James C. Fredenburgh

Evidence for Allosteric Linkage between Exosites 1 and 2 of Thrombin

Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH2Cl (FPR)-thrombin. Hirudin-(54–65) and sF2, a synthetic peptide corresponding to residues 63–116 of prothrombin fragment 2, were used as ligands for exosites 1 and 2 of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPR-thrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF2 and hirudin-(54–65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF2 is titrated with hirudin-(54–65), complete displacement of fluorescein-sF2 is observed. Likewise, when thrombin-bound fluorescein-hirudin-(54–65) is titrated with sF2, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.

Exosites 1 and 2 Are Essential for Protection of Fibrin-bound Thrombin from Heparin-catalyzed Inhibition by Antithrombin and Heparin Cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (γ-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 → Ala, Arg97 → Ala, and Arg101 → Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, α1-antitrypsin Met358 → Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.

Medical device-induced thrombosis: what causes it and how can we prevent it?

Blood‐contacting medical devices, such as vascular grafts, stents, heart valves, and catheters, are often used to treat cardiovascular diseases. Thrombus formation is a common cause of failure of these devices. This study (i) examines the interface between devices and blood, (ii) reviews the pathogenesis of clotting on blood‐contacting medical devices, (iii) describes contemporary methods to prevent thrombosis on blood‐contacting medical devices, (iv) explains why some anticoagulants are better than others for prevention of thrombosis on medical devices, and (v) identifies future directions in biomaterial research for prevention of thrombosis on blood‐contacting medical devices.

Selective depletion of factor XI or factor XII with antisense oligonucleotides attenuates catheter thrombosis in rabbits.

Central venous catheter thrombosis can cause venous obstruction and pulmonary embolism. To determine the extent to which catheter thrombosis is triggered by the contact or extrinsic pathway of coagulation, we used antisense oligonucleotides (ASOs) to selectively knock down factor (f)XII, fXI, or high-molecular-weight kininogen (HK), key components of the contact pathway, or fVII, which is essential for the extrinsic pathway. Knockdown of contact pathway components prolonged the activated partial thromboplastin time and decreased target protein activity levels by over 90%, whereas fVII knockdown prolonged the prothrombin time and reduced fVII activity to a similar extent. Using a rabbit model of catheter thrombosis, catheters implanted in the jugular vein were assessed daily until they occluded, up to a maximum of 35 days. Compared with control, fXII and fXI ASO treatment prolonged the time to catheter occlusion by 2.2- and 2.3-fold, respectively. In contrast, both HK and fVII knockdown did not significantly prolong the time to occlusion, and dual treatment with fVII- and fXI-directed ASOs produced a time to occlusion similar to that with the fXI ASO alone. These findings suggest that catheter thrombosis is triggered via the contact pathway and identify fXII and fXI as potential targets to attenuate this complication.

Oral Direct Factor Xa Inhibitors

Vitamin K antagonists, such as warfarin, have been the mainstay of oral anticoagulation for many decades. Although effective, warfarin has numerous limitations, including a variable dose requirement from patient to patient because of differences in dietary vitamin K intake, common genetic polymorphisms, and multiple drug interactions that affect its pharmacodynamics and metabolism. Consequently, warfarin requires frequent monitoring to ensure that a therapeutic anticoagulant effect has been achieved because excessive anticoagulation can lead to bleeding, and because insufficient anticoagulation can result in thrombosis. Such monitoring is burdensome for patients and physicians and is costly for the health care system. These limitations have prompted the development of new oral anticoagulants that target either factor Xa or thrombin. Although the path to the development of these drugs has been long, the new drugs are at least as effective and safe as warfarin, but they streamline clinical care because they can be administered in fixed doses without routine coagulation monitoring. This article focuses on rivaroxaban, apixaban, and edoxaban, the oral factor Xa inhibitors in the most advanced stages of development. After 20 years of discovery research, these agents are already licensed for several indications. Thus, the long path to finding replacements for warfarin has finally reached fruition. Therefore, development of the oral factor Xa inhibitors represents a translational science success story.

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg184, Lys185, Arg189, Arg192, Arg193, demonstrated a ∼130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates γ-thrombin at a 65-fold slower rate than α-thrombin, (b) hirudin-(54–65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation ∼100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.

Effect of nonspecific binding to plasma proteins on the antithrombin activities of unfractionated heparin, low-molecular-weight heparin, and dermatan sulfate

BACKGROUND Nonspecific binding to plasma proteins decreases the anti-factor Xa (anti-Xa) activity of unfractionated heparin (UFH) but not that of low-molecular-weight heparin (LMWH). However, plasma proteins could influence the anti-thrombin (anti-IIa) activity of LMWH. To explore this possibility, we compared the effects of plasma proteins on the anti-IIa activities of UFH and LMWH. We also examined their effects on the anti-IIa activity of dermatan sulfate (DS) because, like UFH, DS binds to plasma proteins. METHODS AND RESULTS There was almost complete recovery of anti-IIa activity when UFH, LMWH, or DS was added to plasma from each of 20 healthy volunteers. The addition of a chemically modified heparin with low affinity for antithrombin III to plasma containing UFH increased the anti-IIa activity in a concentration-dependent fashion by displacing UFH from plasma proteins. In contrast, addition of low-affinity heparin had no effect on the anti-IIa activity of LMWH. LMWH does not bind to plasma proteins because the bulk of the LMWH chains are < 6000 D, and only heparin fractions > 6000 D bind nonspecifically to plasma proteins. As further evidence that plasma proteins do not influence the anti-IIa activity of LMWH, the rate of thrombin inhibition in plasma in the presence of LMWH is virtually identical to that in buffer containing physiological amounts of the major antithrombins. In contrast, with UFH or DS, the rate of thrombin inhibition is twofold slower in plasma than in buffer. CONCLUSIONS Nonspecific binding of UFH to plasma proteins most likely contributes to the variable anti-IIa response to UFH in patients with thromboembolic disease. Although DS also binds to plasma proteins, the clinical significance of this finding is unclear. In contrast, because LMWH does not bind to plasma proteins, the anti-IIa activity of LMWH should be just as predictable as its anti-Xa activity.

Mechanism of catheter thrombosis: comparison of the antithrombotic activities of fondaparinux, enoxaparin, and heparin in vitro and in vivo

In patients undergoing percutaneous coronary intervention, catheter thrombosis is more frequent with fondaparinux than heparin. This study was undertaken to identify the responsible mechanism and to develop strategies for its prevention. Percutaneous coronary intervention catheter segments shortened plasma clotting times from 971 ± 92 to 352 ± 22 seconds. This activity is factor XII (fXII) dependent because it was attenuated with corn trypsin inhibitor and was abolished in fXII-deficient plasma. Heparin and enoxaparin blocked catheter-induced clotting at 0.5 and 2 anti-Xa U/mL, respectively, whereas fondaparinux had no effect. Addition of fondaparinux to bivalirudin or low-dose heparin attenuated catheter-induced clotting more than either agent alone. In a rabbit model of catheter thrombosis, a 70 anti-Xa U/kg intravenous bolus of heparin or enoxaparin prolonged the time to catheter occlusion by 4.6- and 2.5-fold, respectively, compared with saline, whereas the same dose of fondaparinux had no effect. Although 15 anti-Xa U/kg heparin had no effect on its own, when given in conjunction with 70 anti-Xa U/kg fondaparinux, the time to catheter occlusion was prolonged 2.9-fold. These findings indicate that (1) catheters are prothrombotic because they trigger fXII activation, and (2) fondaparinux does not prevent catheter-induced clotting unless supplemented with low-dose heparin or bivalirudin.

Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Thrombin exosite 1 binds the predominant γA/γA-fibrin form with low affinity. A subpopulation of fibrin molecules, γA/γ′-fibrin, has an extended COOH terminus γ′-chain that binds exosite 2 of thrombin. Bivalent binding to γA/γ′-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from γA/γ′-fibrin clots than from γA/γA-fibrin clots. After 24 h of washing, however, both γA/γ′- and γA/γA-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in γA/γ′-fibrin clots than they were in γA/γA-fibrin clots. This difference reflects bivalent binding of thrombin to γA/γ′-fibrin because (a) it is abolished by addition of a γ′-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the γ′-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with γA/γ′-fibrin than with γA/γA-fibrin clots. Thus, bivalent binding of thrombin to γA/γ′-fibrin protects thrombin from inhibition, raising the possibility that γA/γ′-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex ofd-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin withK d values of 13 and 0.13 μm, respectively. t-PA binds fibrin through two classes of sites withK d values of 0.05 and 2.6 μm, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with ε-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a K d of 0.15 μm. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with aK d of 5.4 μm. Lys-Pg binds to (DD)E and Fg with K d values of 0.03 and 0.23 μm, respectively. t-PA binds to (DD)E and Fg withK d values of 0.02 and 0.76 μm, respectively; interactions were eliminated with ε-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.