Anne Riddell

Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B

BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Enhanced thrombin generation in patients with cirrhosis-induced coagulopathy

Summary.  Background: Prothrombin time (PT) and the international normalized ratio (INR) are still routinely measured in patients with liver cirrhosis to ‘assess’ their bleeding risk despite the lack of correlation with the two. Thrombin generation (TG) assays are global assays of coagulation that are showing promise in assessing bleeding and thrombosis risks. Aim: To study the relationship between the INR and TG profiles in cirrhosis‐induced coagulopathy. Methods: Seventy‐three patients with cirrhosis were studied. All TG parameters were compared with those from a normal control group. Contact activation was prevented using corn trypsin inhibitor. TG was also assayed in the presence of Protac®. The endogenous thrombin potential (ETP) ratio was derived by dividing the ETP with Protac® by the ETP without Protac®. Results: The INR (mean 1.7) did not correlate with the ETP and the velocity of TG (P > 0.05). There was no difference between the lag time and ETP of the two groups (P > 0.05). The velocity of TG was increased in cirrhosis (67.95 ± 34.8 vs. 45.05 ± 25.9 nM min−1; P = 0.016) especially in patients with INRs between 1.21 and 2.0. Both the ETP with Protac® and the ETP ratio were increased in cirrhosis (mean 1074 ± 461.4 vs. 818 ± 357.9 nM min, P = 0.004 and 0.80 ± 0.21 vs. 0.44 ± 0.15, P ≤ 0.0001, respectively). Conclusion: Despite a raised INR, TG parameters are consistent with a hypercoagulable profile in cirrhosis‐related coagulopathy. This confirms that the PT or INR should not be used to assess bleeding risk in these patients, and other parameters, such as TG, need to be explored as clinical markers of coagulopathy.

Evaluation of coagulation abnormalities in acute liver failure

BACKGROUND & AIMS In acute liver failure (ALF), prothrombin time (PT) and its derivative prothrombin time ratio (PTR) are elevated, and are considered predictors of increased bleeding risk. We aimed at determining whether increased PT/PTR reflects the haemostatic potential and bleeding risk in ALF patients. METHODS Twenty consecutive ALF patients were recruited. Samples were analysed on admission for standard laboratory clotting tests (e.g. PT), thromboelastography (TEG), individual pro and anticoagulant factors and thrombin generation (TG) kinetics with and without Protac, a snake venom protein C activator, and microparticle assay. TG was also measured in 20 age and sex matched healthy volunteers. RESULTS PT was significantly raised (50.7s ± 7.2, p=0.0001) but did not correlate with TEG parameters. TEG tracings were consistent with a hypocoagulable state in 20%, normal in 45%, and hypercoagulable in 35% of the patients. There was a concomitant and proportional reduction in plasma levels of both procoagulants and natural anticoagulant proteins, in conjunction with a significant elevation in plasma levels of factors-VIII (FVIII) and Von Willebrand factor, and microparticles, culminating in an overall efficient, albeit reduced, thrombin generation capacity in comparison with healthy individuals. A heparin-like effect (HLE) was also noted in most patients. No significant clinical bleeding complications occurred and no blood transfusions were required. CONCLUSIONS In ALF, despite grossly deranged PT in all patients, estimation of bleeding risk suggests that the coagulation disturbance in ALF patients is complex and heterogeneous for which an individualised approach is required.

Quantifying the Effect of Antiplatelet Therapy A Comparison of the Platelet Function Analyzer (PFA-100 ® ) and Modified Thromboelastography (mTEG ® ) with Light Transmission Platelet Aggregometry

Background: Antiplatelet therapy with aspirin and clopidogrel is known to confer protection against ischemic events. Increasing numbers of patients are presenting for surgery while taking these drugs. This may lead to an increase in perioperative blood loss, particularly in those who have a heightened response to the drugs. Identifying these patients preoperatively would allow us to plan appropriate management. Methods: The antiplatelet effect of aspirin and/or clopidogrel was measured using two point-of-care monitors: the platelet function analyzer (PFA-100®; Dade, Miami, FL) and the modified thromboelastograph (mTEG®; Haemoscope Corp., Niles, IL). This was compared with optical light transmission aggregometry. Results: All people taking aspirin displayed a definitive aspirin effect on aggregometry (n = 20). Ninety percent of these were identified by modified thromboelastography (n = 18). Seventy percent were identified by the platelet function analyzer (n = 14). Fifty percent of people taking clopidogrel displayed a definitive response to the drug on aggregometry. Seventy percent of these were identified on modified thromboelastography (n = 7). None were identified by the platelet function analyzer. There was good agreement between the results of the aggregometry and modified thromboelastography in clopidogrel patients (&kgr; = 0.81). Conclusion: The search for a point-of-care monitor of platelet function has been the focus of much research. This study has shown that the modified thromboelastograph can be used for monitoring the effect of clopidogrel as well as aspirin. It potentially has a wide scope to be used for the monitoring of effectiveness of therapy as well as a possible predictor of perioperative bleeding.

Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

The effects of glycosaminoglycans on coagulation: a thromboelastographic study.

Endogenous heparinoids impair coagulation, evidenced by thrombelastography in cirrhotic patients with bacterial infection, but it is not clear which glycosaminoglycans can be detected by native and heparinase-modified thrombelastography. To assess the effects of different glycosaminoglycans on thrombelastography parameters and the reversibility of these effects by heparinase-I-modified thrombelastography. Twenty volunteers were enrolled. Solutions of heparan sulphate, dermatan sulphate, and chondroitin-4-sulphate were prepared at ‘equivalent’ concentrations, based on the composition and anticoagulant activity of danaparoid. Serial dilutions of each glycosaminoglycan were prepared to achieve 1.0, 0.5, 0.1, and 0.05 U/ml. Native and heparinase-modified thrombelastography, anti-activated factor X activity and heparin cofactor II activity were evaluated at each concentration. A statistically significant heparin-like effect was seen with 1 and 0.5 U/ml heparan sulphate, and 1 and 0.5 U/ml dermatan sulphate, which was completely reversed by heparinase-modified thrombelastography. Anti-activated factor X activity was significantly increased in samples containing heparan and dermatan sulphates. The heparin cofactor II activity decreased with 1.0 and 0.5 U/ml dermatan sulphate and chondroitin-4-sulphate, but not with heparan sulphate. Heparan and dermatan sulphates affect haemostasis when added to whole blood in vitro, detectable by native thrombelastography and completely reversed by heparinase-I-modified thrombelastography. They may therefore be responsible for the heparin-like effect seen by thrombelastography in patients with cirrhosis and bacterial infection.

The effects of unfractionated heparin, low molecular weight heparin and danaparoid on the thromboelastogram (TEG): an in-vitro comparison of standard and heparinase-modified TEGs with conventional coagulation assays.

To investigate the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and danaparoid (DPD) added to whole blood in vitro on standard and heparinase-modified thromboelastogram (TEG) parameters compared with conventional assays of coagulation. The effects of UFH, LMWH and DPD on standard TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-activated factor X (anti-FXa) activity, at concentrations of these anticoagulants ranging from 0.025 to 1 U/ml. In the second part of the study, the effects of very low concentrations (0.005–0.05 U/ml) of UFH, LMWH and DPD on the difference between standard and heparinase-modified TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-FXa activity. Standard TEG parameters were outside the reference range at lower concentrations of UFH, LMWH and DPD than most conventional coagulation assays were able to detect. Only anti-FXa activity was more sensitive to the presence of these anticoagulants than the standard TEG alone. The lowest concentration of UFH, LMWH and DPD used in this study (0.005 U/ml) caused significant differences between the standard and heparinase-modified α-angles of the TEG. In addition, the difference between standard and heparinase-modified TEG parameters distinguished between low concentrations (0.005–0.05 U/ml) of UFH with greater sensitivity than anti-FXa activity, but were less sensitive to LMWH and DPD. The standard TEG is more sensitive to UFH, LMWH and DPD than most conventional coagulation tests, with the exception of anti-FXa activity. Calculation of the difference between standard and heparinase-modified TEG parameters greatly increases the sensitivity of the assay for the effects of these anticoagulants, and is more sensitive to very low quantities of UFH than anti-FXa activity.

Dialyzers Designed to Increase Internal Filtration Do Not Result in Significantly Increased Platelet Activation and Thrombin Generation

Introduction: To increase middle molecule clearances, high-flux dialyzers with increased internal filtration have been developed. However, dialyzer design and structure may affect thrombin generation and platelet activation, thereby risking increased clotting and reduced dialyzer clearances. Methods: Coagulation parameters, platelet, white cell and endothelial activation markers were measured prior to and following dialysis sessions in 12 patients using two different dialyzers designed for increased internal filtration. Results: Prior to dialysis, patients had evidence of activation of coagulation with increased factor VIII:C, thrombin-antithrombin complexes and prothrombin fragment 1+2, increased platelet activation, with raised platelet factor 4, β-thromboglobulin levels and increased fibrinolysis (raised D-dimers). Dialysis was associated with the release of soluble platelet integrin, sP selectin, increased endothelial activation with increased levels of von Willebrand factor (vWF) antigen (vWF:Ag) and vWF propeptide (vWF:pp) and sE selectin. There was no difference in tinzaparin levels at the end of the dialysis session using either dialyzer, as shown by anti-Xa activity – 0.145 ± 0.027 versus 0.11 ± 0.017 IU/ml, respectively. Conclusion: Haemodialysis patients have an inflammatory phenoytype, characterized by increased activation of coagulation, platelets and also fibrinolysis. However, dialyzers designed to increase internal filtration did not significantly increase platelet activation or thrombin generation.

Role of the Thrombelastograph as an adjunctive test in thrombophilia screening.

The Thrombelastograph (TEG) provides a global assessment of haemostatic function. We have investigated the potential role of TEG in thrombophilia testing by studying 87 consecutive patients (all with personal or family history of thrombosis). For each patient, routine thrombophilia screening tests and TEG were performed in parallel. In keeping with previous reports, at least one thrombophilic trait was identified in 34% of the patients. Among those patients with previous thrombosis, the yield from screening was higher (54% with prothrombotic trait). Overall, a hypercoaguable TEG was demonstrated in 45% of patients. However, TEG and standard thrombophilia testing did not necessarily identify the same subset of individuals. Of 39 patients with hypercoaguable TEG, only 17 had a demonstrable thrombophilic trait. Conversely, of 30 patients with proven thrombophilia, 17 also had a hypercoaguable TEG. We conclude that TEG cannot be used as a sole initial screening test in patients referred for thrombophilia testing, as it fails to identify 43% of underlying thrombophilic traits. However, TEG may be a useful adjunctive test, particularly in patients in whom the regular thrombophilia screen proves unremarkable. Prospective studies are warranted to determine whether a hypercoaguable TEG can be used to predict a subset of patients at increased risk for recurrent thrombotic events.

Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay

Summary. This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen‐binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (≤ 0·7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0·7), but the VWF:RCo/VWF:CBA ratios were discordant (≤ 0·7) (P = 0·001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high‐molecular‐weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet‐binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.