Factor VLEIDEN and cardiopulmonary bypass: investigation of haemostatic parameters and the effect of aprotinin using an ex vivo model

Abstract

It has been suggested that aprotinin results in significantly increased risk for perioperative thrombotic complications in patients with Factor V^LEIDEN (F5L) due to its ability to competitively inhibit activated protein C (APC) function in vitro. No clinical studies have been performed to assess the effect of aprotinin on APC function of F5L in vivo. We developed an ex vivo model to mimic the effects of cardiopulmonary bypass with the exclusion of the patient in order to assess APC function. Blood from normal (n = 2) and F5L heterozygous donors (n = 2) was treated with aprotinin or placebo (saline). The blood was heparinized, added to the prime and circulated at 2 l/min through a modified cardiopulmonary bypass circuit. After 60 min of circulation, the heparin was neutralized with protamine sulfate. Blood samples, drawn at specific time points, were analysed for APC ratio. Results showed a decrease in APC ratio for both F5L and normal bloods with the addition of aprotinin (18% and 40%, respectively). APC ratios also decreased with the commencement of extracorporeal circulation for all bloods, resulting in an APC ratio of 1.35 in normal placebo-treated blood and 0.67 in F5L placebo-treated blood. The combined effect of aprotinin and extracorporeal circulation resulted in APC ratios of 0.90 for normal blood and 0.63 for F5L blood, corresponding to a severe dysfunction of APC intraoperatively (reference range 1.9-4.0). The data from this model predict an increased risk of perioperative thrombosis due to inhibition of APC function in cardiac surgical patients heterozygous for the F5L mutation. Aprotinin further compounds the severity of APC dysfunction, though the effect is more severe in normal blood. The ex vivo model employed was an effective tool for the investigation of the haemostatic effect of aprotinin. This model may be exploited for other applications such as the investigation of novel or emerging haemostatic agents prior to clinical trial.

Get full access to this article

View all access options for this article.

References

Bertina RM , Koeleman BP , Koster T et al. Mutation in blood coagulation factor V associated with resistance to activated protein C . Nature 1994; 369: 64-67 .

Heeb MJ , Kojima Y , Greengard JS , Griffin JH. Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V . Blood 1995; 85: 3405-3411 .

Dahlback B , Stenflo J. A natural anticoagulant pathway: proteins C, S, C4b-binding protein and thrombomodulin. In: Bloom AL , Forbes CD , Thomas DP , Tuddenham EGD , eds. Haemostasis and thrombosis. Edinburgh: Churchill Livingstone , 1994: 671-698.

Zoller B , Dahlback B. Resistance to activated protein C caused by a factor V gene mutation . Curr Opin Hematol 1995; 2: 358-364 .

Koster T , Rosendaal FR , de Ronde H , Briet E , Vandenbroucke JP , Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia Study . Lancet 1993; 342: 1503-1506 .

van Bockxmeer FM , Baker RI , Taylor RR. Premature ischaemic heart disease and the gene for coagulation factor V [letter] . Nat Med 1995; 1: 185-185 .

Rosendaal FR , Siscovick DS , Schwartz SM et al. Factor V Leiden (resistance to activated protein C) increases the risk of myocardial infarction in young women . Blood 1997; 89: 2817-2821 .

Stassen JM , Lambeir AM , Matthyssens G. Characterisation of a novel series of aprotinin-derived anticoagulants. In vitro and pharmacological properties . Thromb Haemost 1995; 74: 646-654 .

Espana F , Estelles A , Griffin JH , Aznar J , Gilabert J. Aprotinin (trasylol) is a competitive inhibitor of activated protein C . Thromb Res 1989; 56: 751-756 .

10.

Taby O , Chabbat J , Steinbuch M. Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification . Thromb Res 1990; 59: 27-35 .

11.

Schmaier AH. Aprotinin: can its benefits be offset by harmful effects? [editorial] . Transfusion 1997; 37: 1105-1107 .

12.

Cosgrove DM , Heric B , Lytle DW. Aprotinin therapy for reoperative myocardial revascularization: a placebo controlled study . Ann Thorac Surg 1992; 54: 1031-1038 .

13.

Westaby S. Aprotinin in perspective . Ann Thorac Surg 1993; 55: 1033-1041 .

14.

Umbrain V , Christiaens F , Camu F.

Intraoperative coronary thrombosis: can aprotinin and protamine be incriminated?

J Cardiothorac Vasc Anesth 1994; 8: 198-201 .

15.

van der Meer J , Millege HL , Ascoop CA. Aprotinin in aortocoronary bypass surgery: increased risk of vein-graft occlusion and myocardial infarction? Supportive evidence from a retrospective study . Thromb Haemost 1996; 75: 1-3 .

16.

Jegaden O , Vedrine C , Rossi R. Aprotinin does not compromise arterial graft patency in coronary bypass operations . J Thorac Cardiovasc Surg 1993; 106: 180-181 .

17.

Bidstrup BP , Royston D , Sapsford RN. Reduction in blood loss and blood use after cardiopulmonary bypass with high dose aprotinin (Trasylol) . J Thorac Cardiovasc Surg 1989; 97: 364-372 .

18.

Lemmer JH , Stanford W , Bonney SL. Aprotinin for coronary bypass operations: efficacy, safety, and influence on early saphenous vein graft patency: a multicentre, randomized, double-blinded, placebo controlled study . J Thorac Cardiovasc Surg 1994; 107: 543-553 .

19.

Royston D.

Intraoperative coronary thrombosis: can aprotinin be incriminated?

J Cardiothorac Vasc Anesth 1994; 8: 137-141 .

20.

Sweeney JD , Blair AJ , Dupuis MP , King TC , Moulton AL. Aprotinin, cardiac surgery, and factor V Leiden . Transfusion 1997; 37: 1173-1178 .

21.

Baksaas ST , Videm V , Pedersen T et al. Comparison of three oxygenator-coated and one total-circuit-coated extracorporeal devices . Perfusion 1999; 14: 119-127 .

22.

Baksaas ST , Videm V , Fosse E et al. In vitro evaluation of new surface coatings for extracorporeal circulation . Perfusion 1999; 14: 11-19 .

23.

Videm V , Nilsson L , Venge P , Svennevig JL. Reduced granulocyte activation with a heparin coated device in an in vitro model of cardiopulmonary bypass . Artif Organs 1991; 15: 90-95 .

24.

Fritz R , Wunderer G. Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs . Arzneimittelforschung 1983; 33: 479-494 .

25.

Cardigan RA , Mackie IJ , Machin SJ. The effect of heparin and its neutralisation on functional assays for factor VIIa, factor VII and TFPI . Thromb Res 1996; 84: 237-242 .

26.

Cumming AM , Jones GR , Wensley RT , Cundall RB. In vitro neutralization of heparin in plasma prior to the activated partial thromboplastin time test: an assessment of four heparin antagonists and two anion exchange resins . Thromb Res 1986; 41: 43-56 .

27.

Ouehenberger P , Handler S , Mannhalter C , Kyrle PA , Speiser W. The Factor V (Leiden) test: Evaluation of an assay based on dilute Russell Viper Venom Time for the detection of the Factor V Leiden mutation . Thromb Res 1999; 96: 125-133 .

28.

Rylatt DB , Hohnen-Behrens C , Pilgrim RL , Dickeson LE , Neal M , Exner T. Detecting APC-resistant factor V: a functional method without plasma dilution . Blood Coagul Fibrinolysis 1999; 10: 359-366 .

29.

Dahlback B , Carlsson M , Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C . Proc Natl Acad Sci USA 1993; 90: 1004-1008 .