[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS).
Serum from patients with APS (n = 59), systemic lupus erythematosus (SLE; n = 106), other autoimmune rheumatic disease (ARD; n = 63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (± AT-III).
Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P <0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P <0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P <0.05) and did not correlate with binding to AT-III.
APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy.
[Show abstract][Hide abstract] ABSTRACT: Introduction The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS). Methods Serum from patients with APS (n = 59), systemic lupus erythematosus (SLE; n = 106), other autoimmune rheumatic disease (ARD; n = 63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (± AT-III). Results Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P <0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P <0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P <0.05) and did not correlate with binding to AT-III. Conclusion APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy
Arthritis Research & Therapy 03/2015; · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IntroductionRivaroxaban can affect lupus anticoagulant (LA) testing and antiphospholipid antibodies (aPL) may interfere with rivaroxaban anticoagulant action.AimsTo establish the influence of rivaroxaban on LA detection and of aPL on rivaroxaban anticoagulant action.Methods
Rivaroxaban and 52 IgG preparations (20 LA+ve, 12 LA-ve thrombotic antiphospholipid syndrome [APS] patients, and 20 normal controls [NC]), were spiked into pooled normal plasma (PNP) for relevant studies. LA detection was also studied in APS patients receiving rivaroxaban 20mg once daily.ResultsIn vitro spiking of samples with rivaroxaban showed no false positive LA with Textarin time, Taipan venom time/Ecarin clotting time (TVT/ECT), dilute prothrombin time (dPT) and in-house dilute Russell viper venom time (DRVVT), but false positives in the majority of NC and LA negative IgG with two commercial DRVVT reagents at 250ng/mL but not 50ng/mL rivaroxaban. Ex vivo studies: six LA+ve patients on rivaroxaban remained LA positive with TVT/ECT and DRVVT at peak (162-278ng/mL) and trough (30-85ng/mL) rivaroxaban levels. Six LA-ve patients became (apparently) LA+ve with two DRVVT reagents, test/confirm ratio median [CI]: 1.6 [1.3-1.8], 1.6 [1.4-1.9], but not by TVT/ECT at peak rivaroxaban levels; and remained LA-ve with both DRVVT reagents and TVT/ECT at trough levels. aPL positive IgG spiking of PNP had no effect on rivaroxaban anticoagulant action on thrombin generation or rivaroxaban anti-Xa levels.Conclusions
The TVT/ECT ratio and Textarin time were not affected even at peak rivaroxaban levels, enabling detection of LA ex vivo. aPL had no effects on rivaroxaban anticoagulant action in vitro.This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 03/2015; 13(7). DOI:10.1111/jth.12917 · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Rivaroxaban is non-inferior to warfarin for the treatment of venous thromboembolism, with regard to clinical efficacy and safety. The ex-vivo effects of warfarin versus therapeutic dose rivaroxaban on in-vivo markers of coagulation activation and thrombin generation remain undefined. The aim of this study was to compare the effects of warfarin and therapeutic dose rivaroxaban on ex-vivo thrombin generation (TG), and the in-vivo markers of coagulation activation, prothrombin fragment 1.2 (F1.2), thrombin-antithrombin complex (TAT), and D-dimer.
Eighty-five patients with venous thromboembolism were studied, 45 on warfarin, target INR 2.5 and 40 on rivaroxaban 20mg once daily.
Anticoagulation was in therapeutic range in 71% (32/45) warfarin and 65% (26/40) rivaroxaban treated patients. 8 patients on warfarin and 9 patients on rivaroxaban had subtherapeutic INR and rivaroxaban levels respectively. Both rivaroxaban and warfarin reduced endogenous thrombin potential (ETP) and peak thrombin, and prolonged lag time and time to peak, compared to normal controls (p<0.0001). The lag time and time to peak TG were longer, and peak thrombin was lower in patients receiving rivaroxaban (p<0.0001) compared with warfarin, although warfarin-treated patients had lower ETP (p=0.0008). In-vivo coagulation activation markers were within the normal ranges in all rivaroxaban-treated patients (including those with levels considered to be subtherapeutic) and in 37/45 warfarin-treated patients who had an INR≥2.0. The warfarin-treated patients with subtherapeutic INRs exhibited slightly raised F1.2 and/or TAT.
In conclusion, both rivaroxaban and warfarin provided effective anticoagulation, as assessed by inhibition of TG and makers of in-vivo coagulation activation.
Thrombosis Research 12/2014; 135(2). DOI:10.1016/j.thromres.2014.11.037 · 2.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied.
In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)).
Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P <0.01) but not in the late phase after TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels.
ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD.
Journal of the Neurological Sciences 10/2014; 348(1-2). DOI:10.1016/j.jns.2014.10.035 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accurate diagnosis of obstetric antiphospholipid syndrome (APS) is a prerequisite for optimal clinical management. The international consensus (revised Sapporo) criteria for obstetric APS do not include low positive anticardiolipin (aCL) and anti β2 glycoprotein I (aβ2GPI) antibodies (< 99th centile) and/or certain clinical criteria such as two unexplained miscarriages, three non-consecutive miscarriages, late pre-eclampsia, placental abruption, late premature birth, or two or more unexplained in vitro fertilisation failures. In this review we examine the available evidence to address the question of whether patients who exhibit non-criteria clinical and/or laboratory manifestations should be included within the spectrum of obstetric APS. Prospective and retrospective cohort studies of women with pregnancy morbidity, particularly recurrent pregnancy loss, suggest that elimination of aCL and/or IgM aβ2GPI, or low positive positive aCL or aβ2GPI from APS laboratory diagnostic criteria may result in missing the diagnosis in a sizeable number of women who could be regarded to have obstetric APS. Such prospective and retrospective studies also suggest that women with non-criteria obstetric APS may benefit from standard treatment for obstetric APS with low-molecular-weight heparin plus low-dose aspirin, with good pregnancy outcomes. Thus, non-criteria manifestations of obstetric APS may be clinically relevant, and merit investigation of therapeutic approaches. Women with obstetric APS appear to be at a higher risk than other women of pre-eclampsia, placenta-mediated complications and neonatal mortality, and also at increased long-term risk of thrombotic events. The applicability of these observations to outcomes in women with non-criteria obstetric APS remains to be determined.
Thrombosis and Haemostasis 10/2014; 113(1). DOI:10.1160/TH14-05-0416 · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have reported A disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) deficiency in noncirrhotic intrahepatic portal hypertension (NCIPH) patients of European origin with preserved liver function. We aimed to study ADAMTS13-von Willebrand factor (vWF) imbalance in Indian patients with NCIPH.
Twenty-nine cases with NCIPH [22 males; 29 years (13-58); Child's A, 23; B, 6], 22 disease controls with cryptogenic chronic liver disease [15 males; 46 years (18-74); Child's A, 9; B, 9; C, 4] and 17 healthy controls [14 males; 32 years (27-45)] were enrolled in the study. We measured ADAMTS13 antigen and activity (by collagen binding assay (CBA) and by fluorescence resonance energy transfer [FRET] assay), and vWF antigen levels in plasma of study patients.
ADAMTS13 activity by CBA in NCIPH patients (32 %, 5 % to 100 %; median, range; p-value <0.001) and disease controls (36 %, 5 % to 144 %; p = 0.001) was significantly lower than in healthy controls (87 %; 60 % to 148 %). ADAMTS13 antigen and activity by FRET assay were also lower in cases and disease controls. ADAMTS13 activity (by CBA) to antigen ratio was lower in NCIPH and disease controls than in healthy controls. Of 29 NCIPH patients, 3 (all in Child's A) had severe ADAMTS13 deficiency (<10 % ADAMTS13 activity), and 8 (Child's A, 7; B, 1) had moderate ADAMTS13 deficiency (10 % to 25 % activity). Conversely, vWF antigen and vWF:ADAMTS13 ratio were higher in patients with NCIPH and in disease controls than in healthy controls.
This study validates the finding of ADAMTS13 deficiency in NCIPH despite preserved liver functions in an Indian population suggesting its involvement in pathogenesis of NCIPH.
Indian Journal of Gastroenterology 04/2014; 33(4). DOI:10.1007/s12664-014-0460-4
[Show abstract][Hide abstract] ABSTRACT: Pregnancy is associated with significant haemostatic changes, with a progressive rise in most clotting factors. There is limited data on the changes of factor XIII (FXIII) level during pregnancy. This study assesses changes in FXIII activity during normal pregnancy and establish FXIII reference range during each trimester of pregnancy and immediate postnatal period. This is a cross sectional study of 376 women with normal uneventful pregnancies. Plasma FXIII activity was measured during first (weeks 0-12, n = 116), second (weeks13-28, n = 132), third trimester (weeks 29-42, n = 128) and postnatal (day 0-3; n = 30). Samples were also collected from non-pregnant women (n = 25) as a control group. FXIII was assayed on CS-5100 analyser using chromogenic reagent. The mean ± SD FXIII activity was 112 ± 29 IU dL(-1) during first trimester, 96 ± 26 IU dL(-1) during second trimester, 83 ± 21 IU dL(-1) during third trimester, 90 ± 19 IU dL(-1) during postnatal period, and 113 ± 26 IU dL(-1) in the control. The reference range was calculated during the first (55-169 IU dL(-1) ), second (45-147 IU dL(-1) ), third trimester (42-125 IU dL(-1) ) and postnatal period (61-137 IU dL(-1) ). There was a significant reduction in the mean FXIII activity during the second and third trimester compared to the first trimester and control group (P < 0.0001). During the immediate postnatal period, the mean FXIII activity was not statistically different compared to the third and second trimester levels but was significantly lower compared to the first trimester (P < 0.0001) level and the control group (P = 0.0002). This study establishes the reference range for FXIII activity during the three trimesters of normal pregnancy and immediate postnatal period. Women have a significantly decreased level of FXIII activity during a normal uneventful pregnancy.
[Show abstract][Hide abstract] ABSTRACT: The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser.
We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5-10 μm), epinephrine (0.5-10 μm), collagen (0.5-10 mg/μL), ristocetin (0.75-1.25 mg/mL) and arachidonic acid (0.12-1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument.
CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150-480 x 10(9) /L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2-5 μm ADP MA and slope varying between 3-12%.
Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200-300 x 10(9) /L; aggregation with a PRP count <100 x 10(9) /L showed poor sensitivity. Imprecision and detection of antiplatelet drug effects was similar between the CS-2000i and AggRAM. These data demonstrate that CS-2000i is comparable to a stand-alone aggregometer, although CS-2000i has the advantages of walk-away technology and also required a smaller sample volume than the AggRAM (44% less).
International journal of laboratory hematology 11/2013; 36(4). DOI:10.1111/ijlh.12161 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IntroductionThe Clinical and Laboratory Standards Institute (CLSI) has produced a guideline detailing how to determine the activated partial thromboplastin time's (APTT) sensitivity to clotting factor deficiencies, by mixing normal and deficient plasmas. Using the guideline, we determined the factor sensitivity of two APTT reagents. MethodsAPTTs were performed using Actin FS and Actin FSL on a Sysmex CS-5100 analyser. The quality of factor-deficient and reference plasmas from three commercial sources was assessed by assaying each of the clotting factors within the plasmas and by performing thrombin generation tests (TGT). ResultsTesting samples from 50 normal healthy subjects gave a two-standard deviation range of 21.8-29.2s for Actin FS and 23.5-29.3s for Actin FSL. The upper limits of these ranges were subsequently used to determine APTT factor sensitivity. Assay of factor levels within the deficient plasmas demonstrated that they were specifically deficient in a single factor, with most other factors in the range 50-150iu/dL (Technoclone factor VII-deficient plasma has 26iu/dL factor IX). APTTs performed on mixtures of normal and deficient plasmas gave diverse sensitivity to factor deficiencies dependent on the sources of deficient plasma. TGT studies on the deficient plasmas revealed that the potential to generate thrombin was not solely associated with the levels of their component clotting factors. Conclusion
Determination of APTT factor sensitivity in accordance with the CLSI guideline can give inconsistent and misleading results.
International journal of laboratory hematology 05/2013; 35(6). DOI:10.1111/ijlh.12109 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ADAMTS13 activity assays are sometimes useful in confirming the clinical diagnosis or to distinguish different thrombotic microangiopathies (TMA). We investigated the commonly used clinical assays for ADAMTS13 activity. 159 samples from normal subjects or acquired TMA patients were studied in collagen binding (CBA), Fret and chromogenic peptide substrate assays. Frozen aliquots of pooled normal plasma gave similar values by CBA, Fret-VWF73 peptide, Fret-VWF86 and chromogenic VWF73 ELISA (chr-VWF73). Two lyophilised commercial calibrants gave lower ADAMTS13 activity by CBA than peptide substrate assays. The addition of solid HEPES to normal plasma caused a significant fall in CBA, but not Fret-VWF73 activity and might partly explain the differences, since lyophilised plasmas are often HEPES buffered. Normal plasmas showed good agreement between CBA and Fret assays, although chr-VWF73 gave slightly higher values. In acquired TMA, there was reasonable agreement between assays for samples with <11% ADAMTS13 activity (83% of samples showed agreement between CBA, Fret-VWF73 and chr-VWF73), but samples with moderate deficiency frequently showed lower CBA levels (only 41-52% agreement). However, there were also some discrepancies among the peptide substrate assays, with Fret-VWF86 sometimes giving slightly higher values than the VWF73 substrate assays. An International reference plasma might improve standardization, but is not the only problem. It is unclear which assay has greatest clinical utility, this may depend on the nature of the sample. If the activity does not match the clinical picture, an alternative method should be performed. Where therapeutic monitoring is required, the same activity assay should be used throughout.
Thrombosis and Haemostasis 01/2013; 109(3). DOI:10.1160/TH12-08-0565 · 4.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrand's Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE(®) VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet-based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited 'HIL' (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS-analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra- and inter-assay imprecision (over a 31-day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL(-1) , CV < 3.0%) and pathological (Mean 36.1 IU dL(-1) , CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3-753 IU dL(-1) ) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL(-1) . Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE(®) VWF Ac assay was shown to be reliable and precise.
[Show abstract][Hide abstract] ABSTRACT: We propose that porto-pulmonary hypertension (PPH) may arise as a consequence of deficiency of ADAMTS13 (a plasma metalloprotease that regulates von Willebrand factor size and reduces its platelet adhesive activity) and provide a clinical case history to support our hypothesis A patient with non-cirrhotic intrahepatic portal hypertension (NCIPH), ulcerative colitis and celiac disease developed symptoms of PPH which had advanced beyond levels which would have made her an eligible candidate for liver transplantation (mean pulmonary artery pressure (PAP) 49 mm Hg). She was known to have severe ADAMTS13 deficiency, which we considered to be causative of, or contributory to her NCIPH. We postulated that increasing porto-systemic shunting associated with advancing portal hypertension would make the next encountered vascular bed, the lung, susceptible to the pathogenic process that was previously confined to the portal system, with pulmonary hypertension as its consequence. Her pulmonary artery pressures fell significantly during the next year on weekly replacement of plasma ADAMTS13 by infusions of fresh frozen plasma and conventional drug treatment of her pulmonary hypertension. Her pulmonary artery pressures had fallen to acceptable levels when, in response to platelet infusion, it rose precipitously and dangerously. The sequence strongly supports our hypothesis that PPH is a consequence of ADAMTS13 deficiency and is caused by platelet deposition in afferent pulmonary vessels.
Journal of Hepatology 11/2012; 58(4). DOI:10.1016/j.jhep.2012.11.003 · 11.34 Impact Factor