An activation-specific platelet inhibitor that can be turned on/off by medically used hypothermia.

OBJECTIVE: Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. METHODS AND RESULTS: An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a ß-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. CONCLUSIONS: This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.

Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene: new insights into the function of the protein.

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.