Inhibition of Thrombin-Induced Platelet Aggregation Using Human Single Chain Fv Antibodies Specific for TREM-Like Transcript-1.

Platelets play a duel role in our cardiovascular system. They are important in maintaining our vascular integrity. They also facilitate the progression of cardiovascular diseases (CVD) such as arteriosclerosis. In more severe cases, displaced platelet aggregates can lead to myocardial infarction or stroke. Because of their prominent role, anti-platelet therapies are used to reduce mortality associated with CVD. However, the use of these therapies is sometimes associated with bleeding complications, thrombocytopenia or non-responsiveness and the search for new therapeutic targets continues. Some of the recent treatment strategies include molecules that target platelet membrane receptors. The success shown by this class of therapeutics has prompted us to evaluate the recently characterized murine platelet receptor, TREM like transcript 1 (TLT-1), as a therapeutic target. Murine studies suggest that TLT-1 is a platelet specific protein found in α-granules of platelets and megakaryocytes. Here we asked if human TLT-1 was also linage specific and if platelet aggregation could be modulated through interaction with TLT-1 specific antibodies. We evaluated human TLT-1 (hTLT-1) for pattern of expression in the peripheral blood and we found that hTLT-1, like its mouse counterpart, is platelet specific. To address TLT-1 function we panned a naïve human library of single chain fragments (scFvs) with a TLT-1 extracellular domain/human fc fusion protein to produce monoclonal antibodies specific for hTLT-1. We isolated six scFv clones using ELISA to detect reactivity to the fusion protein and confirmed their specificity by flow cytometry on cells transfected with TLT-1 and purified human platelets. These scFvs were then tested to see if they would disrupt or enhance platelet aggregation. Here we demonstrate, for the first time, inhibition of thrombin induced platelet activation using scFv that are specific for TLT-1. Taken together these results suggest TLT-1 is a potential target for therapeutic intervention of cardiovascular disease.