Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, ...

A Cu-catalysed macrocyclisation was performed to obtain a macrocyclic coumarin-containing tripeptide for use in thrombin activity measurements.

Antithrombin I (fibrin) is an important inhibitor of thrombin generation that functions by sequestering thrombin in the forming fibrin clot, and also by reducing the catalytic activity of fibrinbound thrombin. Thrombin binding to fibrin takes place at two classes of non-substrate sites: 1) in the fibrin E domain (two per molecule) through interaction with thrombin exosite 1; 2) at a single site...

The therapeutically important anticoagulant heparin catalyzes inactivation of thrombin by antithrombin III via formation of an intermediary ternary thrombin-heparin-antithrombin III complex that is subsequently converted to a stable thrombin-antithrombin III complex with the release of heparin. Point mutations at Arg-180, Arg-245, Lys-248, and Lys-252 in thrombin markedly reduced the efficiency...

BACKGROUND The multimeric size and platelet-tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). In vitro ADAMTS-13 is susceptible to proteolytic inactivation by thrombin. OBJECTIVES In this study, we aimed to characterize the inactivation of ADAMTS-13 by th...

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproteren...

Thrombin and factor Xa (FXa) are agonists for G protein-coupled receptors (GPRCs) and may contribute to vascular lesion formation by stimulating proliferation of vascular smooth muscle cells (SMCs). Mitogenic signaling of GPCRs requires transactivation of receptor tyrosine kinases (RTKs). In rat SMCs, thrombin transactivates the epidermal growth factor receptor (EGFR) via a pathway that involve...

Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have be...

To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin s...

Thrombin and fibrinogen powders are the active components of advanced surgical hemostasis products including the EVARREST Fibrin Sealant Patch. Measuring the enzymatic activity of thrombin in the presence of fibrinogen is challenging, as hydration of the powders in a neutral aqueous environment will cause the enzyme to rapidly react with the fibrinogen to form a fibrin clot, which in turn binds...