Novel Assay Reveals Optimal Treatment in Rare Blood Disorder

Liam Davenport

April 04, 2019

GLASGOW — Combining prophylactic infusions and anti-platelet therapy normalises thrombus formation in patients with congenital thrombotic thrombocytopenic purpura (TTP) under shear flow conditions, reveals a novel assay developed by UK researchers that could revolutionise both the diagnosis and management of this rare condition.

Congenital TTP is an inherited deficiency in ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the blood glycoprotein von Willebrand factor (VWF).

Even in the presence of normal blood counts, patients can experience lethargy, headaches and abdominal pain, and the condition can lead to neurological, cardiac and renal complications.

Novel Shear Flow Assay

As current assays are unable to examine clot formation in normal blood flow, clinicians have been left unsure as to the best way to approach treatment.

Dr Ferras Alwan, from the Department of Haematology at University College London Hospital, therefore developed a novel shear flow assay that can assesses thrombus coverage under physiological blood flow conditions.

The study, presented at the British Society for Haematology Annual Scientific Meeting in Glasgow, and conducted as part of the UK TTP Registry, showed that giving anti-platelet therapy such as daily aspirin prior to prophylactic infusion reduced thrombus formation to normal levels.

Dr Alwan said: "Anti-platelet therapy decreases thrombus formation and works synergistically to improve thrombus surface coverage before and after prophylaxis, and this newly developed assay can be used as a biomarker to assess treatment options and efficacy in congenital TTP."

In addition, the team found that the recombinant ADAMTS13 (BAX 930, Shire), which is currently the subject of a phase 3 trial assessing its safety and efficacy, may be able to normalise thrombus formation in the presence of anti-platelet therapy, regardless of the dose given.

More Work to Be Done

Session co-chair Dr Keith Gomez, from the Haemophilia Centre and Thrombosis Unit at Royal Free Hospital, London, said that, when he began as a junior doctor, TTP "was probably the most serious non-malignant condition, with the highest rate of mortality".

"What we’ve seen through the work in the TTP Registry is that, by bringing together resources into a specialist centre, we’ve improved, first of all, our survival, mostly through treatment options."

However, he told Medscape News UK that "there’s still a lot of work to be done".

Dr Gomez said: "As haematologists we often focus on treatment, which is important," but he believes that Dr Alwan’s research showed "beautifully" the importance of diagnostics.

He added: "Unless we tackle that side of things...we’re not going to make the next leap into how we’re going to use new treatments like recombinant ADAMTS13.

"I think it shows how translational research is not just about therapeutic products, but it’s also about diagnostics."

Very Rare Condition

Dr Alwan told the audience that the overall incidence of TTP is estimated to be 2 to 6 people per million per year, of which between 2% and 10% are congenital cases.

These classically present during childhood but there is also a peak during pregnancy.

Congenital TTP results in the presence of ultra-large VWF multimers in the blood and, it is presumed, the subsequent formation of circulating platelet rich microthrombi.

Dr Alwan explained that there is increasing evidence that prophylactic therapy can improve long-term outcomes and reduce stroke incidence.

However, there is little consensus on when to start treatment, how much to give and how often, and whether there should be a target ADAMTS13 level. If there is a target, there is no consensus over whether it should be at peak or trough levels.

He said that this absence of agreement is due to "a lack of evidence and a lack of monitoring options" in congenital TTP.

The issue with monitoring is that, in addition to platelet and haemoglobin levels being typically normal, the assays for ADAMTS13 are insufficiently sensitive to detect low levels.

Moreover, many of the effects seen in congenital TTP, including VWF multimer attachment, vWF bond cleavage by ADAMTS13, and the impact of VWF and ADAMTS13 mutations, are seen only under shear flow.

These are undetected by current assays, however, as they test blood samples only under static conditions.

Research Details

To get around this problem, Dr Alwan and colleagues set out to develop a novel shear flow assay to determine thrombus formation in congenital TTP in the presence of normal blood counts.

They took a microfluid system (Venaflux, Cellix) that is able to provide shear flow and mimic the blood circulation, coating the microchannels with type 1 collagen and mounting it on an epifluorescence microscope.

To examine shear flow, they treated whole blood samples with DiOC6 to stain the platelets and perfused the blood over the microchannels at a predetermined rate.

As the blood passed through the channels, thrombi formed, and the researchers measured the total surface area coverage by thrombus.

Before using patient samples, the team determined the normal range of thrombus coverage by perfusing blood samples from 50 healthy controls, revealing a surface coverage range of 6% to 39%, at a median of 23%.

Next, the team obtained blood samples from 22 patients with congenital TTP, of whom 19 were female. The median age of the patients was 33 years, and their median platelet count was 266.

The total surface coverage by thrombus was significantly higher in patients than in controls, at a median of 89% and a range of 47% to 100%.

In addition, the researchers found that the thrombi formed by congenital TTP patients were larger than those formed by normal controls, due to increased adhesion of VWF to collagen.

Not only that, but the VWF strings were twice as long in patients as in controls, and six times more VWF was seen per field.

To examine the impact of prophylaxis, the team repeated the assay in 17 patients who received a plasma infusion and five patients who were given BPL-8Y (intermediate purity factor VIII), with blood samples taken 30 minutes before and after prophylaxis.

Plasma infusion was associated with a significant reduction in total thrombi surface coverage, from 89% pre-treatment to 44% post-treatment (p=0.0005).

The improvement with BPL-8Y was not significant, at 65% and 41% pre- and post-treatment (p=0.095), although Dr Alwan pointed out that the starting total was lower than in patients given plasma infusion.

He also noted that the degree of thrombus coverage post-treatment was not as low as that seen in normal controls.

Moreover, while 50% of patients normalised their surface coverage with prophylactic treatment, 100% were back up to abnormal levels by the time of their next infusion.

Hypothesising that anti-platelet treatment would have a synergistic effect with prophylaxis, 19 patients started aspirin 75 mg once daily, with three switched to clopidogrel 75 mg once daily due to gastrointestinal effects.

Before prophylaxis, anti-platelet therapy for at least 10 days reduced total thrombus surface coverage significantly from 89% to 50% (p<0.0001).

When these anti-platelet-treated patients were then given prophylactic therapy, they saw their total thrombus coverage decrease further to a level significantly less than that seen with prophylactic therapy alone, at 19% versus 44% (p=0.0011).

Notably, total thrombus surface coverage returned to the normal range in 100% of the patients given both treatments, which was significantly more than the 50% seen with prophylactic therapy alone (p=0.04) and they remained in the normal range until their next infusion.

Finally, the researchers looked at the effect of recombinant ADAMTS13 in vitro on pre-treatment blood samples, testing it at 47 IU/dL, 93 IU/dL, and 140 IU/dL doses.

They found dose-dependent responses, with the percentage of patients achieving normalised thrombus coverage increasing from 36% to 45% and 64%, respectively, with the three doses.

When recombinant ADAMTS13 was added to samples taken from patients treated with anti-platelet drugs, the team found that all samples corrected to the normal range, regardless of the dose given.

Improving Symptoms

In the post-presentation discussion, Dr Alwan said that, alongside the improvements in thrombus coverage, patients treated with anti-platelets alongside prophylactic therapy experienced improvements in their symptoms.

He explained that: "we know that giving plasma infusions, for example, improves symptoms, but what you often see is they’ll have an improvement for the first 10 days and then they start to feel groggy towards the end".

"What we found is, giving an anti-platelet to these patients, they tended to have much fewer symptoms for the duration, and I think personally that will end up becoming the norm for patients with congenital TTP."

Asked about whether the assay could be used in patients with acquired TTP, Dr Alwan replied: "To be honest, right now we’ve got it working in the congenitals and not working so well in the acquired patients...probably because thrombus formation is more than just ADAMTS13 and platelets."

He pointed out that patients with acquired TTP are typically thrombocytopenic and anaemic, "so lots of the factors which will go into making clots that are demonstrated in this assay won’t be there".

Dr Alwan believes that, currently, the best role for the assay in acquired TTP is in patients who go into clinical remission but have ongoing ADAMTS13 deficiency.

"What’s really interesting in these patients is that it looks like there’s patients who have low ADAMTS13 ongoing and have normal flow assays, and there’s patients who have ongoing ADAMTS13 deficiency and don’t have normal flow assays," he said.

"I think this is where it’s going to differentiate between those patients."

The recombinant ADAMTS-13 was provided by Shire, who also provided an unrestricted educational grant to fund Alwan’s PhD.

BSH 2019: Abstract BSH19-OR-033. Presented April 3.


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