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To Understand the Therapeutic Target of Thrombotic Thrombocytopenia Purpura

Introduction to TTP

Thrombotic thrombocytopenic purpura (TTP) is an acute, rare blood disease characterized by microthrombosis, thrombocytopenia, mechanical hemolysis, and multiple organ damage. Its pathological mechanism usually involves the deficiency or dysfunction of a metalloproteinase called ADAMTS13. ADAMTS13 is responsible for cleaving the vWF in plasma. If the activity of ADAMTS13 is reduced, the vWF molecules will form microthrombi in the blood vessels, leading to platelet aggregation and inducing thrombocytopenia. Microthrombi can further trigger the rupture of red blood cells in the blood vessels (mechanical hemolysis), resulting in anemia.

TTP can be divided into the following according to the pathogenesis: (1) rare hereditary congenital TTP (cTTP) and TTP caused by ADAMTS13 gene mutation; (2) acquired autoimmune TTP (iTTP), caused by self-deficient ADAMTS13.

VWF Protein

VWF is a plasma glycoprotein produced by vascular endothelial cells and megakaryocytes, and plays an important role in platelet aggregation and coagulation. The VWF gene is located on chromosome 12, is about 180kb in length, and contains 52 exons and 51 introns. The VWF gene is mainly expressed in vascular endothelial cells and megakaryocytes.

The VWF protein contains the A1 domain and the A2 domain. The A1 domain binds to the platelet membrane glycoprotein Ibα (GPIbα) and participates in the platelet adhesion process; the A2 domain contains the ADAMTS13 cleavage site. VWF binds to ADAMTS13, cleaves the specific peptide bond of the A2 domain, and cleaves the super-large VWF multimer into VWF multimers with smaller molecular weight, avoiding the accumulation of super-large multimers (>20,000 kDa). Since PPT patients lack ADAMTS13, they cannot cleave VWF protein, causing VWF to recruit too many platelets in the blood and form aggregates, leading to the occurrence of microvascular thrombosis.

TTP pathogenesis-Tekbiotech.jpeg

Fig.1 TTP pathogenesis

VWF Clinical Manifestations

The most classic clinical symptoms of VWF are anemia and thrombocytopenia, but some patients will have neurological symptoms such as headache, transient cerebral ischemia, stroke and loss of consciousness. A small number of patients will have symptoms of fever, nausea, abdominal pain and mild increase in creatinine levels.

Based on clinical manifestations, it is impossible to distinguish between iTTP and hTTP, but ADAMTS13 conformation (conformation index>0.5) can be used as a basis to judge iTTP.

VWF Antibody

The global mortality rate of TTP is still high, and the targeted strategy of VWF has great prospects for the treatment of TTP. The therapeutic targets of TTP include ADAMTS13, anti-ADAMTS13 autoantibodies and VWF, among which VWF targets have attracted much attention.

Using VWF as a therapeutic strategy is a current research hotspot, which blocks the formation of microthrombi by preventing VWF from binding to platelets.

Caplacizumab, an anti-VWF aptamer, is a nano-antibody that blocks the binding of the A1 domain of VWF to platelet GPIbα, thereby preventing the formation of microthrombi. Caplacizumab has high safety and short-term efficacy.

Acetylcysteine is a reductase that changes the size of VWF polymers by reducing the disulfide bonds between polypeptide chains, resulting in a decrease in the ability to adhere to platelets, and is suitable for plasma replacement.

VWF Antibody Case

Studies have reported that natural fucoidan and hydrolyzed fucoidan have certain effects on platelet aggregation. Wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIb-deficient mouse and human platelets were designed and obtained. Natural fucoidan was hydrolyzed with hydrochloric acid and characterized by chromatography, UV-visible spectroscopy and fluorescence spectroscopy. Flow cytometry was used to detect platelet activation markers (P-selectin expression, PAC-1 and fibrinogen binding) and platelet-VWF A1 interaction. Enzyme-linked immunosorbent assay was used to evaluate GPIbα-VWF A1 interaction. Western blot was used to detect GPIb-IX-induced signal transduction.

The amino acid residues EDRLPR in the ADAMTS13 domain were mutated one by one by point mutation technology (mutants M1~M7), and transfected into human embryonic kidney HEK293 cells to observe the ability of wild-type and mutant ADAMTS13 to cleave vWF under denaturing conditions, shear stress and ADAMTS13 antibody treatment. The results showed that the wild-type and mutant ADAMTS13 were detected by Western blotting, and the relative molecular weight of wild-type and mutant ADAMTS13 was analyzed by Odyssey imaging system. Fluorescence resonance energy transfer (FRET) detection showed that the ADAMTS13 mutants M4 (R635A) and mutants M7 (R638A) had reduced FRET-vWF73 cleavage ability, indicating that there are multiple binding sites between the C-terminus of ADAMTS13 and vWF.

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