Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare blood clotting disorder. Currently, there is no method to assess the disease’s severity or monitor the treatment impact.
Alwan and co-workers (2021) utilized a dynamic high shear flow assay from Cellix to understand the disease better - reporting increased in vitro platelet adhesion and aggregation in cTTP patients; and additionally demonstrating significantly decreased thrombi following Plasma Infusion (PI), with levels in the normal range achieved in patients taking additional antiplatelet therapy. Here we´ll briefly discuss their main findings, highlighting how they used Cellix's VenaFlux Solutions in their study.
What is congenital thrombotic thrombocytopenic purpura (cTTP)?
Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare genetic disorder consisting of blood clot formation in the small vessels of the body. Under normal conditions, von Willebrand factor (VWF) aids the recruitment of platelets to damaged blood vessels. VWF is a large multimeric glycoprotein, which is usually cut into smaller pieces by an enzyme called ADAMTS13 - this prevents spontaneous recruitment of platelets in healthy blood vessels.
In TTP patients, the presence of antibodies inhibits normal ADAMTS13 function, blocking it from cleaving the VWF protein.
This results in an accumulation of ultra-large VWF strands which promote platelet recruitment, thereby increasing blood clot formation. The formation of these blood clots in small blood vessels (i.e. thrombosis) causes a reduction of platelets leading to a low platelet count which is known as thrombocytopenia. When these small blood vessels are damaged or burst, they give rise to blood spots known as purpura.
Symptoms: Usually develop in early childhood due to hemolytic anemia, low platelets (thrombocytopenia), and neurological dysfunction. The patient may have fatigue, paleness, shortness of breath, and a rapid heart rate. The abnormal clotting can cause headaches, confusion, seizures, and cardiovascular, renal, and gastrointestinal problems .
Treatment: Fresh frozen plasma (FFP) infusion (PI) decreases the frequency of acute events. However, evidence shows that cTTP should be treated as a chronic disease since untreated patients often report stroke and cardiac and renal sequelae . A regular prophylactic treatment decreases these morbidities .
Why use Cellix's VenaFlux platform to study TTP?
Current ADAMTS13 activity assays operate under static conditions . However, shear flow is crucial to this process as the VWF A2 domain bond cleaved by ADAMTS13 is only exposed by shear stress . Existing assays also do not consider the effect of flow on different mutations of ADAMTS13, which can influence VWF binding, or the impact of VWF levels, which affect thrombus formation [6,7].
To address these limitations, Alwan and co-workers (2021) utilized a dynamic high shear flow assay from Cellix (VenaFlux platform) to model VWF-mediated platelet adhesion and aggregation, potentially reflecting the in vivo impact in cTTP patients who describe non-overt symptoms. The aim was to understand the disease pathophysiology better and evaluate the effectiveness of current treatment response and therapeutic options.
The study included patients with cTTP under the care of a single tertiary center for one year.
Researchers used FRETS VWF-73 to measure ADAMTS13 activity, ELISA to quantify anti-ADAMTS13 IgG antibodies, and Sanger sequencing to identify genetic mutations in the samples.
Shear flow-based assays
Researchers used Cellix's VenaFlux microfluidic platform to mimic hemostatic capacity under shear flow. Citrated whole blood samples from cTTP patients was used to assess VWF adsorption to type I collagen, subsequent platelet adhesion and aggregation.
The blood samples were treated with DiOC6, a fluorescent dye, before passing through a biochip microchannel (Vena8 Fluoro+) coated with equine type I collagen at a rate of 80dynes/cm2. As blood passed over the channel under shear flow, platelet thrombi formed, allowing the measurement of the total surface coverage by thrombus, with the help of an epifluorescence microscope. VWF analysis consisted of taking composite pictures of formed thrombi highlighting VWF and platelets to compare the difference in thrombi in cTTP patients before and after treatment.
The prophylactic treatment consisted of either intermediate purity factor VIII concentrate at approximately 15–20 IU/kg given fortnightly or PI at a volume of 10 mL/kg given every 1–2 weeks depending on laboratory parameters and patient symptoms. Citrated whole blood samples were taken 30 mins before and after treatment and repeated measurements were undertaken for patients on aspirin or clopidogrel.
Platelet surface coverage
The surface coverage by thrombus formation was significantly higher in patients with cTTP than in normal controls. The pretreatment surface coverage was 89% (range: 47–100%) versus 23% in normal controls (range: 6–39%).
Also, ADAMTS13 activity levels, VWF activity levels, and VWF antigen levels influenced surface coverage.
Thrombi formed in cTTP patients were larger than those formed in controls. There was also an increase in VWF adhesion to collagen and VWF size.
The patients who received PI had a decrease in the surface coverage of thrombus formation. The pre-treatment coverage was 89% versus 16% post-treatment, as seen in the example below.
The addition of an antiplatelet agent to the regular PI prophylaxis reduced pre-treatment surface coverage further (median 50% versus 89% without antiplatelet therapy). This reduction persisted after treatment. However, all patients treated with antiplatelet combined with PI saw surface coverage return to the normal range. The antiplatelet therapy plus PI prophylaxis resolved non-overt symptoms such as headache, lethargy, and abdominal pain in 100% of patients compared to 74% with PI alone.
cTTP patients had higher VWF activity than normal controls (184% vs. 102%). VWF antigen levels were also significantly higher (123% in cTTP vs. 103% in controls). The VWF activity levels in cTTP patients did not change with PI, BPL-8Y, or antiplatelet therapy.
The researchers concluded that:
Increased in vitro platelet-VWF adhesion occurs despite normal blood counts in cTTP patients outside of acute episodes suggesting an increased potential to form microvascular thrombi.
Prophylactic therapy with ADAMTS13 rich products helps lessen thrombi, but the dose and frequency need optimizing, for which this assay, in conjunction with ADAMTS 13 assays, could tailor therapy.
Consideration should also be given to starting patients on low-dose antiplatelet agents, which were well tolerated and led to symptomatic improvement when combined with ADAMTS13 replacement therapy.
 NIH National Center for Advancing Translational Sciences. Congenital thrombotic thrombocytopenic purpura.
 Koki Mise et al. Long term follow up of congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome) on hemodialysis for 19 years: a case report. BMC Nephrol. 2013 Jul;14:156. doi: 10.1186/1471-2369-14-156
 José Barbot et al. Ten years of prophylactic treatment with fresh-frozen plasma in a child with chronic relapsing thrombotic thrombocytopenic purpura as a result of a congenital deficiency of von Willebrand factor-cleaving protease. Br J Haematol. 2001 Jun;113(3):649–51. doi: 10.1046/j.1365-2141.2001.02808.x
 Seiji Kato et al. Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity. Transfusion. 2006 Aug;46(8):1444–52. doi: 10.1111/j.1537-2995.2006.00914.x
 Weiqiang Gao et al. Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity. Blood [Internet]. 2008 Sep 1;112(5):1713–9. doi: 10.1182/blood-2008-04-148759
 Emma Kraus et al. Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13. Thromb Res. 2014 Dec;134(6):1285–91. doi: 10.1016/j.thromres.2014.08.013
 Hideo Yagi et al. Highly elevated plasma level of von Willebrand factor accelerates the formation of platelet thrombus under high shear stress in plasma with deficient ADAMTS13 activity. Thromb Res. 2017 Nov;159:91–5. doi: 10.1016/j.thromres.2017.10.007