Sriram Krishnaswamy

Exosite-driven substrate specificity and function in coagulation

Summary.  Macromolecular substrate recognition and serine proteinase specificity lie at the heart of the tightly regulated hemostatic response. Mechanisms established for the less specific serine proteinases of digestion have played a dominant role in guiding investigations of the basis for the narrow specificities exhibited by the coagulation enzymes. These concepts have also dominated the development of specific inhibitors of coagulation for therapeutic purposes. Studies of the enzymology and physical biochemistry of prothrombinase challenge these prevailing ideas by establishing a principal role for exosites within the enzyme in determining substrate recognition and directing the action of the enzyme on its biological substrate. Mechanisms by which narrow protein substrate specificity is achieved by prothrombinase also apply to several other reactions of coagulation. These strategies are increasingly evident in the action of other families of enzymes that act with high specificity on protein substrates. Exosite‐driven enzymic function probably represents a widely employed biological strategy for the achievement of high macromolecular substrate specificity.

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of ≈10, the rate of specific cleavage at the Tyr1605–Met1606 bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by ADAMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the high-affinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.

Regions Remote from the Site of Cleavage Determine Macromolecular Substrate Recognition by the Prothrombinase Complex

The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, α-thrombin, was a linear competitive inhibitor of prethrombin 2 activation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and α-thrombin compete for binding to the exosite without restricting access to the active site of factor Xa within prothrombinase. Inhibition by α-thrombin was not altered by saturating concentrations of low molecular weight heparin. Furthermore, proteolytic removal of the fibrinogen recognition site in α-thrombin only had a modest effect on its inhibitory properties. Both α-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp148 and separated into component domains. The C-terminal-derived ζ2 fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the ζ1 fragment was without effect. As the ζ2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the fibrinogen recognition or heparin-binding sites, and spatially removed from structures surrounding the scissile bond.

Role of the Activation Peptide Domain in Human Factor X Activation by the Extrinsic Xase Complex

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1–52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase γ (ARHγ), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1–49 and a mixture of Xdes 1–34 and Xdes 1–43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russells viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1–49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1–49 of the activation peptide or with carbohydrate structures attached to these residues.

New insights into the spatiotemporal localization of prothrombinase in vivo.

The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological milieu to affect hemostatic plug formation remains unaddressed. We used confocal fluorescence microscopy to image the distribution of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster arteriole. These proteins bound to the injury site extend beyond the platelet mass to the surrounding endothelium. Although bound FVa and FXa may have been present on the platelet core at the nidus of the injury, bound proteins were not evident on platelets adherent even a small distance from the injury site. Manipulations to drastically reduce adherent platelets yielded a surprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation. Thus, platelets adherent to the site of vascular injury do not play the presumed preeminent role in supporting prothrombinase assembly and thrombin formation. Rather, the damaged/activated endothelium and possibly other blood cells play an unexpectedly important role in providing a procoagulant membrane surface in vivo.

Extrinsic pathway proteolytic activity

Publisher Summary This chapter outlines the methodology for studying the catalytic properties of the human extrinsic “Xase” complex with respect to its biological substrates, human factors IX and X, and a synthetic fluorescent substrate for factor VIIa. It discusses the reconstitution of tissue factor (TF) apoprotein into membranes and describes the factor IX activation. Functional TF can be reconstituted into mixed detergent–phospholipid micelles by incubating TF with preformed small unilamellar vesicles composed of phosphatidylcholine (75%) and phosphatidylserine (PCPS) (25%) in the presence of calcium ions and Tween 80. Detergent dialysis techniques are modified to incorporate TF into PCPS membranes. These procedures rely on the removal of octylglucoside at a controlled rate from detergent-solubilized phospholipid solutions in the presence of TF. Factor X activation is assessed by the increase in factor Xa-specitic amidolytic activity following the quenching of extrinsic Xase activity with ethylenediaminetetraacetic acid (EDTA).

Exosites determine macromolecular substrate recognition by prothrombinase.

The prothrombinase complex, composed of factor Xa and factor Va assembled on a membrane surface, catalyzes the proteolytic formation of thrombin during blood coagulation. The molecular basis for the macromolecular substrate specificity of prothrombinase is poorly understood. By kinetic studies of prethrombin 2 cleavage by prothrombinase in the presence or absence of fragment 1.2, we show that occupation of the active site of the catalyst by inhibitors or alternate peptidyl substrates does not alter the affinity for prethrombin 2. Productive recognition of the macromolecular substrate therefore results from an initial interaction at enzymic sites (exosites) distinct from the active site, which largely determines substrate affinity. This interaction at exosites is evident even in the absence of activation peptide domains responsible for mediating the binding of the substrate to membranes or factor Va. Interactions at the active site with structures surrounding the scissile bond then precede bond cleavage and product release. The second binding step, which appears unfavorable, does not affect substrate affinity but contributes to the maximum catalytic rate. Therefore, binding specificity of prothrombinase for the macromolecular substrate is determined by exosites on the enzyme. We show that competitive inhibition of prethrombin 2 cleavage can be accomplished by interfering with the exosite binding step without obscuring the active site of the enzyme. These findings suggest limitations to the common approach of inferring the basis of factor Xa specificity with active site mutants or the targeting the active site of factor Xa with reversible inhibitors for therapeutic purposes. The achievement of distinctive macromolecular substrate specificities through exosite interactions and modulation of maximum catalytic rate through binding steps may also underlie the reactions catalyzed by the other coagulation complexes containing trypsin-like enzymes.

The transition of prothrombin to thrombin.

The proteolytic conversion of prothrombin to thrombin catalyzed by prothrombinase is one of the more extensively studied reactions of blood coagulation. Sophisticated biophysical and biochemical insights into the players of this reaction were developed in the early days of the field. Yet, many basic enzymological questions remained unanswered. I summarize new developments that uncover mechanisms by which high substrate specificity is achieved, and the impact of these strategies on enzymic function. Two principles emerge that deviate from conventional wisdom that has otherwise dominated thinking in the field. (i) Enzymic specificity is dominated by the contribution of exosite binding interactions between substrate and enzyme rather than by specific recognition of sequences flanking the scissile bond. Coupled with the regulation of substrate conformation as a result of the zymogen to proteinase transition, novel mechanistic insights result for numerous aspects of enzyme function. (ii) The transition of zymogen to proteinase following cleavage is not absolute and instead, thrombin can reversibly interconvert between zymogen‐like and proteinase‐like forms depending on the complement of ligands bound to it. This establishes new paradigms for considering proteinase allostery and how enzyme function may be modulated by ligand binding. These insights into the action of prothrombinase on prothrombin have wide‐ranging implications for the understanding of function in blood coagulation.

Ligand Binding Shuttles Thrombin along a Continuum of Zymogen- and Proteinase-like States

The critical and multiple roles of thrombin in blood coagulation are regulated by ligands and cofactors. Zymogen activation imparts proteolytic activity to thrombin and also affects the binding of ligands to its two principal exosites. We have used the activation peptide fragment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogenicity of thrombin by isothermal titration calorimetry. We show that F12 binding is sensitive to subtle aspects of proteinase formation beyond simply reporting on zymogen cleavage. Large thermodynamic differences in F12 binding distinguish between a series of thrombin species poised along the transition of zymogen to proteinase. Active-site ligands transitioned a zymogen-like state to a proteinase-like state. Conversely, removal of Na+ converted proteinase-like thrombin to a more zymogen-like form. Thrombin mutants, with deformed x-ray structures, previously considered to be emblematic of specific regulated states of the enzyme, are instead shown to be variously zymogen-like and can be made proteinase-like by active-site ligation. Thermodynamic linkage between anion-binding exosite 2, the Na+-binding site, and the active site arises from interconversions of thrombin between a continuum of zymogen- and proteinase-like states. These interconversions, reciprocally regulated by different ligands, cast new light on the problem of thrombin allostery and provide a thermodynamic framework to explain the regulation of thrombin by different ligands.

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.