◽  
Generation Test ◽  
Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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