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Fragment-based lead discovery is a usual strategy in drug discovery to identify innovative lead compounds. The success of this approach strongly relies on the capacity to detect weak binders and characterize their binding site. NMR and X-ray crystallography are the conventional technologies used to tackle this challenge. However, their large protein consumption and the cost of equipment reduce their accessibility. Here, an affinity capillary electrophoresis methodology was developed that enables the detection of mM binders, the determination of dissociation constants, and the characterization of the fragment binding site. On the basis of multiple equilibrium theory, dissociation constants in the μM-mM range were determined, and a new methodology is proposed to establish graphically if two fragments bind the same protein pocket. The applicability of this methodology was demonstrated experimentally on coagulation factor XIIa by evaluating pairs of fragments with expected behavior. This study reinforces the significance of using affinity capillary electrophoresis to gather valuable information for medicinal chemistry projects.

Human coagulation factor XIIa (FXIIa) is a trypsin-like serine protease that is involved in pathologic thrombosis. As a potential target for designing safe anticoagulants, FXIIa has received a great deal of interest in recent years. In the present study, we employed virtual high-throughput screening of 500,064 compounds within Enamine database to acquire the most potential inhibitors of FXIIa. Subsequently, 18 compounds with significant binding energy (from -65.195 to -15.726 kcal/mol) were selected, and their ADMET properties were predicted to select representative inhibitors. Three compounds (Z1225120358, Z432246974, and Z146790068) exhibited excellent binding affinity and druggability. MD simulation for FXIIa-ligand complexes was carried out to reveal the stability and inhibition mechanism of these three compounds. Through the inhibition of activated factor XIIa assay, we tested the activity of five compounds Z1225120358, Z432246974, Z45287215, Z30974175, and Z146790068, with pIC50 values of9.3∗10−7,3.0∗10−5,7.8∗10−7,8.7∗10−7, and1.3∗10−6 M, respectively; the AMDET properties of Z45287215 and Z30974175 show not well but have better inhibition activity. We also found that compounds Z1225120358, Z45287215, Z30974175, and Z146790068 could be more inhibition of FXIIa than Z432246974. Collectively, compounds Z1225120358, Z45287215, Z30974175, and Z146790068 were anticipated to be promising drug candidates for inhibition of FXIIa.

Procoagulant activity of surface-immobilized coagulation factor XIIa (activated Hageman factor) is reported. Activity of FXIIa immobilized onto the surfaces of three silanized-glass procoagulants spanning a wide range of wettability was assayed in normal and FXII-deficient plasmas. Previously published mathematical models were used to characterize the procoagulant activity of protein-immobilized materials and soluble enzymes. Results show that FXIIa activity is unrelated to underlying procoagulant surface chemistry and is similar to soluble FXIIa activity. The uninfluential role of the surface on FXIIa suggests that the solid surface activates FXII in biomaterial-induced blood coagulation but is not otherwise involved in FXIIa activity as described by the classical mechanism.

Activated coagulation Factor XII (FXIIa) infusion increases peripheral resistance (TPR) and mean arterial pressure (MAP) of Brown Norway but not plasma kininogen deficient Brown Norway Katholiek (BNK) rats. FXIIa concentrations are elevated in hypertensive end-stage renal disease patients receiving conventional haemodialysis (CHD). Conversion to nocturnal haemodialysis (NHD) lowers peripheral resistance and MAP.To determine whether the plasma coagulation FXIIa-kallikrein-kinin axis contributes to the hypertension of chronic kidney disease (CKD).Plasma FXIIa and haemodynamic data were acquired in 11 CHD patients before and after 2 months of NHD. Cardiac and systemic haemodynamics of Brown Norway and BNK rats rendered hypertensive and uremic by 5/6 nephrectomy (NX) were determined before and after acute FXIIa inhibition.FXIIa was increased three-fold in CHD patients relative to control plasma (P0.05). After conversion to NHD, both ΔMAP and ΔTPR correlated with ΔFXIIa. In rats, plasma FXIIa was three-fold higher in both NX groups than respective SHAM controls (all P0.05), but MAP (147 ± 4 vs. 133 ± 2 mmHg; P0.05) and TPR (2.8 ± 0.2 vs. 2.3 ± 0.2 units; P0.05) were greater in Brown Norway NX (n = 16) than in BNK (n = 15) NX rats. FXIIa correlated with MAP only in Brown Norway NX, and plasma catecholamines were increased relative to SHAM only in Brown Norway NX (P0.05). In Brown Norway NX rats, FXIIa inhibitor infusion decreased MAP (-12 mmHg) and TPR (-0.5 Units) (both P0.05), and halved catecholamines (P0.05). No such changes occurred in BNK NX rats.FXIIa-kininogen mediated vasoconstriction contributes significantly to CKD hypertension in Brown Norway rats; this novel mechanism may be active in humans with CKD.

Plasma coagulation Factor XII is a zymogen of a trypsin-like serine protease, Factor XIIa, whose activation results in prekallikrein, factor XI, plasminogen and complement C1 activation. Factor XII has the unique ability to autoactivate when exposed to negatively charged, artificial surfaces. As a result of this phenomenon, Factor XII was thought to be an initiator of blood-plasma coagulation reactions, because when whole blood or plasma is added to a glass tube, a cascade of proteolytic reactions occurs that result in thrombin formation and the proteolysis of fibrinogen leading to clot formation. However, when factor XII deficient patients were recognized, the deficiency is not associated with bleeding. Therefore, the notion that activated forms of factor XII initiated physiologic hemostasis was dropped. Similarly, activated forms of factor XII were thought to be the initiator of activation of the plasma kallikrein/kinin system. However, recent studies showing that there is an unique prekallikrein, but not factor XII, activator associated with endothelial cells indicate that factor XII is activated secondarily to prekallikrein activation on cells …

An extended polymorphism of the coagulation factor XIIIA can routinely be detected in human plasma samples and white cell lysates by isoelectric focusing in polyacrylamide gels containing 3 M urea in the pH range 5-8. Analyses of 184 families with 513 children confirmed the formal model proposed by Suzuki et al. [Am J Hum Genet 1988;43:170-174]. Four common alleles, F XIIIA*1A, 1B, 2A, 2B, at an autosomal locus control the expression of ten phenotypes. On the basis of the population sample from southwest Germany the frequencies of the common alleles F XIIIA*1A, 1B, 2A, 2B were calculated as 0.175, 0.609, 0.011, and 0.205, respectively.

In this paper, we report a simple rapid method for simultaneous determination of Coagulation Factor XIIIA (F13A) and plasminogen (PLG) phenotypes by PAGIF with a nominal pH range of 3.5 to 10. followed by immunofixation and silver stain. Critical considerations concerning the conditions of molecular separation and detection strategies are also presented.

Recently, FXIIa was highlighted as an original attractive target for the development of new anticoagulant drugs with low rates of therapy-related hemorrhages. In this work, we describe the development of a new series of 3-carboxamide-coumarins that are the first potent and selective nonpeptidic inhibitors of FXIIa.