Epitope analysis of autoantibodies to ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Introduction

Autoantibodies to ADAMTS13 have a pivotal role in the pathogenesis of acquired thrombotic thrombocytopenic purpura (TTP). By decreasing the function of ADAMTS13, autoantibodies impair the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers into smaller sizes, leading to lethal platelet-VWF thrombi in the microcirculation. We therefore aimed to determine the sites of autoantibody recognition on ADAMTS13.

Materials and Methods

In this study, IgG purified from 13 acquired TTP patients were examined to determine their binding sites on ADAMTS13. Immobilized IgG on microtiter plate or proteinG beads was screened by phage library expressing various peptides of ADAMTS13.

Results

In screening, diverse peptide sequences were obtained from almost all of the ADAMTS13 domains, including the spacer domain, which is considered a major binding site. In particular, we detected an identical amino-acid sequence in the C-terminus of the spacer domain from Gly662 to Val687 that was recognized by autoantibodies from 5 TTP patients. The specific autoantibody was expected to be associated with the plasma levels of the ADAMTS13 antigen or activity, and with the quantity of ADAMTS13 autoantibodies or the inhibitory autoantibody titer in TTP patient plasma. These measurements, however, did not seem to be related to the presence or absence of the specific autoantibody.

Conclusions

These findings indicate that the specific autoantibody might be a feature of acquired TTP, although its clinical significance remains to be elucidated.     

Elevated plasma neutrophil elastase concentration is associated with disease activity in patients with thrombotic thrombocytopenic purpura

Introduction

Genetic and autoimmune risk factors contribute to the development of thrombotic thrombocytopenic purpura (TTP) but triggers are needed to bring about acute disease.The aim of the study was to investigate the association of neutrophil activation with acute TTP, to assess whether neutrophil activation changes during plasma exchange therapy and to show if complement- and neutrophil activation are parallel, characteristic processes in acute TTP.

Materials and Methods

Altogether 49 EDTA-plasma samples of 21 TTP patients with acute disease and 17 in remission were investigated along with 20 healthy controls.A stable complex of PMNE-proteinase-inhibitor was measured by ELISA (Calbiochem, Merck-Millipore, Darmstadt, Germany).

Results

Acute disease was associated with significantly increased PMNE levels, the group medians were similarly low in TTP patients in remission and in healthy controls. Increased PMNE levels were characteristic for hematologically active and ADAMTS13 deficient form of TTP. PMNE concentration inversely correlated to disease activity markers platelet count (r = − 0.349, p = 0.032) and hemoglobin levels (p = − 0.382 p = 0.018). Achievement of remission was associated with significant reduction of plasma PMNE levels (p = 0.031, Wilcoxon test). There was positive correlation between PMNE levels and complement activation markers C3a and Bb.

Conclusions

We report increased PMNE levels in acute TTP and showed its association to activity markers of acute TTP and complement activation. Effective treatment of an acute TTP episode resulted in marked decrease in PMNE levels. Our data support and extend previous observations that neutrophil extracellular traps may be released in acute TTP and potentially contribute to the pathophysiology of this disease.     

Risk factors and clinical profile of thrombotic thrombocytopenic purpura in systemic lupus erythematosus patients. Is this a distinctive clinical entity in the thrombotic microangiopathy spectrum?: A case control study

Introduction

The association of thrombotic thrombocytopenic purpura (TTP) with systemic lupus erythematosus (SLE) is rare. It is associated with high morbidity and mortality. Information about risk factors and clinical outcomes is scant.

Material and Methods

A retrospective case-control study was performed in a referral center in Mexico City between 1994 and 2013. Patients were diagnosed with TTP if they fulfilled the following criteria: microangiopathic haemolytic anaemia, thrombocytopenia, high LDH levels, normal fibrinogen and negative Coombs’ test. Patients with SLE were diagnosed with ≥ 4 ACR criteria. We included three study groups: group A included patients with SLE-associated TTP (TTP/SLE; cases n = 22, TTP events n = 24); patients with non-autoimmune TTP (NA-TTP; cases n = 19, TTP events n = 22) were included in group B and patients with SLE without TTP (n = 48) in group C.

Results

After multivariate analysis, lymphopenia < 1000/mm3 [OR 19.84, p = 0.037], high SLEDAI score three months prior to hospitalisation [OR 1.54, p = 0.028], Hg < 7 g/dL [OR 6.81, p = 0.026], low levels of indirect bilirubin [OR 0.51, p = 0.007], and less severe thrombocytopenia [OR 0.98, p = 0.009] were associated with TTP in SLE patients. Patients with TTP/SLE received increased cumulative steroid dose vs. NA-TTP (p = 0.006) and a higher number of immunosuppressive drugs (p = 0.015). Patients with TTP/SLE had higher survival than NA-TTP (p = 0.033); however, patients hospitalised for TTP/SLE had a higher risk of death than lupus patients hospitalised for other causes

Conclusions

Lymphopenia is an independent risk factor for TTP/SLE. It is likely that patients with TTP/SLE present with less evident clinical features, so the level of suspicion must be higher to avoid delay in treatment.     

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

Introduction

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells’ surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage.

Material and Methods

VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13.

Results

To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity.

Conclusions

Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.     

D-dimer,FVIII and thrombotic burden in the acute phase of deep vein thrombosis in relation to the risk of post-thrombotic syndrome

Background

Post-thrombotic syndrome (PTS) is the most common complication of deep vein thrombosis (DVT), but few data are available on the risk factors for PTS.

Aims

To assess whether the time-course of D-dimer, FVIII, and thrombotic burden are related to PTS development.

Methods

Patients (n = 59) with proximal DVT of the lower limbs (age 64; range:20-88 years; male 56%) were enrolled on the day of diagnosis (D0) and all received heparin for 5-7 days, overlapped and followed by vitamin K antagonists (VKA) for 3 months. Whole-leg compression ultrasound examination was conducted on D0 and 7 (D7), 30 (D30), and 90 (D90) days afterwards, when blood samples were also taken for D-dimer (STA Liatest) and FVIII (chromogenic assay) testing. Thrombotic burden was defined at each time point according to a score, which considered thrombosis extent and occlusion degree. Villalta score was evaluated at D30, D90, and D180.

Results

At D90, 12 patients developed PTS (Villalta score ≥ 5) and the median Villalta score was 1 (IQR 0.3-3.0) and was not correlated with either D-dimer or FVIIII time course. At D180, 13 patients had PTS and they had similar thrombotic score at D0, D30 to those without PTS, but higher at D90 (7.6 ± 5.1 vs. 3.2 ± 3.6; p = 0.011). Thrombotic score at D90 was correlated with Villalta score at D90 (rho = 0.374, p = 0.009) and at D180 (rho = 0.436, p = 0.006).

Conclusions

Thrombotic burden after 90 days of VKA is correlated with PTS.     

High Shear Dependent von Willebrand Factor Self-assembly Fostered by Platelet Interaction and Controlled by ADAMTS13

Introduction

The paradigm of activation induced platelet aggregation has recently been refuted under blood flow conditions with shear rates exceeding 20,000 s^- 1. These lead to reversible rolling platelet aggregates, which were dependent on the presence of immobilized and soluble von Willebrand factor.

Material and Methods

In vitro experiments using direct fluorescence video-microscopy were performed in wall parallel and stagnation point flow chambers with shear rates raised from 20,000 to 50,000 s^- 1. Washed blood cell suspension containing recombinant von Willebrand factor (rVWF) was perfused over rVWF or collagen coated surfaces.

Results

Here we show for the first time with the visualization of rVWF that not only colloid and polymer, i.e. platelets and VWF, form a composite, but that VWF itself is capable of entirely reversible self-assembly. On a collagen surface the platelet-VWF-conglomerates did not roll but VWF nets bound permanently to the collagen fibers and captured and immobilized platelets from the flow. Lowering the shear rate below the threshold of 20,000 s^- 1 no longer dissolved these deposits. Ultralarge multimer containing rVWF was most effective compared to normal sized rVWF. The presence of ADAMTS13 limited rolling aggregate and platelet-VWF-conglomerate formation to a time window of 7-8 minutes. Changing wall parallel flow to stagnation point flow halved the required shear rate threshold.

Conclusion

We conclude that flow dynamics can trigger reversible von Willebrand factor self-assembly and platelet-VWF-conglomerate accrual, which are regulated by ADAMTS13 to a time span needed by coagulation to stabilize it, e.g. in case of vessel injury.     

Thrombospondin-1 and ADAMTS13 competitively bind to VWF A2 and A3 domains in vitro

Introduction

ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motif. 13) is the major metalloprotease for VWF degradation. ADAMTS13 deficiency causes the accumulation of uncleaved VWF and might lead to a lethal thrombotic thrombocytopenic purpura (TTP). Thrombospondin-1 (TSP1) is considered as a reductase of VWF (von Willebrand factor) which can mildly downregulate the size of VWF by targeting on disulfide bond between VWF dimers. It was reported that TSP1 might protected VWF from cleaving by ADAMTS13, yet the underlying mechanism of this VWF protection has remained unknown.

Materials and Methods

Full-length ADAMTS13 and different domains (A1,A2,A3) of human VWF were constructed and expressed respectively. The binding ability of TSP1 or ADAMTS13 with each VWF domain or full-length VWF was investigated by using enzyme linked immunosorbent assay. The inhibition of ADAMTS13 activities by the different concentrations of TSP1 were observed by western blot and residual-collagen binding assay (R-CBA) under the denaturing condition.

Results

We found that ADAMTS13 interacted with the rVWF A1, A2, A3 domains and full-length VWF, while TSP1 also bound to three A domains, especially to A2 and A3 domains. We observed that TSP1 partially blocked ADAMTS13 binding to A2 domain, A3 domain and full length VWF. The results of our assays showed that TSP1 could restrain ADAMTS13 activity up to 70%.

Conclusions

Our study suggested that TSP1 played competitively inhibitory role in ADAMTS13 binding and cleaving of VWF, and the potential competition might happen within A2 and A3 domains.     

Changes in plasma von Willebrand factor and ADAMTS13 levels associated with left atrial remodeling in atrial fibrillation

Introduction

Previous studies have shown raised plasma von Willebrand factor (VWF) levels in patients with atrial fibrillation (AF). However, little is known about changes of VWF associated with VWF-cleaving protease (ADAMTS13) in AF. The aim of this study was to examine the relationship between changes in plasma VWF and ADAMTS13 levels, and left atrial remodeling in AF patients.

Materials and Methods

We measured plasma VWF and ADAMTS13 antigen levels in 70 paroxysmal AF (PAF) patients, 56 chronic AF (CAF) patients, and 55 control subjects.

Results

Plasma VWF levels (mU/ml) were significantly higher in CAF and PAF patients compared with the controls (2103 ± 743, 1930 ± 676, 1532 ± 555, respectively, P < 0.0001 in CAF vs. controls, P = 0.001 in PAF vs. control), while ADAMTS13 levels (mU/ml) were significantly lower in CAF and PAF patients compared with the controls (795 ± 169, 860 ± 221, 932 ± 173, respectively, P = 0.0002 in CAF vs. controls, P = 0.04 in PAF vs. control). The VWF/ADAMTS13 ratio was significantly higher in patients with CAF than PAF or controls (2.81 ± 1.30, 2.34 ± 0.92, 1.73 ± 0.83, respectively; P = 0.01 in CAF vs. PAF, P < 0.0001 in CAF vs. controls). There was a significant correlation between the VWF/ADAMTS13 ratio and left atrial diameter (positive correlation; r = 0.275, P = 0.0002) and left atrial appendage flow velocity (negative correlation; r = - 0.345, P = 0.0018).

Conclusions

These findings suggest that the imbalance between plasma VWF and ADAMTS13 levels caused by left atrial remodeling might be closely associated with intra-atrial thrombus formation in AF patients.     

Elevated plasma levels of soluble platelet glycoprotein VI (GPVI) in patients with thrombotic microangiopathy

Background

Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets. This study examined the soluble platelet glycoprotein VI (sGPVI) levels in patients with TMA to evaluate the activation of platelets in thrombotic states.

Materials and Methods

The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in patients with TMA.

Results

The plasma levels of sGPVI were significantly higher in postoperative patients, patients with TMA and those with disseminated intravascular coagulation (DIC) than in those without thrombosis. The plasma levels of sGPVI were the highest in patients with TMA without markedly reduced ADAMTS13 and those were significantly reduced after plasma exchange.

Conclusion

The measurement of sGPVI level is therefore considered to be important for the diagnosis and evaluation of TMA.     

Identification of epitopes on ADAMTS13 recognized by a panel of monoclonal antibodies with functional or non-functional effects on catalytic activity

Introduction

von Willebrand factor (VWF) cleavage by ADAMTS13 is mediated by multi-step interactions between their multi-domain structures. To clarify the relationship between inhibitory effects of monoclonal antibodies and epitopes on each ADAMTS13 domain, we analyzed how each ADAMTS13 domain contributes to catalyze VWF using a mouse anti-ADAMTS13 monoclonal antibody panel.

Materials and Methods

FRETS-VWF73 assay was used to examine the effects of 14 anti-ADAMTS13 monoclonal antibodies on the catalytic activity of plasma ADAMTS13. Epitope mapping was performed using phage surface display. Libraries expressing peptide fragments of ADAMTS13 were screened with the monoclonal antibodies.

Results

Eleven epitopes of 14 monoclonal antibodies were successfully defined. Three monoclonal antibodies recognizing metalloprotease or disintegrin-like domains strongly inhibited the catalytic activity and their epitopes were on Gln159-Asp166, Tyr 305-Glu327, and Asn308-Glu376. Five monoclonal antibodies recognizing TSP1-3 to -7 repeats showed weak inhibitory effects, and their epitopes were on Pro744-Ala806, Pro856-Cys864, Gln892-Gly940, Cys1007-Cys1072, and Gln1163-Asn1185. Four monoclonal antibodies recognizing the TSP1-1, TSP1-2, CUB1 or CUB2 domains had no inhibitory effects, and their epitopes, except that for TSP1-1, were Pro682-Cys742, Thr1200-Cys1213, and Gln1409-Glu1414. Two monoclonal antibodies recognizing cysteine-rich and spacer domains showed moderate inhibitory effects, but their epitopes were not determined.

Conclusions

We revealed the epitopes of 11 monoclonal anti-ADAMTS13 antibodies on each of the domains and clarified their association with inhibitory effects on VWF catalysis under static conditions. Catalytic activity correlated strongly with the epitopes on metalloprotease and disintegrin-like domains, weakly with those on TSP1-3 to -7 repeats, and negatively with those on TSP1-1, -2, and CUB domains.     

Procoagulant activity and cellular origin of microparticles in human amniotic fluid

Introduction

Amniotic fluid contains various procoagulant factors involved in intro-vascular amniotic fluid-induced coagulopathies. During the progression of normal pregnancy, microparticles would be shed off from cells and accumulate in amniotic fluid over time. In this study, our aims were to investigate the cellular origin and procoagulant entity of these microparticles.

Materials and methods

Twenty amniotic fluid samples from healthy parturient women were collected, and the microparticles were isolated and stained with phycoerythrin-labeled antibodies to CD138, CD41a, CD144 and CD11b to identify their cellular origin. Their phosphatidylserine and tissue factor expression levels were quantified with fluorescein isothiocyanate-labeled annexin V and anti-tissue factor antibody staining. Their procoagulant activity was tested with plasma coagulation assay and factor Xase and prothrombinase assays.

Results

Phenotypic analysis showed 36.8% and 33.8% of amniotic fluid microparticles positive for CD138 and CD11b, respectively, indicating their epithelial cell or leukocyte origin. Of these microparticles, 66.3 ± 5.9% expressed phosphatidylserine while 37.4 ± 4.1% expressed tissue factor. In addition, amniotic fluid microparticles could significantly shorten the plasma coagulation time and increase the production of factor Xa and thrombin. Inhibition assays with annexin V and anti-tissue factor antibody confirmed the coagulation effects of amniotic fluid microparticles.

Conclusion

The microparticles derived from epithelial and leukocytes may be a mechanism of amniotic fluid-induced coagulopathies.     

Prothrombin fragment 1 + 2 in urine as a marker on coagulation activity in patients with suspected pulmonary embolism

Introduction

We have recently reported that increased levels of urine prothrombin fragment 1 + 2 reflected radiologically verified deep vein thrombosis. In this study we evaluated whether urine prothrombin fragment 1 + 2 was associated with pulmonary embolism in non-selected patients.

Materials and methods

Patients with clinical suspected pulmonary embolism were interviewed on comorbidities and medications. Urine was collected from each patient before radiological examination and snap frozen until analysed on urine prothrombin fragment 1 + 2 with an ELISA kit. Imaging of the pulmonary arteries were conducted with contrast enhanced computer tomography.

Results

Pulmonary embolism was diagnosed in 44/197 patients. Non-significantly higher urine prothrombin fragment 1 + 2 levels were found in non-selected patients with pulmonary embolism vs. those without (p = 0.324). Significantly higher urine prothrombin fragment 1 + 2 levels were found in the pulmonary embolism positive patients without comorbidities (n = 13) compared to the control group (n = 28) (p = 0.009). The calculated sensitivity, specificity and negative predictive value using the lowest detectable urine prothrombin fragment 1 + 2 level was 82%, 34% and 87%, respectively.

Conclusions

There was no significant urine prothrombin fragment 1 + 2 level difference in patients with and without pulmonary embolism. In non-comorbide pulmonary embolism positive patients the urine prothrombin fragment 1 + 2 levels were significantly higher compared to the control group. The negative predictive value found in this study indicates that uF1 + 2 has the potential to identify patients with a low risk of PE.     

Simvastatin Increases ADAMTS13 Expression in Podocytes

Introduction

ADAMTS13 is a specific von Willebrand factor–cleaving protease. Severe deficiency of ADAMTS13 is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released from hepatic stellate cells and endothelial cells, but is also expressed in other cells, including kidney podocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has a beneficial effect on atherosclerosis and also has anti-inflammatory and antithrombotic properties. A recent study indicates that ADAMTS13 reduces inflammatory plaque formation during early atherosclerosis in mice. In our study, we investigated the effects of simvastatin on inflammatory cytokines–induced ADAMTS13 expression in podocytes.

Materials and Methods

A conditionally immortalized mouse podocyte cell line was utilized to study the expression of ADAMTS13 in podocytes. The influence of TNF-α, IL-4, IL-6 and simvastatin on ADAMTS13 was investigated. ADAMTS13 mRNA levels in podocytes were measured by using real-time PCR and protein levels were detected by Western blotting.

Results

Simvastatin significantly up-regulated the expression levels of ADAMTS13 mRNA and protein in podocytes. IL-6 decreased ADAMTS13 expression, and TNF-α had no significant effects on ADAMTS13 expression in podocytes. IL-4 reduced ADAMTS13 mRNA expression but not its protein level. Simvastatin was able also reversed the inhibitory effect of IL-6.

Conclusions

We demonstrate that simvastatin increases the expression of ADAMTS13 in a dose-dependent manner in podocytes, which likely contributes to the antithrombotic property of statin. Different inflammatory cytokines have different effects on the levels of ADAMTS13 mRNA expression and protein within podocytes.     

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock

Introduction

The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis.

Materials and methods

MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay.

Results

MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2–2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r_s = − 0.84), time to peak (r_s = − 0.83), peak (r_s = 0.85) and ETP (r_s = 0.83).

Conclusions

MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.     

Anti-Xa Activity After Enoxaparin Prophylaxis In Hospitalized Patients Weighing Less Than Fifty-Five Kilograms

Introduction

Low-molecular-weight heparins are commonly used for the prophylaxis of thromboembolic disease. In contrast to therapeutic doses, recommended prophylactic doses are fixed (i.e., 40 mg/day enoxaparin). Dosing of patients with extreme body weights has not been well studied, especially dosing of low weight patients.

Objectives

To establish the anti-Xa activity that results from 40 mg/day enoxaparin in inpatients with body weight ≤ 55 kg.

Patients/Methods

Cross-sectional study including inpatients older than 18 years, with body weight ≤ 55 kg, and whose treating physician found indication for 40 mg/day enoxaparin. We excluded patients with renal failure and those using oral anticoagulants. Anti-Xa activity was measured 3 hours after the second dose of enoxaparin. Statistical analyses were conducted to determine the effect of body weight on anti-Xa levels.

Results

Average age was 72.5 years (interquartile range, 30) and median body weight was 49.7 kg (interquartile range, 7). Twenty-five percent of patients weighed ≤ 45 kg, 37.5% weighed 46–50 kg, and 37.5% weighed 51–55 kg. The mean anti-Xa activity was 0.54 ± 0.18 IU/ml, and 60% of the patients exhibited activity ≥ 0.5 IU/ml. Weight and anti-Xa activity inversely correlated (Spearman’s rho = − 0.428, p = 0.001). Patients weighing ≤ 45 kg exhibited higher anti-Xa activity (0.61 ± 0.18 IU/ml, p = 0.008) than heavier patients and an odds ratio of 8 for anti-Xa level ≥ 0.5 IU/ml (95% CI: 1.42-45.06).

Conclusions

Anti-factor Xa activity rises significantly when body weight decreases. Patients of low weight, especially those weighing < 45 kg, exhibited an anti-Xa activity higher than the desired range for thromboembolic prophylaxis.     

Effects of naturally occurring mutations in CUB-1 domain on synthesis, stability, and activity of ADAMTS-13

Introduction

Upon stimulation, endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and hyperactive forms that are rapidly cleaved by ADAMTS-13. Mutations in the ADAMTS13 gene result in ULVWF-mediated thrombosis found in patients with familial thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 fits in the consensus of the ADAMTS family metalloproteases, but also contains two unique C- terminal CUB domains. Studying mutations in CUB domains could provide insights into the functional role of these domains.

Methods

Three naturally occurring mutations (C1213Y, W1245del and K1256FS) in the CUB-1 domain found in patients with TTP were expressed in Hela cells. The secretion, stability and VWF-cleaving activity of the mutants under static and flow conditions were examined.

Results

The mutations impaired secretion of ADAMTS-13 to apical surface, but not to extracellular matrix of transfected Hela cells. C1213Y and K1256FS also accelerated, whereas W1245del delayed, extracellular degradation of the mutants. The mutations also resulted in a moderate decrease in cleaving plasma VWF under static conditions. However, the mutated ADAMTS-13 bound to VWF substrate similarly as the wild-type metalloprotease and remained active in cleaving (UL)VWF under flow conditions.

Conclusions

The CUB-1 domain is critical for ADAMTS-13 secretion and stability upon secretion. ADAMTS-13 deficiency found in TTP patients could be resulted from reduced ADAMTS-13 secretion and, in the case of C1213Y and K1256FS accelerated degradation. W1245del is highly resistant to degradation and active in cleaving VWF.     

A serendipitously identified novel small molecule procoagulant compound giving rise to a high-throughput screening assay based on human plasma

Introduction

Oral treatment is lacking for haemophilia, the rare bleeding disorders, and some severe forms of von Willebrand’s disease. We have serendipitously identified a small molecule procoagulant compound (AZ10047130). This publication describes some characteristics of AZ10047130 and a systematic search for novel hits using a, human plasma-based, high-throughput screening (HTS) assay.

Material and Methods

Coagulation, thrombin generation, chromogenic assays and surface plasmon resonance (SPR) experiments were used to characterise AZ10047130. A 1536-well formatted human plasma coagulation assay for HTS was developed.

Results

In the plasma clot assay (re-calcified plasma with low tissue factor) AZ10047130 shortened time to coagulation with an EC₅₀ value of 3.9 μM (assay concentration). AZ10047130 was similarly effective in immunodepleted human and haemophilia A plasmas. SPR and chromogenic substrate experiments indicated that AZ10047130 binds to the heparin binding site of several coagulation factors. The HTS screened in excess of one million compounds. It generated some hits belonging to the same pharmacophore as AZ10047130 but also some entirely novel hits.

Conclusion

These novel small molecule procoagulant compounds may serve as templates for discovery of oral procoagulant drugs.     

Human platelets do not express tissue factor

Background

The controversy about the expression of tissue factor (TF) in platelet after de novo synthesis prevail despite many groups recognize that platelet isolation, assays and reagents, particularly non-specific antibodies, may account for the diversity. In this study the potential of TF expression was evaluated using immune-purified human platelets and employing a very sensitive and highly specific TF activity assay.

Methods

Isolated platelets in plasma anti-coagulated with Fragmin were subjected to stimulation by LPS plus PMA, IgG antibody or TRAP and tested for TF activity.

Results

Platelets stimulated with LPS plus PMA for 4 hours expressed trace amounts of TF like activity (PCA), not inhibited by anti-TF antibody (0.2 ± 0.1 mU/ml blood). Platelets, not immune-adsorbed to remove monocytes, showed significant TF activity (2.0 ± 0.9 mU/ml blood) that was nearly abolished by anti-TF antibody. IgG antibody from patient with lupus anticoagulant failed to enhance the trace amount of PCA as compared to the control in contrast to high TF activity induced in monocytes (0.4 ± 0.1 mU/ml blood versus 27.5 ± 10.5 mU/10⁶ cells) showing that activation of complement is not mediating TF expression. Platelet subjected to TRAP activation for 10 min possessed only trace amounts of PCA that was not inhibited by anti-TF antibody and slightly enhanced by anti-TFPI antibody.

Conclusions

It is concluded that platelets free of monocytes do not express TF activity when stimulated by LPS or activated complement factors, implying no role for Toll like receptor (TLR4) as suggested recently. There is no evidence of TF activity associated with platelets as a result of rapid and dynamic process.     

Association between ADAMTS13 polymorphisms and risk of cardiovascular events in chronic coronary disease

Introduction

Association between ADAMTS13 levels and cardiovascular events has been described recently. However, no genetic study of ADAMTS13 in coronary patients has been described.

Materials and Methods

Based on related populations frequencies and functional studies, we tested three ADAMTS13 polymorphisms: C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 560 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function. The incidence of the 5-year end-points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for each polymorphim's allele, genotype and haplotype. Risk was assessed with the use of logistic regression and Cox proportional-hazards model and multivariable adjustment was employed for possible confounders.

Results

Clinical characteristics and received treatment of each genotype group were similar at baseline. In an adjusted model for cardiovascular risk variables, we were able to observe a significant association between ADAMTS13 900V variant and an increased risk of death (OR: 1,92 CI: 1,14-3,23, p = 0,015) or death from cardiac cause (OR:2,67, CI: 1,59-4,49, p = 0,0009). No association between events and ADAMTS13 Q448E or P618A was observed.

Conclusions

This first report studying the association between ADAMTS13 genotypes and cardiovascular events provides evidence for the association between ADAMTS13 900V variant and an increased risk of death in a population with multi-vessel CAD.     

Open-label,randomized study of the effect of rivaroxaban with or without acetylsalicylic acid on thrombus formation in a perfusion chamber

Introduction

Rivaroxaban, a direct factor Xa inhibitor, has demonstrated effectiveness for the management of both venous and arterial thrombosis. This study was designed to investigate the antithrombotic effect of rivaroxaban, with or without acetylsalicylic acid (ASA), in an ex vivo perfusion chamber at both low and high shear rates.

Materials and methods

Healthy subjects (N = 51) were enrolled in a randomized, crossover (rivaroxaban 5, 10 or 20 mg with or without ASA), and parallel-group (compared with ASA plus clopidogrel) study. Thrombi formed on pig aorta strips were measured after a 5-minute perfusion at low and high shear rates with blood from the subjects by measuring D-dimer concentration (for fibrin deposition) and P-selectin content (for platelet deposition).

Results

ASA alone had no impact on thrombus D-dimer levels, whereas rivaroxaban alone at peak concentrations decreased D-dimer levels by 9%, 84% and 65% at low shear rate and 37%, 73% and 74% at high shear rate after doses of 5, 10 and 20 mg, respectively. Steady-state ASA plus rivaroxaban 5 mg caused a greater reduction in D­dimer levels (63%) than monotherapy at low shear rate. Co-administration of ASA with clopidogrel was associated with a 30% decrease in D-dimer levels at low shear rate and a 14% decrease at high shear rate. No conclusive effect on P-selectin content was observed across the treatment groups.

Conclusions

Rivaroxaban dose-dependently inhibited ex vivo thrombus formation under low and high shear rates. Co-administration of ASA had an additional effect on the antithrombotic action of low-dose rivaroxaban.