"Trojan Horse" Enzyme Delivery Could Potentially Treat Thrombotic Thrombocytopenic Purpura

Treating the blood clotting disorder thrombotic thrombocytopenic purpura (TTP) might be possible through the use of a therapeutic enzyme, according to new findings. TTP manifests as sudden and nonspecific blood clots, typically in the places like the brain, heart, pancreas, and kidneys, and can cause damage to those organs.

Investigators from the University of Alabama created this proof of concept experiment with “Trojan horse” like design in order to create a potential novel therapeutic treatment for TTP. First, a little bit of background information from the study authors. TTP is caused by a patient lacking the enzyme ADAMTS13, which normally assists the blood clotting process along with a protein called the von Willebrand factor. Missing this ADAMTS13 enzyme, which is carried in platelets, causes the micro-clots.

Symptom onset usually occurs in the middle of the night, and can include a headache, blurred vision, malaise, and abdominal pain, the investigators said. The in-hospital death rate is about 20%. Patients can die in 1 to 2 days if they are misdiagnosed, they added.

In previous work, the investigative team created transgenic mice that had human ADAMTS13 expressed in their platelets. The mice with ADAMTS13 expression blocked arterial thrombosis and also prevented TTP in those mice.

This time, the investigators took their findings 4 steps further in a multifaceted experiment using both ex vivo experiments with human blood and also in vivo experiments with mice models of TTP.

First, they isolated human platelets to observe their ADAMTS13 intake at varying temperatures and concentrations. They learned that ADAMTS13 was taken up at both 25 and 37 degrees Celsius, but not 4 C, through endocytosis, depending on the concentration of each amount.

Then, the team proved that ADAMTS13 taken up by the human platelets could remain intact and functional when interacting with von Willebrand factor. It still functioned after the investigators tested it in conditions that might cause the platelets to break, such as in simulations with microfluidic chemicals.

The “Trojan horse” method comes into play as the ADAMTS13 enzyme is taken in by the platelets, which are about 5 times bigger than the enzyme. The platelet acts as a protective delivery vehicle with the enzyme successfully smuggled into the blood.

In their third phase, the investigators used those microfluidic chemicals that simulate the arteriole flow and offers a surface for blood clotting. They showed that the additional ADAMTS13 platelets prevented in vitro thrombus formation in arterial flow in TTP patient simulations and reconstituted TTP blood simulations.

“The most surprising findings are that platelets can uptake recombinant ADAMTS13 like a sponge and properly stored it in alpha granules, which is releasable upon stimulation under flow or shear,” study leader Dr. Long Zheng told Rare Disease Report^®. “Such tiny amount of recombinant ADAMTS13 delivered by platelets to the site of vascular injury demonstrates a dramatic anti-thrombotic effect ex vivo and in vivo.”

Finally, they gave ADAMTS13 platelets via transfusion in mice that lacked the enzyme and showed that in vitro thrombus formation in abdominal arterioles was inhibited there, as well.

“These findings suggest that transfusion of platelets loaded with recombinant ADAMTS13 may be developed as a novel therapy for immune-mediated TTP, hopefully, to replace daily therapeutic plasma exchange, the current standard of care, to reduce mortality,” Dr. Zheng said.

The paper, titled “Transfusion of platelets loaded with recombinant ADAMTS13 is efficacious for inhibiting arterial thrombosis in mice and in human,” was published in the journal Arteriosclerosis, Thrombosis and Vascular Biology.