A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice.

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

A unique methionine-rich protein-aragonite crystal complex: Structure and mechanical functions of the Pinctada fucata bivalve hinge ligament.

The bivalve hinge ligament holds the two shells together. The ligament functions as a spring to open the shells after they were closed by the adductor muscle. The ligament is a mineralized tissue that bears no resemblance to any other known tissue. About half the ligament is composed of a protein-rich matrix, and half of long and extremely thin segmented aragonite crystals. Here we study the hinge ligament of the pearl oyster Pinctada fucata. FIB SEM shows that the 3D organization is remarkably ordered. The full sequence of the major protein component contains a continuous segment of 30 repeats of MMMLPD. There is no known homologous protein. Knockdown of this protein prevents crystal formation, demonstrating that the integrity of the matrix is necessary for crystals to form. X-ray diffraction shows that the aragonite crystals are more aligned in the compressed ligament, indicating that the crystals may be actively contributing to the elastic properties. The fusion interphase that joins the ligament to the shell nacre is composed of a prismatic mineralized tissue with a thin organic-rich layer at its center. Nanoindentation of the dry interphase shows that the elastic modulus of the nacre adjacent to the interphase gradually decreases until it approximates that of the interphase. The interphase modulus slightly increases until it matches the ligament. All these observations demonstrate that the ligament shell complex is a remarkable biological tissue that has evolved unique properties that enable bivalves to open their shell effectively innumerable times during the lifetime of the animal. STATEMENT OF SIGNIFICANCE: The hinge ligament shell complex is a unique functioning structural tissue whose elastic properties enable the shell to open without expending energy. Methionine-rich proteins are not known elsewhere raising fundamental questions about secondary and tertiary structures contributing to its elastic properties. The segmented and extremely thin aragonite crystals embedded in this matrix may also have unexpected elastic materials properties as they flex during compression. The structure of the interphase comprises a fascinating biological joint that connects two very different materials. The interphase materials, including the nacre, are graded with respect to elastic modulus so as to approximately match the connecting components. The interphase incorporates a thin organic rich layer that presumably functions as a gasket. This study raises many fundamental questions relevant to the diverse fields of protein chemistry, biomineralization and biological materials.

Structural and functional analyses of a TIMP and MMP in the ligament of Pinctada fucata.

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods. PfTIMP consists of 143 amino acid residues and has a molecular weight of 13,580.4. To investigate the activity of PfTIMP, inhibition of matrix metalloproteinase (MMP) activity was measured. PfTIMP strongly inhibited human MMP13 and MMP9. Eight MMP homologs were identified from a P. fucata genomic database by BLAST search. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double stranded (ds) RNA targeting MMP54089 was injected into living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.