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.

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.

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.

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.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

FACTOR IX CONCENTRATES ARE THROMBOGENIC AT HIGH DOSES IN DOGS

1987 ◽  
Author(s):  
J Ferguson ◽  
J Dawes ◽  
C V Prowse ◽  
P R Foster ◽  
P A Feldman ◽  
...  
Keyword(s):  
Platelet Count ◽  
Fibrinogen Level ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Fibrinopeptide A ◽  
Blood Products ◽  
Elevated Plasma ◽  
High Doses

A canine model has recently been established to assess the potential thrombogenicity of intravenously infused blood products. Elevated plasma levels of fibrinopeptide A (FpA) were identified as the most sensitive indicator of a thrombogenic response, and this was the only parameter to change significantly when issued batches of factor IX (II + X) concentrate were infused at a dose of 100 iu/kg. After infusion of 200 iu/kg, however, plasma FpA concentrations and FDP titres rose, the APTT was prolonged, and the platelet count and fibrinogen level fell. At this dose, therefore, FIX concentrates which were not identified by in vitro tests as potentially thrombogenic induced a response when infused into dogs.A batch of FIX concentrate which failed the criteria for in vitro thrombogenicity and was therefore not issued for routine use was infused at 100 iu/kg. Plasma FpA levels rose as did the FDP titre, and fibrinogen concentrations fell, but the APTT was only slightly prolonged. The thrombogenic response to 200 iu/kg of issued FIX concentrate was at least as severe as that following infusion of 100 iu/kg of this rejected batch.Thus, there is a threshold dose of FIX concentrate above which a severe thrombogenic response can ensue, and the current in vitro tests may not be a reliable indicator of potential thrombogenicity when FIX concentrates are infused at high doses. This should be taken into account when administering unusually high doses, particularly to patients who may have reduced levels of circulating protease inhibitors.

Immunochemical Characterisation Of Two Human Factor IX Inhibitors And Treatment With Activated Concentrate

1981 ◽  
Author(s):  
J C Giddings ◽  
A L Bloom
Keyword(s):  
Exchange Chromatography ◽  
Factor Ix ◽  
Therapeutic Trial ◽  
Igg Subclass ◽  
Human Igg ◽  
Heavy Chains ◽  
Human Factor Ix ◽  
Activity Unit ◽  
Factor Ix Deficiency

Plasma was obtained from two unrelated patients (A and B) with severe hereditary factor IX deficiency who had developed specific inhibitors of factor IX. Immunoglobulin (Ig) was fractionated by salt precipitation and ion exchange chromatography and resulted in 90% recovery of the factor IX inhibitor. Inhibitor plasma or Ig fractions were mixed with specific antisera to ig chains, incubated and centrifuged. Supernatant was assayed for residual inhibitor. Antisera, specific for the heavy chains of human IgG, IgA, IgM, IgD and IgE and the subclasses 1 to 4 of human IgG were used together with antisera specific for the kappa and lambda light chains of IgG. Results of inhibitor neutralisation assays were the same whether plasma or Ig fractions were used. Inhibitor from patient A was completely neutralised by anti IgG and by anti-lambda serum, and was largely neutralised by antiserum to IgG subclass 4. Other antisera had no effect. Inhibitor from patient B was completely neutralised by anti IgG and partially neutralised by anti-kappa and anti-lambda serum. Partial neutralisation of inhibitor was also seen with antisera to IgG subclass 1 and IgG subclass 4. Other antisera again had no effect. Inhibitor A inactivated factor IX instantaneously to a titre of 1 in 100. Neither conventional factor IX concentrate (Oxford DE) at 1 u/ml nor activated‘factor IX’ concentrate (Feiba, Immuno) at 1 u/ml (‘activity’ unit) corrected the kaolin cephalin clotting time (KCCT) of patient A in vitro. However after intravenous administration of Feiba (25 u/kg) progressive shortening of the KCCT occurred from 127 sec. to 60 sec. at 60 min. and was followed by rapid resolution of a haemarthrosis. It is concluded that factor IX antibodies may be of monoclonal or oligoclonal immunoglobulin class and that on development, therapeutic trial of activated factor IX-prothrombin concentrates is indicated for the control of bleeding.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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