Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substance(s) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor Vlla and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXadependent inhibition of fVIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world.These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing ofAcAP reveals no homology to any previously identified anticoagulant or protease inhibitor.Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic 14 for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K d ϳ1 nM) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K d ‫؍‬ 260-500 nM) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.The proteolytic activation of prothrombin is catalyzed by the prothrombinase complex of coagulation (2-5). Prothrombinase assembles through membrane-dependent interactions between the serine proteinase, factor Xa, and the protein cofactor, factor Va (2, 3). Although solution-phase Xa is a competent enzyme, its incorporation into prothrombinase yields a profound increase in the rate of thrombin formation (2, 3).Prothrombin is the only known protein substrate cleaved efficiently by prothrombinase (2, 6). Such stringent selectivity is not evident in the action of factor Xa on oligopeptidyl substrates, nor is the rate of peptidyl substrate hydrolysis significantly enhanced upon assembly of factor Xa into prothrombinase (7,8). Thus, the narrow and defined specificity of prothrombinase toward its protein substrate is unlikely to be solely explained by the specific recognition of residues surrounding the scissile bond by the active site of factor Xa within the enzyme complex.Mechanistic studies of bovine prothrombinase function have borne out this suggestion (9 -11). A series of studies indicate that recognition of the biological substrate is achieved through stepwise interactions of the protein substrate at an extended macromolecular recognition site (exosite) in prothrombinase removed from the catalytic site of Xa, followed b...

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa⅐TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa⅐TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K D ‫؍‬ 1-10 nM), and similarly supported the inhibition of fVIIa⅐TF (K i * ‫؍‬ ϳ10 pM). The roles of phospholipid membrane composition in the inhibition of fVIIa⅐TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF S ), or reconstituted into membranes, containing phosphatidylcholine (TF PC ) or a mixture of phosphatidylcholine and phosphatidylserine (TF PCPS ). In the absence of the fX derivative, inhibition of fVIIa⅐TF was similar for all three conditions (K i ϳ1 M), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF S , TF PC , and TF PCPS . The removal of the ␥-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa⅐TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa⅐TF⅐fX(a) complex assembled on an appropriate phospholipid surface.The blood coagulation response to vascular injury or inflammation results from a series of amplified reactions, in which several specific zymogens of serine proteases in plasma are sequentially activated by limited proteolysis (1). The serine protease factor VIIa (fVIIa) 1 present in the blood specifically binds to tissue factor (TF), a transmembrane receptor glycoprotein bound to subendothelial structures or present on the surface of monocytic or other inflammatory cells, which accumulate at the site of injury (2). The exposure of TF to circulating blood is the triggering event that results in the formation of a catalytic complex (fVIIa⅐TF) that initiates the amplified cascade of proteolytic events leading to the formation of the serine protease thrombin (3). The action of thrombin coupled with the particular rheological environment found in diseased or damaged vascular beds, result in thrombi with compositions that vary from platelet-rich, a characteristic of the arterial vasculature, to fibrin-rich, platelet-poor clots, typical of the venous vasculature (4).The pathway leading from the formation of the fVIIa⅐TF complex to thrombin proceeds through the serine protease factor Xa (fXa). Factor Xa is formed by the proteolytic activation of the zymogen factor X (fX) either by the fVIIa⅐TF complex or by the catalytic complex composed of the serine protease factor IXa and its nonenzymatic cof...

The interaction of factor Xa with factor Va on a membrane surface results in the assembly of the prothrombinase complex. The highly specific and multistep interaction between recombinant tick anticoagulant peptide (rTAP) and factor Xa was used to probe perturbations in the macromolecular interaction sites of factor Xa that accompany prothrombinase assembly. Steady-state kinetic studies indicated that the incorporation of factor Xa into prothrombinase resulted in a modest 3-fold increase in the rate constant for inhibition by rTAP. However, the overall dissociation constant for the enzyme-inhibitor interaction (Ki*) was decreased approximately 30-fold to 5.3 pM. This finding was verified by fluorescence stopped-flow studies of the multistep reaction between rTAP and solution-phase factor Xa or prothrombinase by using 4-aminobenzamidine. The second-order rate constant for the binding or rTAP to the protease (k + 1 = 3.35 x 10(6) M-1.s-1) was increased approximately 2-fold (k + 1 = 6.6 x 10(6) M-1.s-1) following the assembly of prothrombinase, while the rate constant for the subsequent slow displacement of the fluorophore from the active site of factor Xa was decreased by 20-fold. Therefore, factor Va alters macromolecular interaction sites on factor Xa; which leads to the stabilization of intermediates in the reaction of the protease with rTAP and an increased overall affinity for the inhibition of factor Xa. Fluorescence measurements of prothrombinase assembly using factor Xa modified with dansylglutamylglycinylarginine chloromethyl ketone (DEGR-Xa) indicated that the preformed rTAP-Xa binary complex bound to factor Va more tightly (Kd = 30.7 +/- 6.2 pM) than factor Xa alone (Kd = 1.25 +/- 0.29 nM). The 30-fold higher affinity of the rTAP-Xa complex for factor Va can completely account for the increased affinity of rTAP for prothrombinase and implies adequate thermodynamic description of the reactions involved. Collectively, the data suggest that the interaction of factor Xa with factor Va on a membrane surface alters macromolecular recognition sites on factor Xa involved in binding rTAP. As a result of this conformational change, the inhibition of factor Xa by rTAP is thermodynamically favored when the enzyme is assembled in the prothrombinase complex.