Human coagulation factor V (FV) is an essential coagulation protein with functions in both the pro- and anticoagulant pathways. Failure to express and control FV functions can either lead to bleeding, or to thromboembolic disease. Both events may develop into a life-threatening condition. Since the first description of APC resistance, and in particular the description of the so-called factor V(Leiden) mutation, in which a prominent activated protein C cleavage site in FV has been abolished through a mutation in the FV gene, FV has been in the center of attention of thrombosis research. In this review we describe how the functions of FV are expressed and regulated and provide an extensive description of the role that FV plays in the etiology of thromboembolic disease.

Most orally bioavailable drugs on the market are competitive inhibitors of catalytic sites, but a significant number of targets remain undrugged, because their molecular functions are believed to be inaccessible to drug-like molecules. This observation specifically applies to the development of small-molecule inhibitors of macromolecular interactions such as protein-membrane interactions that have been essentially neglected thus far. Nonetheless, many proteins containing a membrane-targeting domain play a crucial role in health and disease, and the inhibition of such interactions therefore represents a very promising therapeutic strategy. In this study, we demonstrate the use of combined in silico structure-based virtual ligand screening and surface plasmon resonance experiments to identify compounds that specifically disrupt protein-membrane interactions. Computational analysis of several membrane-binding domains revealed they all contain a druggable pocket within their membrane-binding region. We applied our screening protocol to the second discoidin domain of coagulation factor V and screened >300,000 drug-like compounds in silico against two known crystal structure forms. For each C2 domain structure, the top 500 molecules predicted as likely factor V-membrane inhibitors were evaluated in vitro. Seven drug-like hits were identified, indicating that therapeutic targets that bind transiently to the membrane surface can be investigated costeffectively, and that inhibitors of protein-membrane interactions can be designed.computational chemistry ͉ discoidin domain ͉ surface plasmon resonance

Human blood coagulation factor V (FV)2 is as a single-chain glycoprotein that consists of a mosaic domain structure composed of three homologous A-type domains, two smaller C-type domains, and a large B-domain (A1-A2-B-A3-C1-C2) (1). FV, which has little or no intrinsic procoagulant cofactor activity, is activated by thrombin or factor Xa (FXa) through limited proteolysis (2). Proteolytic activation of FV results in the removal of the FV B-domain and the exposure of regions in the FV molecule that are important for the expression of its procoagulant activity (3, 4). Activated FV (FVa) is a heterodimer that consists of a heavy chain (A3-A2 domains) and a light chain (A1-C1-C2 domains) that are noncovalently associated in a calcium-dependent manner (5, 6). FVa is the essential nonenzymatic cofactor of the prothrombinase complex, which upon complex formation with FXa accelerates FXa-catalyzed prothrombin activation in the presence of calcium ions and a phospholipid membrane surface by several orders of magnitude (2, 7).Although FV can be activated by a number of proteases (8, 9), thrombin is the most potent physiological activator of FV. Activation of FV by thrombin is achieved through limited proteolysis at Arg 709 , Arg 1018 , and Arg 1545 , of which the cleavage after Arg 1545 is required for full expression of FVa activity (10). Thrombin-catalyzed activation of FV follows a kinetically preferred order of bond cleavage, in which cleavage at Arg 709 occurs first, followed closely by cleavage at Arg 1018 and slow cleavage at Arg 1545 , which results in formation of the FVa light chain (11,12).A characteristic feature of thrombin are two distinct electropositive surface regions, termed exosite I and exosite II, that contribute to the specificity of thrombin by mediating the recognition of its substrates, inhibitors and receptors (13,14). Although both exosites have been implicated in FV activation (15-18), their role in the recognition of the individual thrombin cleavage sites is still unclear. In this study, we have used FV cleavage site mutants in combination with specific thrombin exosite I and II inhibitors to elucidate the importance of each exosite for the individual FV activation cleavages.Furthermore, we have used FV B-domain deletion mutants to probe the structural requirements for thrombin exosite interactions located in the B-domain and compared them with the structural requirements for FV activation by RVV-V and LVV-V, which are snake venom proteases that are known to activate FV by a single cleavage at Arg 1545 (19 -21

The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.