Inhibits Coagulation Factor Xa Research Articles

A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism

AbstractInhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all β-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo–electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Preparation, Characterization and Ex vivo Permeability Study of Transdermal Apixaban O/W Nanoemulsion Based Gel

This study designed to prepare ultrafine apixaban (APX) o/w nanoemulsion (NE) based gel with droplet size below 50 nm as a good method for transdermal APX delivery without using permeation enhancer, alternatively, the formulation components itself act as permeation enhancer. APX, a potent oral anticoagulant drug that selectively and directly inhibit coagulation factor Xa, was selected as a good candidate for transdermal delivery as it displays poor water solubility (0.028 mg/mL) and low bioavailability (50%). APX-NE gel was prepared using triacetin, triton-x-100 and carbitol as oil phase, surfactant and cosurfactant respectively, while Carbopol 940 used as a gelling agent. Ex vivo permeation of APX-NE gel through human stratum corneum reveal

Apixaban Ultrafine O/W Nano Emulsion Transdermal Drug Delivery System: Formulation, In Vitro and Ex Vivo Characterization -

Apixaban (APX) is a potent oral anticoagulant drug that directly inhibit coagulation factor Xa for prevention of venous thromboembolism (VTE) following total hip or knee replacement surgery. Orally APX has poor water solubility (0.028 mg/mL) and relative low bioavailability (50%). Transdermal APX delivery was utilized as a convenient alternative route to control oral limitations. This study designed to formulate ultrafine APX o/w nanoemulsion with self-permeation enhancing properties through skin barrier utilizing the ultrafine (> 50 nm) nanosized droplets as well as nanoemulsion components themselves to act as a permeation enhancer. Solubility study result in selecting triacetin oil, triton-x-100 and carbitol as oil phase, surfactant and cosurfactant respectively, while pseudoternary phase diagram construct nanoemulsion area for choosing formulations. Twenty-one o/w nanoemulsions prepared and characterized for droplet size, pH values, percent transmittance, electroconductivity, APX content, in vitro APX release, and ex vivo permeation through Albino Wistar rat abdominal skin to simulate human skin. Among formulations, ten preparations demonstrate ultrafine APX o/w nanoemulsions with high percent transmittance and electroconductivity, pH values appropriate for skin application, ultrafine droplet sizes (> 50 nm) and accepted APX content. In vitro release studies reveal significant (p ≤ 0.05) increase in APX dispersibility and diffusion through dialysis membrane. Ex vivo APX permeation through rat abdominal skin was significantly (p ≤ 0.05) increased in comparison with pure drug as assured by significant (p ≤ 0.05) enhancement in permeation parameters Jss, KP and ER with shorter Tlag, which could be attributed to permeation enhancing properties of nanoemulsion formulation itself.

How can we reverse bleeding in patients on direct oral anticoagulants?

The direct oral anticoagulants (DOACs), or non-vitamin K antagonist oral anticoagulants (NOACs), including dabigatran, which inhibits thrombin, as well as rivaroxaban, apixaban, edoxaban, and betrixaban, which inhibit coagulation factor Xa, are as-sociated with similar or lower risk of bleeding compared with warfarin. The need for reversal of their anticoagulant effect may occur in patients with life-threatening bleeding or those requiring urgent surgery. Currently, the only specific reversal agent for dabigatran, idarucizumab, is widely available, while andexanet alfa, which reverses factor Xa inhibitors, was approved in the United States in May 2018. Ciraparantag, which has been designed to reverse all DOACs and other anticoagulants, is being investigated in clinical trials. In the absence of licensed reversal agents for the oral factor Xa inhibitors, prothrombin complex concentrates are suggested in patients with life-threatening bleeding. Vitamin K and fresh frozen plasma should not be used to reverse DOACs. This review presents the current evidence regarding bleeding risk on DOACs and the reversal strategies to provide guidance on the management of patients treated with DOACs, who experience serious bleeding.

Reversal agents for non-vitamin K antagonist oral anticoagulants.

The non-vitamin K antagonist oral anticoagulants (NOACs) include dabigatran, which inhibits thrombin, and apixaban, betrixaban, edoxaban, and rivaroxaban, which inhibit coagulation factor Xa. Although clinical studies of NOACs were conducted without antidotes, patient outcomes with major bleeding when receiving NOACs were no worse than those in patients treated with a vitamin K antagonist. Nonetheless, in patients with life-threatening bleeding or requiring urgent surgery, the capacity for rapid NOAC reversal is likely to increase patient safety. Three NOAC reversal agents are in various stages of development: idarucizumab, a specific reversal agent for dabigatran; andexanet alfa, which reverses factor Xa inhibitors; and ciraparantag, which is purported to reverse all NOACs. Idarucizumab is licensed in many countries, andexanet is under consideration by regulatory agencies, and ciraparantag is undergoing phase III evaluation. In the absence of licensed reversal agents for the oral factor Xa inhibitors, prothrombin complex concentrates are often used in patients taking these agents who present with life-threatening bleeding. In this Review, we summarize the approved indications for the NOACs, outline how to measure their anticoagulant effects, describe the mechanism of action of the reversal strategies, assess the preclinical and clinical data supporting their use, provide guidance on potential indications for reversal, and offer a management approach for patients treated with NOACs who present with serious bleeding or require urgent surgery.

Zymogen-like FXa Is a Potent Bypassing Agent for Reversal of Direct FXa Inhibitors in Vivo

The pharmacokinetic challenges of warfarin therapy have led to the development of new oral anticoagulants (NOACs) that directly inhibit coagulation factor Xa (FXa). While these new drugs (including rivaroxaban and apixaban) have many benefits over warfarin, no approved strategy exists to reverse their anticoagulant effects in the event of life-threatening bleeding or emergent need for surgery. The most promising reversal strategy in clinical development is a catalytically inactive form of FXa (Gla-domainless FXaS195A, GD-FXaS195A) that binds the NOAC with high affinity to relieve inhibition of endogenous FXa. In principle, removing the inhibitor through molecular engagement is attractive, especially when administered before bleeding begins (e.g. pre-surgery), and there is in vivo evidence to support the efficacy of GD-FXaS195A as a pre-injury antidote for direct FXa inhibitors. However, since GD-FXaS195A is a scavenger of the inhibitor and not a pro-hemostatic agent, GD-FXaS195A may not be effective if given after a bleeding episode has begun. Moreover, its mechanism of action necessitates a 1:1 ratio of GD-FXaS195A to neutralize the inhibitor. Thus, high doses (hundreds of milligrams of protein) will likely be required in humans.We hypothesized that a pro-hemostatic bypassing agent might be able to reverse the effects of direct FXa inhibitors no matter when it is administered, and with high potency. To evaluate this, we used a variant of FXa (FXaI16L) that is more zymogen-like than wild-type (wt)-FXa. This “zymogen-like” variant has lower catalytic activity in in vitro assays compared to wt-FXa due to impaired active site maturation. It is also resistant to active site inhibitors including plasma protease inhibitors, resulting in an extension of its plasma half-life (>30 minutes vs ~1 minute for wt-FXa). Importantly, its activity is rescued upon binding to its cofactor FVa in vivo, and we have previously shown that FXaI16L bypasses the intrinsic pathway defect in hemophilic mice. Here we evaluated whether FXaI16L might also be able to bypass direct FXa inhibitors using in vitro thrombin generation assays (TGAs) and two in vivo injury models in mice, and directly compared FXaI16L to GD-FXaS195A. In TGA experiments, 500 nM rivaroxaban decreased peak thrombin generation to 23% of that observed in normal human plasma (NHP). This decreased thrombin generation could be reversed by the addition of 3 nM human (h)FXaI16L which normalized peak thrombin generation. In comparative studies, hFXaI16L was 300-fold more potent than GD-FXaS195A in TGA assays.These data highlight that differences in mechanism of action (pro-hemostatic vs. scavenger) will have a huge impact on amounts of protein needed to revive thrombin generation. This was further reflected in in vivo studies. In invivo experiments with wild-type mice using 7.5% FeCl3 to induce carotid artery thrombosis, 1 mg/kg rivaroxaban prevented formation of occlusive thrombi. 30 minutes after the initial FeCl3 injury to rivaroxaban-treated mice, infusion of 0.25 mg/kg murine (m)FXaI16L normalized the phenotype, causing rapid occlusion at the injury site within 5 minutes. In contrast, we did not observe carotid artery occlusion with administration of up to 25 mg/kg GD-FXaS195A 30 minutes after the injury to rivaroxaban-treated mice (Fig. 1). When both rivaroxaban and the reversal agent were given prior to the injury, both mFXaI16L (0.5 mg/kg) and GD-FXaS195A (25 mg/kg) were effective at reversing the inhibition. Furthermore, using intravital microscopy to examine thrombus formation at the site of laser injury to mouse cremasteric arterioles, we found that 1 mg/kg rivaroxaban completely abrogated fibrin deposition and substantially reduced platelet accumulation. Treatment with1 mg/kg mFXaI16L substantially increased platelet and fibrin deposition at the site of laser injury. [Display omitted] Taken together, these data provide strong support for the in vivo efficacy and potency of FXaI16L as a potential pro-hemostatic bypass agent to reverse direct FXa inhibitors. The data also illustrate that antidotes like GD-FXaS195A may not be as effective as pro-hemostatic agents like FXaI16L following an injury, and we predict that effective reversal of NOACs will require a nuanced approach that uses an antidote or a bypassing agent depending on the clinical scenario. DisclosuresJasuja:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding.

Rivaroxaban in der Prävention und Therapie thromboembolischer Erkrankungen

Rivaroxaban (Xarelto(®)) is a new anticoagulant for the prevention and treatment of thromboembolic disorders. Rivaroxaban inhibits coagulation factor Xa directly, has high oral bioavailability, shows low propensity for drug-drug interactions and requires no routine coagulation monitoring. In patients undergoing elective knee or hip replacement surgery rivaroxaban (10 mg/d) is highly effective to prevent venous thromboembolism. In patients with non-valvular atrial fibrillation rivaroxaban (20 mg/d) has been approved to prevent stroke or systemic embolism. The favourable benefit-risk profile of rivaroxaban in the treatment of deep vein thrombosis (DVT) was shown in EINSTEIN-DVT and led to its clinical approval (twice daily 15 mg for 3 weeks, followed by 20 mg/d). Based on ATLAS-ACS-TIMI-51 which has shown that rivaroxaban (2.5 mg twice daily) reduced thrombotic cardiovascular events and mortality in patients with a recent acute coronary syndrome, the approval of low dose rivaroxaban has been submitted for this indication. Taken together, rivaroxaban may become an effective alternative to standard anticoagulants in the prevention and treatment of thromboembolic disorders.

Effect of New Direct Oral Anticoagulants on Ex Vivo Platelet and Fibrin Thrombus Formation in Blood Flowing Over Fibrillar Type I Collagen

Abstract 2318 Background.Studies have shown that 40–85% of patients undergoing total knee replacement develop venographically confirmed deep vein thrombosis (DVT) if not given post-operative thromboprophylaxis; approximately 0.1 to 1.7% of these patients will suffer fatal pulmonary embolism (PE). Oral anti-vitamin K anticoagulants are effective for the prevention and treatment of venous thrombosis, but have limitations. In particular, they have multiple food and drug interactions as well as variable pharmacokinetics and pharmacodynamics, such that regular laboratory monitoring and dose adjustments are required to maintain an optimal therapeutic range as defined with the International Normalized Ratio (INR). New oral agents that inhibit coagulation factor Xa or thrombin have been developed and shown to be effective and safe without requiring laboratory monitoring. In view of the relevance of the latter point, we have studied patients treated with an oral anti factor Xa agent (Rivaroxaban) or Coumadin, and evaluated the antithrombotic efficacy of the respective drugs by measuring platelet aggregation and fibrin deposition in patient blood perfused over fibrillar collagen type I. Material and Methods. Blood drawn from an antecubital vein and containing 0.011 M trisodium citrate as anticoagulant was recalcified with 5 mM calcium chloride and immediately perfused through a rectangular chamber mounted on the stage of a confocal microscope and presenting a surface coated with fibrillar collagen type I under laminar flow conditions at the wall shear rate of 300 1/s. Platelets and fibrin were specifically detected in situ through distinct fluorochromes. We tested 8 normal controls, 8 patients treated with Coumadin and a stable INR value between 1.94 and 2.90 (mean 2.34; standard deviation 0.34), and 7 patients treated with Rivaroxaban at between 8 and 16 days (mean 12.14; standard deviation 2.48 days) from the initiation of therapy. The volume of platelet aggregates and fibrin deposited onto the collagen fibrils was measured distinctly from stacks of confocal sections by integrating surface coverage of each thrombus component in consecutive optical planes separated by 2 micrometers in height. Results and Discussion. There was no significant difference in the volume of platelet and fibrin aggregates formed in blood of normal control and patients treated with either Coumadin or Rivaroxaban. This result was surprising because the patients treated with Coumadin had a laboratory demonstration of significantly retarded coagulation. We reasoned, however, that coagulation tests are typically performed in platelet-poor plasma, while in the perfusion assay coagulation occurs in whole blood and on a surface onto which flowing platelets are fully activated, thus increasing the local procoagulant potential. For this reason, we performed a series of experiments in which a variable amount of a highly specific thrombin inhibitor, lepirudin, was titrated into the recalcified blood before perfusion. We thus determined that with 50 nM lepirudin added to blood there was no decrease in the volume of platelet aggregates and fibrin deposited onto collagen in blood of normal individuals, while the volume of fibrin was decreased in patients receiving either Coumadin or Rivaroxaban. The corresponding values for normal controls, Coumadin-treated and Rivaroxaban-treated patients, in the order, were (mean volume ± standard error of the mean in cubic micrometers): Platelet aggregates = 28,592±3,354; 36,959±4,973; 44,448±7,110; Fibrin = 84,190±9,740; 47,298±7,308; 35,780±5,091. The differences in platelet aggregate volumes were not significant, while fibrin volume was significantly smaller in the anticoagulant-treated patients as compared to normal (p<0.01 for Coumadin and p<0.001 for Rivaroxaban); the difference between patients treated with one or the other anticoagulant was not significant. These results show that Rivaroxaban and Coumadin at therapeutically effective dosage have comparable effect in reducing thrombin generation, as evidenced by the reduced volume of fibrin formed in flowing blood exposed to collagen. This, however, is accompanied by an increased volume of platelet aggregates on the highly thrombogenic collagen surface. The relevance of these experimental results with respect to prevention of arterial as opposed to venous thrombosis deserves further investigation. Disclosures:No relevant conflicts of interest to declare.

Ancylostoma ceylanicum anticoagulant peptide-1: role of the predicted reactive site amino acid in mediating inhibition of coagulation factors Xa and VIIa.

Hookworm infection is a leading cause of gastrointestinal blood loss and iron deficiency anemia in developing countries. Ancylostoma hookworms secrete potent anticoagulants, which have been shown to target coagulation factors Xa and the factor VIIa/Tissue Factor complex, respectively. The goal of these experiments was to determine the mechanism of action of three recombinant hookworm anticoagulants using in vitro assays. Three hookworm coagulation inhibitors were expressed and purified, along with site directed mutants targeting each of the predicted P1 inhibitory reactive site amino acid residues. Using chromogenic assays, it has been confirmed that Ancylostoma caninum Anticoagulant Peptide 5 (AcAP5) inhibits coagulation factor Xa (fXa) by a canonical, substrate-like mechanism. In contrast, Ancylostoma ceylanicum Anticoagulant Peptide-1 (AceAP1) binds to and inhibits fXa by both active site and non-active site mediated interactions. Data from in vitro studies also demonstrates that AceAP1 inhibits the factor VIIa/Tissue complex (fVIIa/TF) and displays a distinct pattern of fXa binding. Together, these data suggest that the human hookworm A. ceylanicum has evolved a single anticoagulant that targets multiple components of the mammalian coagulation response, effectively mimicking the concerted action of the two related inhibitors from A. caninum. Despite the amino acid sequence similarity, AceAP1 appears to interact with coagulation proteases fXa and fVIIa by a novel mechanism, perhaps explaining its spectrum of inhibitory activity.

FONDAPARINUX (ARIXTRA®): A NEW ANTICOAGULANT

SUMMARYFondaparinux is a promising new antithrombotic agent. This pentasaccharide selectively and specifically inhibits coagulation factor Xa, and requires antithrombin as co‐factor. It is entirely synthetic, in contrast to conventional heparin and low molecular heparins which are derived from animal tissues. Fondaparinux exhibits a high bioavailability and is convenient to use as it only needs to be given once daily by subcutaneous injection. Peak plasma levels are achieved within two hours of dosing and the plasma half‐life of fondaparinux is approximately 17 hours. There is no specific antidote for fondaparinux: it is not neutralised by protamine sulphate. Fondaparinux shows no cross‐reactivity with antibodies associated with heparin‐induced thrombocytopenia. Several randomised, double‐blind studies have demonstrated superiority with respect to a low molecular weight heparin (enoxaparin) in preventing venous thromboembolism in the setting of orthopaedic surgery. The results of clinical trials of fondaparinux in the treatment of deep vein thrombosis and acute coronary syndrome are also presented.

The synthetic pentasaccharide fondaparinux: first in the class of antithrombotic agents that selectively inhibit coagulation factor Xa.

Fondaparinux (Arixtra), a synthetic pentasaccharide, is the first in a new class of antithrombotic agents that selectively inhibit coagulation factor Xa. In vitro experiments demonstrated that it is a selective inhibitor of factor Xa. In plasma, fondaparinux selectively binds to antithrombin, catalyzes factor Xa inhibition, and thereby inhibits thrombin generation. Its antithrombotic efficacy has been demonstrated in various animal models mimicking venous and arterial thrombosis. In humans, its pharmacokinetic profile is favorable, with a rapid onset of antithrombotic activity and an elimination half-life allowing a convenient once-daily dosing regimen. In several clinical trials, fondaparinux was more effective than the reference drug, enoxaparin, in preventing venous thromboembolism after hip fracture, major knee, and elective hip replacement surgeries. The overall reduction in the risk of venous thromboembolism ranged between 26.3 and 56.4%, depending on the trial. This superior efficacy was achieved without increasing the risk of clinically relevant bleeding. Fondaparinux also showed promising results in the treatment of patients with venous thromboembolism and acute coronary syndromes. Thus, it is now established that selective factor Xa inhibition is an efficient way to prevent venous thrombosis. The advent of fondaparinux offers an opportunity to improve substantially the management of venous thromboembolism.

Recombinant full-length tissue factor pathway inhibitor fails to bind to the cell surface: implications for catabolism in vitro and in vivo

AbstractTissue factor pathway inhibitor (TFPI) plays a key role in the regulation of tissue factor-initiated blood coagulation secondary to loss of the integrity of the blood vessel wall. TFPI is a naturally occurring Kunitz-type protease inhibitor that inhibits coagulation factor Xa and, in a factor Xa-dependent manner, mediates feedback inhibition of the factor VIIa/tissuefactor catalytic complex. In vivo full-length TFPI is thought to be primarily bound to the vascular endothelium and the high affinity binding requires an intact carboxy terminus. Here we describe a full-length TFPI molecule, expressed in mouse C127 cells (TFPIC127), which exhibits virtually no cellular binding yet contains the intact carboxy terminus. This TFPI (TFPIC127) is neither internalized nor degraded via the TFPI endocytic receptor, LDL-receptor–related protein. Pharmacokinetic studies of TFPIC127 in vivo demonstrate a 10-fold prolongation in the plasma half-life, compared with that of bacterial recombinant TFPI.

The Low Density Lipoprotein Receptor-related Protein Can Function Independently from Heparan Sulfate Proteoglycans in Tissue Factor Pathway Inhibitor Endocytosis

Tissue factor pathway inhibitor (TFPI) is a plasma serine protease inhibitor that directly inhibits coagulation factor Xa and regulates blood coagulation via inhibition of factor VIIa-tissue factor enzymatic activity. We previously demonstrated that >90% of TFPI bound to a single population of low affinity binding sites on hepatoma cells (2 x 10(6) sites/cell, Kd = 30 nM), and, that following binding, the low density lipoprotein receptor-related protein (LRP) mediated TFPI uptake and degradation. We subsequently reported heparan sulfate proteoglycans (HSPGs) constitute a second receptor system involved in TFPI catabolism. In the present study, mouse embryonic fibroblasts heterozygous and homozygous-negative for disruption of the LRP gene were used to further examine the roles of LRP and HSPGs in TFPI endocytosis. We demonstrate that LRP is absolutely required for degrading 125I-TFPI. LRP heterozygous and homozygous-negative cells bind 125I-TFPI similarly, and the 39-kDa protein, an inhibitor of all known ligand interactions with LRP, does not alter 125I-TFPI binding to these cells. TFPI can be cross-linked to LRP on [35S]cysteine-labeled hepatoma and LRP-heterozygous cells but not LRP-negative cells. When HSPGs are blocked with protamine, 125I-TFPI binds in a 39-kDa protein-inhibitable manner to 41,000 high affinity sites/hepatoma cell (Kd = 2.3 nM). Blockade of HSPGs with protamine results in significantly more 125I-TFPI degradation by LRP-positive cells. TFPI can be cross-linked to LRP in the absence and presence of protamine. However, in the presence of protamine, relative to the total pool of cross-linked proteins, 5-fold more TFPI is cross-linked to LRP. Finally, we show TFPI inhibits 125I-alpha2-macroglobulin-methylamine binding to hepatoma cells and that carboxyl-terminal residues 115-319 of the 39-kDa protein inhibit both 125I-TFPI degradation and binding when binding conditions contain protamine. Together, our results suggest that while the majority of TFPI binds to cell surface HSPGs, LRP can function independently from HSPGs in the binding and uptake of TFPI.

The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo.

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor-related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma cells, a mutant of TFPI lacking the third Kunitz-type domain and basic carboxy terminus was generated. We found that this mutant, TFPI-160, did not compete with full-length 125I-TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4 degrees C. At 37 degrees C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-TFPI binding to hepatoma cells could be inhibited > 90% by heparin and other highly charged molecules. Since TFPI, but not TFPI-160, was capable of effectively binding to cultured hepatoma cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI-160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was approximately 30 s whereas the half-life of 125I-TFPI-160 was approximately 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125I-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

Design of Variants of the Second Domain of Urinary Trypsin Inhibitor (R-020) with Increased Factor Xa Inhibitory Activity

The second domain (R-020) of human urinary trypsin inhibitor (UTI) exerts similar inhibitory activities on trypsin, alpha-chymotrypsin, leukocyte elastase, and plasmin to those of UTI itself, and additionally inhibits coagulation factor Xa (FXa) and plasma kallikrein, on both of which UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R-020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was performed with reference to X-ray analysis of the complex of bovine pancreatic trypsin inhibitor (BPTI) and bovine trypsin to determine the site of amino acid modification. The expression plasmids into which R-020 genes with base substitution were inserted were prepared and introduced into Escherichia coli to express R-020 variants. The resulting variants were purified and their enzyme inhibitory activities were measured. The FXa-inhibitory activity was increased in four variants with single amino acid substitution. With another four variants having two amino acid substitutions involving combinations of the above single amino acid substitutions, the FXa-inhibitory activity was further increased. Because the electrostatic interaction within R-020-FXa complex seemed stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding between the enzyme and the inhibitor.