Interactions between antiphospholipid antibodies, oral anticoagulants and haemostasis activation ...

190 Pages·2015·1.96 MB·English

Interactions between antiphospholipid antibodies,
oral anticoagulants and haemostasis activation in
thrombotic antiphospholipid syndrome

Deepa Jayakody Arachchillage MBBS, MRCP, FRCPath

Thesis submitted to University College London (UCL) for the degree of
Doctor of Medicine (Research)

University College London 2015

1
Declaration
I, Deepa Jayakody Arachchillage, confirm that the work presented in this thesis is my
own. Where information has been derived from other sources, I confirm that has
been indicated in the thesis.

-----------------------------------

2
Abstract
Antiphospholipid antibodies (aPL) are associated with an increased thromboembolic
risk but the mechanism is unclear. Warfarin anticoagulation has traditionally been
employed in thrombotic antiphospholipid syndrome (APS), but direct oral
anticoagulants such as rivaroxaban have recently become available.

The aims of this thesis were to:
1. Investigate the frequency, mechanism and clinical associations of activated protein
C resistance (APCr) in thrombotic APS
2. Assess the effects of rivaroxaban on lupus anticoagulant tests
3. Investigate the efficacy of rivaroxaban in terms of thrombin generation (TG) and
haemostasis activation markers in thrombotic patients with or without APS

Thrombotic APS patients had greater APCr than disease controls. Nearly 50% of
APS patients had anti-protein C (anti-PC) antibodies; those with high avidity
antibodies had significantly greater APCr using either exogenous activated PC or
activation of endogenous PC with Protac®. High avidity anti-PC antibodies were
strongly associated with a severe thrombotic phenotype in APS. There was a wide
variation in the sensitivity of thromboplastin reagents to rivaroxaban. Of the six
commonly used thromboplastin reagents studied, Neoplastin®R was the most
sensitive while Innovin® and Thromborel®S were the least sensitive. False positive
dilute Russell’s viper venom time occurred in patients with therapeutic levels of
rivaroxaban. Taipan /Ecarin venom clotting time ratio and Textarin time were not
affected irrespective of the rivaroxaban level, enabling accurate detection of LA. In
vitro studies showed that aPL did not influence the anticoagulant activity of
rivaroxaban as measured by TG and anti-Xa assays. Both rivaroxaban and warfarin
achieved effective anticoagulation, as assessed by inhibition of TG and in-vivo
coagulation activation markers in patients with and without APS.

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Table of Contents
Declaration ................................................................................................................ 2
Abstract ................................................................................................................ 3
List of Tables.............................................................................................................. 10
List of Figures ............................................................................................................ 12
List of abbreviations ................................................................................................... 15
Acknowledgements .................................................................................................... 19
Chapter 1 Introduction .......................................................................................... 20
1.1 Normal haemostasis .................................................................................... 20
1.1.1 Initiation ............................................................................................... 20
1.1.2 Amplification ....................................................................................... 20
1.1.3 Propagation .......................................................................................... 23
1.2 Role of phospholipids in haemostasis ......................................................... 24
1.3 Antiphospholipid syndrome ........................................................................ 25
1.3.1 Pathogenesis ......................................................................................... 26
1.3.2 Diagnosis of antiphospholipid syndrome and the identification of
relationships between assay results and clinical manifestations ........................ 30
1.3.3 Clinical manifestations of APS ............................................................ 32
1.3.4 Management of thrombosis in antiphospholipid syndrome ................. 35
1.3.5 Warfarin ............................................................................................... 37
1.3.6 Low molecular weight heparin............................................................. 38
1.3.7 Non vitamin K antagonist oral anticoagulants ..................................... 39
1.3.8 Management of CAPS ......................................................................... 43
1.3.9 Management of asymptomatic carriers of aPL .................................... 44
4
1.3.10 Alternatives to anticoagulants in the management of the
antiphospholipid syndrome ................................................................................ 45
1.3.11 Treatment of obstetric APS .................................................................. 48
1.4 Aims of my thesis ........................................................................................ 49
1.4.1 Frequency and nature of activated protein C resistance (APCr) and
resistance to activation of endogenous protein C activation .............................. 49
1.4.2 Interactions between rivaroxaban, aPL and haemostasis activation .... 50
Chapter 2 Methods ................................................................................................ 52
2.1 Ethical Committee Approval ....................................................................... 52
2.2 Blood sample collection .............................................................................. 52
2.3 Prothrombin time/ International Normalised Ratio (INR) .......................... 53
2.4 Rivaroxaban anti- Xa assay (Chromogenic assay for rivaroxaban level) ... 53
2.5 Dilute Prothrombin Time (dPT) in KC4A .................................................. 54
2.6 Dilute Russell Viper Venom Time (DRVVT) (in house method) .............. 55
2.7 Dilute Russell Viper Venom Time (DRVVT) (IL ACL TOP 500 Method 57
2.8 Dilute Russell Viper Venom Time (DRVVT) (CS-5100 / CS-2000i
analyser: Sysmex UK Ltd, Milton Keynes, UK) ................................................... 57
2.9 Taipan venom test /Ecarin clotting time (KC4A method)........................... 58
2.10 Textarin® /Ecarin ratio to detect lupus anticoagulants ........................... 59
2.11 TVT and ECT in Sysmex CS-5100i /analyser (Sysmex UK Ltd) ........... 60
2.11.1 TVT ...................................................................................................... 61
2.11.2 ECT ...................................................................................................... 61
2.12 D dimer .................................................................................................... 61
2.13 Prothrombin fragments 1.2 (F1.2) ........................................................... 62
2.14 Thrombin-Antithrombin complexes (TAT) ............................................. 62
2.15 Thrombin generation test ......................................................................... 62
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2.16 Activated protein C resistance determined by thrombin generation........ 65
2.16.1 The assessment of resistance to activation of endogenous protein C
using Protac® ..................................................................................................... 68
2.17 Chromogenic Protein C assay .................................................................. 71
2.18 Free protein S antigen assay .................................................................... 72
2.19 ELISA for Protein C Antibodies .............................................................. 73
2.20 Detection of protein S antibodies (anti-protein S IgG ELISA) ............... 75
2.21 Determination of the avidity of IgG protein C and protein S antibodies . 76
2.22 Purification, Dialysis, and concentration of IgG ..................................... 77
2.23 Quantification of IgG (IgG ELISA)......................................................... 78
2.24 Rivaroxaban preparation for in vitro experiments ................................... 81
2.25 Effect of antiphospholipid antibodies on rivaroxaban in thrombin
generation and anti-Xa assay ................................................................................. 82
2.26 Statistical analysis .................................................................................... 83
Chapter 3 Activated protein C resistance (APCr) and resistance to activation of
endogenous protein C activation determined using thrombin generation patients in
with thrombotic antiphospholipid syndrome. ............................................................ 84
3.1 Introduction ................................................................................................. 84
3.2 Intra and inter-assay imprecision of thrombin generation with buffer
control, rhAPC and Protac ..................................................................................... 84
3.2.1 Results .................................................................................................. 85
3.3 Patients and normal controls ....................................................................... 86
3.3.1 Inclusion criteria................................................................................... 86
3.3.2 Exclusion criteria ................................................................................. 86
3.4 Antiphospholipid antibodies and INR ......................................................... 87
3.5 Resistance to rhAPC and activation of endogenous protein C by thrombin
generation ............................................................................................................... 88
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3.5.1 Results .................................................................................................. 88
3.6 Anti-protein C antibodies ............................................................................ 90
3.7 Anti-protein S antibodies ............................................................................. 91
3.8 Determination of the avidity of anti-protein C and anti-protein S antibodies .
..................................................................................................................... 92
3.9 Anti-protein C antibodies and activated Protein C resistance ..................... 94
3.10 Anti-protein S antibodies and activated Protein C resistance .................. 94
3.11 Evaluation of the presence of anti-protein C antibodies according to APS
classification categories ......................................................................................... 94
3.11.1 PC activity and free PS antigen level ................................................... 95
3.12 Discussion ................................................................................................ 96
Chapter 4 Sensitivity of commonly used thromboplastin reagents to rivaroxaban ..
............................................................................................................ 101
4.1 Introduction ............................................................................................... 101
4.2 Patients and blood sampling ...................................................................... 102
4.3 Rivaroxaban levels .................................................................................... 102
4.4 Prothrombin time ....................................................................................... 103
4.5 Prothrombin time ratio .............................................................................. 105
4.6 Discussion ................................................................................................. 108
Chapter 5 Detection of lupus anticoagulant in the presence of rivaroxaban and
effects of antiphospholipid antibodies on rivaroxaban anticoagulant action ........... 112
5.1 Patients, sample collection and preparation .............................................. 113
5.1.1 In vitro studies: ................................................................................... 113
5.1.2 Ex vivo studies .................................................................................... 115
5.1.3 Prothrombin time (PT) ....................................................................... 116
5.1.4 Rivaroxaban anti-Xa levels ................................................................ 117
5.1.5 Lupus anticoagulant ........................................................................... 117
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5.1.6 Studies to define the effects of aPL on rivaroxaban anticoagulant action
............................................................................................................ 118
5.2 Results ....................................................................................................... 119
Lupus anticoagulant studies ............................................................................. 119
5.2.1 In vitro studies: lupus anticoagulant .................................................. 119
5.2.2 Ex vivo studies .................................................................................... 128
5.2.3 Effects of aPL on rivaroxaban anticoagulant action .......................... 131
5.3 Discussion ................................................................................................. 134
Chapter 6 Thrombin generation and in vivo coagulation activation markers in
patients without APS, treated with therapeutic dose rivaroxaban or warfarin for
venous thromboembolism ........................................................................................ 137
6.1 Introduction ............................................................................................... 137
6.2 Patients and Methods ................................................................................. 138
6.2.1 Patients ............................................................................................... 138
6.2.2 Blood sampling .................................................................................. 139
6.3 Coagulation assays .................................................................................... 140
6.3.1 Prothrombin time (PT) and International normalised ratio ................ 140
6.3.2 Rivaroxaban levels ............................................................................. 140
6.3.3 Ex vivo thrombin generation .............................................................. 141
6.4 Results ....................................................................................................... 141
6.5 In-vivo coagulation activation markers: .................................................... 145
6.6 Bleeding and thrombotic episodes............................................................. 147
6.7 Discussion ................................................................................................. 147
6.7.1 Limitations of this study .................................................................... 151
6.8 Conclusion ................................................................................................. 151
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Chapter 7 In vivo markers of coagulation activation in patients with
antiphospholipid syndrome treated with rivaroxaban or warfarin following venous
thromboembolism .................................................................................................... 152
7.1 Patients and blood sample collection ........................................................ 152
7.2 Results ....................................................................................................... 153
7.2.1 Warfarin group ................................................................................... 153
7.2.2 Rivaroxaban group ............................................................................. 157
7.2.3 Comparison of warfarin and Rivaroxaban arms on day 42................ 158
7.3 Discussion ................................................................................................. 159
Chapter 8 General discussion and future directions ........................................... 162
Publications arising from this thesis ........................................................................ 168

9
List of Tables
Table 1-1 Associations of antiphospholipid antibodies ............................................. 26
Table 1-2 Proposed mechanisms for development of thrombosis in APS ................. 29
Table 1-3 Proposed mechanisms for aPL-mediated fetal loss or complications ....... 30
Table 1-4 Clinical manifestations in patients with aPL ............................................. 32
Table 1-5 Comparison of the pharmacological characteristics of NOAC ................. 41
Table 1-6 Development status of rivaroxaban, apixaban, edoxaban and dabigatran . 43
Table 2-1 Preparation of increasing concentration of rhAPC for thrombin generation
.................................................................................................................................... 66
Table 2-2 Preparation of increasing concentration of Protac® for thrombin
generation ................................................................................................................... 69
Table 2-3 Preparation rivaroxaban concentrations .................................................... 82
Table 3-1 clinical features of the three groups of subjects studied ............................ 87
Table 3-2 PC activity and free PS antigen level in patients with APS and non-APS 96
Table 4-1 Geometric mean normal PT for different thromboplastin reagents ......... 104
Table 5-1 Clinical features and APS classification categories of the 30 APS patients
(I, IIa, IIb, ................................................................................................................. 114
Table 5-2 dPT ratios with APS LA+ve IgG and negative IgG with rivaroxaban
50ng/mL or 250ng/mL. (Normal Screen/confirm ratio >1.2) ................................. 123
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