David Gailani

Sulfated Pentagalloylglucoside Is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced V(MAX) of FXIa hydrolysis of chromogenic substrate without affecting the K(M), suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance.

Protease-activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)–stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP–stimulated platelets. PAR4-AP–mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP–mediated FV secretion and microparticle generation to PAR1-AP–mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP–stimulated platelets, compared with PAR1-AP–stimulated platelets. Rho-kinase inhibition reduced PAR4-AP–mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP–mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

Disposition of tissue factor pathway inhibitor during cardiopulmonary bypass.

Summary.u2002 Background:u2002The tissue factor (TF) factor (F) VIIa complex activates coagulation FIX and FX to initiate coagulation, and also cleaves protease activated receptors (PARs) to initiate inflammatory processes in vascular cells. Tissue factor pathway inhibitor (TFPI) is the only specific inhibitor of the TF‐FVIIa complex, regulating both its procoagulant and pro‐inflammatory properties. Upon heparin infusion during cardiopulmonary bypass (CPB), a heparin releasable pool of endothelial associated TFPI circulates in plasma. Following protamine neutralization of heparin, the plasma TFPI level decreases, but does not return completely to baseline, suggesting that during CPB a fraction of the plasma TFPI becomes heparin‐independent. We have investigated the structural and functional properties of plasma TFPI during CPB to further characterize how TFPI is altered during this procedure. Methods:u2002We enrolled 17 patients undergoing first‐time cardiac surgery involving CPB. Plasma samples were obtained at baseline, 5u2003min and 1u2003h after start of CPB (receiving heparin), 10u2003min after protamine administration (off CPB) and 24u2003h following surgery. Samples were analyzed for full‐length and free (non‐lipoprotein bound) TFPI antigen by enzyme‐linked immunosorbent assay (ELISA) and for TFPI anticoagulant activity using an amidolytic assay. Western blot analysis was used to identify TFPI species of varying molecular weights in three additional patients. Dunnetts test for post hoc comparisons was used for statistical analysis. Results:u2002The ELISA and Western blot data indicated that an increase in full‐length TFPI accounted for most of the heparin releasable TFPI. Following heparin neutralization with protamine, the full‐length TFPI antigen returned to baseline levels while the free TFPI antigen and the total plasma TFPI activity remained elevated. This was associated with the appearance of a new 38u2003kDa form of plasma TFPI identified by Western blot analysis. The 38u2003kDa form of TFPI did not react with an antibody directed against the C‐terminal region of TFPI indicating it has undergone proteolysis within this region. All TFPI measurements returned to baseline 24u2003h following CPB. Conclusions:u2002During CPB the full‐length form of TFPI is the predominant form in plasma because of its prompt release from the endothelial surface following heparin administration. Upon heparin neutralization with protamine, full‐length TFPI redistributes back to the endothelial surface. However, a new 38u2003kDa TFPI fragment is generated during CPB and remains circulating in plasma, indicating that TFPI undergoes proteolytic degradation during CPB. This degradation may result in a decrease in endothelium‐associated TFPI immediately post‐CPB, and may contribute to the procoagulant and proinflammatory state that often complicates CPB.

Discovery of allosteric modulators of factor XIa by targeting hydrophobic domains adjacent to its heparin-binding site.

To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the Vmax of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site (HBS) of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggests the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-element strategy may be widely applicable for discovering SAMs of other GBPs.

Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for triple mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants

Recent development of sulfated non-saccharide glycosaminoglycan mimetics, especially sulfated pentagalloyl glucopyranoside (SPGG), as potent inhibitors of factor XIa (FXIa) (J. Med. Chem. 2013; 56:867-878 and J. Med. Chem. 2014; 57:4805-4818) has led to a strong possibility of developing a new line of factor XIa-based anticoagulants. In fact, SPGG represents the first synthetic, small molecule inhibitor that appears to bind in site remote from the active site. Considering that allosteric inhibition of FXIa is a new mechanism for developing a distinct line of anticoagulants, we have studied SPGGs interaction with FXIa with a goal of evaluating its pre-clinical relevance. Comparative inhibition studies with several glycosaminoglycans revealed the importance of SPGGs non-saccharide backbone. SPGG did not affect the activity of plasma kallikrein, activated protein C and factor XIIIa suggesting that SPGG-based anticoagulation is unlikely to affect other pathways connected with coagulation factors. SPGGs effect on APTT of citrated human plasma was also not dependent on antithrombin or heparin cofactor II. Interestingly, SPGGs anticoagulant potential was diminished by serum albumin as well as factor XI, while it could be reversed by protamine or polybrene, which implies possible avenues for developing antidote strategy. Studies with FXIa mutants indicated that SPGG engages Lys529, Arg530 and Arg532, but not Arg250, Lys252, Lys253 and Lys255. Finally, SPGG competes with unfractionated heparin, but not with polyphosphates and/or glycoprotein Ibα, for binding to FXIa. These studies enhance understanding on the first allosteric inhibitor of FXIa and highlight its value as a promising anticoagulant.

Nucleic acids as cofactors for factor XI and prekallikrein activation: Different roles for high-molecular-weight kininogen

The plasma zymogens factor XI (fXI) and prekallikrein (PK) are activated by factor XIIa (fXIIa) during contact activation. Polyanions such as DNA and RNA may contribute to thrombosis and inflammation partly by enhancing PK and fXI activation. We examined PK and fXI activation in the presence of nucleic acids, and determine the effects of the cofactor high molecular weight kininogen (HK) on the reactions. In the absence of HK, DNA and RNA induced fXI autoactivation. Proteases known to activate fXI (fXIIa and thrombin) did not enhance this process appreciably. Nucleic acids had little effect on PK activation by fXIIa in the absence of HK. HK had significant but opposite effects on PK and fXI activation. HK enhanced fXIIa activation of PK in the presence of nucleic acids, but blocked fXI autoactivation. Thrombin and fXIIa could overcome the HK inhibitory effect on autoactivation, indicating these proteases are necessary for nucleic acid-induced fXI activation in an HK-rich environment such as plasma. In contrast to PK, which requires HK for optimal activation, fXI activation in the presence of nucleic acids depends on anion binding sites on the fXI molecule. The corresponding sites on PK are not necessary for PK activation. Our results indicate that HK functions as a cofactor for PK activation in the presence of nucleic acids in a manner consistent with classic models of contact activation. However, HK has, on balance, an inhibitory effect on nucleic acid-supported fXI activation and may function as a negative regulator of fXI activation.

Allosterism-based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα-binding and heparin-binding site of thrombin

Essentials Allosteric inhibition is a promising method for reducing bleeding risk associated with anticoagulants. Sulfated β‐O4 lignin (SbO4L) targets exosite 2 of thrombin to induce antithrombotic effects. SbO4L exhibits dual anticoagulant and antiplatelet effects without increasing tail bleeding time. This work presents a novel molecule exploiting a novel mechanism of anticoagulation.

An update on factor XI structure and function

Factor XI (FXI) is the zymogen of a plasma protease, factor XIa (FXIa), that contributes to thrombin generation during blood coagulation by proteolytic activation of several coagulation factors, most notably factor IX (FIX). FXI is a homolog of prekallikrein (PK), a component of the plasma kallikrein-kinin system. While sharing structural and functional features with PK, FXI has undergone adaptive changes that allow it to contribute to blood coagulation. Here we review current understanding of the biology and enzymology of FXI, with an emphasis on structural features of the protein as they relate to protease function.

Generation and characterization of aptamers targeting factor XIa

BACKGROUNDnThe plasma protease factor XIa (FXIa) has become a target of interest for therapeutics designed to prevent or treat thrombotic disorders.nnnMETHODSnWe used a solution-based, directed evolution approach called systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that target the FXIa catalytic domain.nnnRESULTSnTwo aptamers, designated 11.16 and 12.7, were identified that bound to previously identified anion binding and serpin bindings sites on the FXIa catalytic domain. The aptamers were non-competitive inhibitors of FXIa cleavage of a tripeptide chromogenic substrate and of FXIa activation of factor IX. In normal human plasma, aptamer 12.7 significantly prolonged the aPTT clotting time.nnnCONCLUSIONSnThe results show that novel inhibitors of FXIa can be prepared using SELEX techniques. RNA aptamers can bind to distinct sites on the FXIa catalytic domain and noncompetitively inhibit FXIa activity toward its primary macromolecular substrate factor IX with different levels of potency. Such compounds can be developed for use as therapeutic inhibitors.