Comparative effects of enoxaparin and unfractionated heparin in healthy volunteers on prothrombin consumption in whole blood during coagulation, and release of tissue factor pathway inhibitor

In a randomized crossover study twelve healthy male volunteers (23.5 +/- of 4.8 years, 73.0 +/- 6.4 kg, 180.8 +/- 5.7 cm) received one subcutaneous injection of either enoxaparin (EN) at 40 mg or 1 mg kg-1, or unfractionated heparin (UH) at 5,000 IU at one week intervals. Area under curves (AUC) of Anti-Xa and Anti-IIa activities correlated with EN dose. The relative effectiveness of EN versus UH 5,000 U as assessed by AUC ratio (EN/UH) was 7 and 15 for Anti-Xa activity, 1.3 and 3.1 for Anti-IIa activity after sc injection of EN 40 mg (4,000 Anti-Xa IU and 1,200 Anti-IIa U) and 1 mg kg-1 (7,300 +/- 640 Anti-Xa IU and 2,190 +/- 290 Anti-IIa IU) respectively. In volunteers receiving EN, a dose dependent inhibition of thrombin generation rate in platelet depleted plasma (PDP), measured with a new and simple chromogenic thrombin generation assay, was observed when compared with baseline values. Similarly, intrinsic prothrombin activation in whole blood, evidenced by measuring residual factor II in serum 2 hours after clotting (prothrombin consumption test: PC), was inhibited in a dose dependent manner. In UH treated volunteers, although the inhibition of thrombin generation rate in PDP was similar to that observed with EN 40 mg, prothrombin consumption in whole blood was not significantly modified. Tissue factor pathway inhibitor (TFPI) activity release was increased similarly for UH and EN 40 (1.4 fold increase above baseline values) and 1.9 fold for the higher dose of EN. The discrepancy between prothrombin consumption in whole blood and inhibition of thrombin generation rate in PDP in the UH and not in the EN group strongly suggests that UH and not EN is influenced by the presence of a platelet component. This could be formed during thrombin induced platelet activation. Platelet factor 4 is a possible candidate. Another hypothesis involves the role of TFPI-UH complex anticoagulant activity which might be inhibited more during whole blood coagulation than the TFPI-EN complex.     

Immobilized hirudin and hirudin-based peptides used for the purification of recombinant human thrombin prepared from recombinant human prothrombin

A single step solid phase radioimmunoassay (SS-SPRIA) has been developed for human chorionic gonadotropin (hCG) using monoclonal antibodies (MAb) from culture media adsorbed immunochemically on plastic tubes. The assays have been found to be very simple in terms of operation and do not demand purification of MAbs. Several MAbs which do not show any displacement in liquid phase RIA and ELISA provide a satisfactory SS-SPRIA. Our investigations revealed that the assumption regarding the stability of the primary Mab-Ag complex during incubation and washing steps in ELISAs is not strictly valid for dissociable MAbs. A comparison of different assay systems suggests that the single step SPRIA offers additional advantages over conventionally used multistep ELISA procedures and provides a quantitative probe for the analysis of epitope-paratope interactions.     

Does antithrombotic therapy influence residual thrombus after thrombolysis of platelet-rich thrombus? Effects of recombinant hirudin, heparin, or aspirin

BACKGROUND: Thrombolysis to normal flow in patients with acute myocardial infarction preserves left ventricular function and decreases mortality. Failure of early reperfusion, reocclusion, or residual thrombus may be due to concurrent activation of the platelet-coagulation system. Thus, we hypothesized that the best concomitant antithrombotic therapy (recombinant [r]-hirudin, heparin, or aspirin) will maximally accelerate thrombolysis by r-tissue-type plasminogen activator (rTPA) and reduce residual thrombus. METHODS AND RESULTS: Occlusive thrombi were formed in the carotid arteries of 29 pigs (by balloon dilatation followed by endarterectomy at the site of injury-induced vasospasm) and matured for 30 minutes before rTPA was started, with or without antithrombotic therapy. Thrombolysis was assessed with the use of angiography and measurement of residual thrombus. Pigs were allocated to one of five treatments: placebo, rTPA, rTPA plus r-hirudin, rTPA plus heparin, or rTPA plus intravenous aspirin. No placebo-treated pig reperfused. Two of six animals treated with rTPA alone reperfused compared with seven of seven animals treated with rTPA plus r-hirudin (reperfusion time, 33 +/- 10 minutes), six of seven animals treated with rTPA plus heparin (reperfusion time, 110 +/- 31 minutes), and two of six animals with rTPA plus aspirin. The activated partial thromboplastin time was prolonged in only the rTPA plus r-hirudin group (25 +/- 0.1 times baseline) and the rTPA plus heparin group (5.3 +/- 0.2 times baseline). Residual 111In-platelet and 125I-fibrin(ogen) depositions were lower in the heparin-treated group and lowest in the r-hirudin-treated group (heparin versus hirudin, respectively; incidence of residual macroscopic thrombus was six of six animals versus two of seven [P = .01]; 125I-fibrin(ogen), 170 +/- 76 versus 48 +/- 6 x 10(6) molecules/cm2 [P = .02]; 111In-platelets, 47 +/- 15 versus 13 +/- 2 x 10(6)/cm2, P = .10). No pigs developed spontaneous bleeding. CONCLUSIONS: Thrombin inhibition with heparin or r-hirudin significantly accelerated thrombolysis of occlusive platelet-rich thrombosis, but only the best antithrombotic therapy (r-hirudin) eliminated or nearly eliminated residual thrombus.     

Two-chain factor VIIa generated in the pericardium during surgery with cardiopulmonary bypass : relationship to increased thrombin generation and heparin concentration

The effect of changes in medullary extracellular tonicity on mRNA expression for aldose reductase (AR), sorbitol dehydrogenase (SDH), Na+/Cl-/betaine (BGT) and Na+/myo-inositol (SMIT) cotransporter in different kidney zones was studied using Northern blot analysis and non-radioactive in situ hybridization in four groups of rats: controls, acute diuresis (the loop diuretic furosemide was administered), chronic diuresis (5 days of diuresis), and antidiuresis [5 days of diuresis followed by 24 h deamino-Cys1, d-Arg8 vasopressin (dDAVP)]. Acute administration of the loop diuretic furosemide significantly reduced AR, SMIT and BGT gene expression in the inner and outer medulla compared with controls. Administration of dDAVP to chronically diuretic rats raised the expression of these three mRNAs in the inner but not the outer medulla compared with the chronically diuretic rats. None of these alterations in medullary tonicity significantly changed SDH expression. The in situ hybridization studies showed AR, BGT and SMIT mRNAs to be expressed in both epithelial and non-epithelial cells of the outer and inner medulla. The various cell types (epithelial, endothelial and interstitial cells) differed in their expression pattern and intensity of AR, SDH, BGT and SMIT mRNA, but the inner medullary cells responded uniformly to a decrease in extracellular tonicity with a reduction, and to an increase with enhancement of their AR, BGT and SMIT expression.     

Distinct effects of recombinant desulfatohirudin (Revasc) and heparin on plasma levels of fibrinopeptide A and prothrombin fragment F1.2 in unstable angina. A multicenter trial

BACKGROUND: Thrombin plays an important role in the pathogenesis of acute coronary thrombosis. We studied the effects of a direct thrombin inhibitor, recombinant desulfatohirudin, and heparin on plasma levels (at 0, 4, 12, and 24 hours) of fibrinopeptide A (FPA), which reflects thrombin action, and prothrombin fragment F1.2, which reflects thrombin generation, in patients with unstable angina. METHODS AND RESULTS: Patients were randomized to one of two doses of heparin (n = 50) (target activated partial thromboplastin time, 65 to 90 seconds or 90 to 110 seconds) or one of four doses of r-hirudin (n = 113) (0.05, 0.10, 0.20, or 0.30 mg.kg-1.h-1 by infusion). r-Hirudin induced a dose-dependent decline in plasma FPA. At 24 hours, FPA levels with 0.1- to 0.3-mg.kg-1.h-1 r-hirudin regimens were significantly lower than with 0.05 mg.kg-1.h-1 r-hirudin; levels with 0.1- to 0.2-mg.kg-1.h-1 r-hirudin regimens were lower than with both heparin regimens. Plasma F1.2 did not decline significantly during therapy with heparin or hirudin except at 0.3 mg.kg-1.h-1 hirudin. At 24 hours, they were higher with the 0.05-mg.kg-1.h-1 r-hirudin regimen than with other regimens. For comparable levels of thrombin generation (F1.2 levels), FPA levels were higher in heparin patients than in hirudin patients. For the same FPA values, the corresponding F1.2 values were higher in the hirudin group. CONCLUSIONS: Our findings provide evidence for distinct in vivo effects of the two agents and suggest that r-hirudin is a relatively more potent inhibitor of thrombin action but a less effective inhibitor of thrombin generation than heparin. The lower FPA levels in hirudin patients may reflect its ability to inactivate clot-bound thrombin. The relative clinical efficacies of the two agents need to be defined by clinical trials in progress.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

Effect of monoclonal antibody against human tissue factor pathway inhibitor on plasma coagulation time

We investigated the possibility of identification of human skin left under various conditions using our original enzyme immunoassay (EIA) for squamous cell carcinoma-related (SCC) antigen. The antigen could be detected in specimens under the following conditions : purified or dried at room temperature for at least 12 months, immersed in fresh water at room temperature for 3 weeks and heated at 100 degrees C for 72 h.     

The solution structure of human coagulation factor VIIa in its complex with tissue factor is similar to free factor VIIa: a study of a heterodimeric receptor-ligand complex by X-ray and neutron scattering and computational modeling

Factor VIIa (FVIIa) is a soluble four-domain plasma serine protease coagulation factor that forms a tight complex with the two extracellular domains of the transmembrane protein tissue factor in the initiating step of blood coagulation. To date, there is no crystal structure for free FVIIa. X-ray and neutron scattering data in solution for free FVIIa and the complex between FVIIa and soluble tissue factor (sTF) had been obtained for comparison with crystal structures of the FVIIa-sTF complex and of free factor IXa (FIXa). The solution structure of free FVIIa as derived from scattering data is consistent with the extended domain arrangement of FVIIa seen in the crystal structure of its complex with sTF, but is incompatible with the bent, less extended domain conformation seen in the FIXa crystal structure. The FVIIa scattering curve is also compatible with a subset of 317 possible extended structures derived from a constrained automated conformational search of 15 625 FVIIa domain models. Thus, the scattering data support extended domain models for FVIIa free in solution. Similar analyses showed that the solution scattering derived and crystal structures of the FVIIa-sTF complex were in good agreement. An automated constrained search for allowed structures for the complex in solution based on scattering curves showed that only a small family of compact models gave good agreement, namely those in which FVIIa and sTF interact closely over a large surface area. The general utility of this approach for structural analysis of heterodimeric complexes in solution is discussed. Analytical ultracentrifugation data and the modeling of these data were consistent with the scattering results. It is concluded that in solution FVIIa has an extended or elongated domain structure, which allows rapid interaction with sTF over a large surface area to form a high-affinity complex.     

Differential effect of unfractionated heparin and low molecular weight heparin on intravascular tissue factor pathway inhibitor: evidence for a difference in antithrombotic action

PURPOSE: Successful endovascular repair of an abdominal aortic aneurysm (AAA) requires the creation of a hemostatic seal between the endograft and the underlying aortic wall. A short infrarenal aortic neck may be responsible for incomplete aneurysm exclusion and procedural failure. Sixteen patients who had an endograft positioned completely below the lowest renal artery and 37 patients in whom a porous portion of an endograft attachment system was deliberately placed across the renal arteries were studied to identify if endograft positioning could impact on the occurrence of incomplete aneurysm exclusion. METHODS: Fifty-three patients underwent aortic grafting constructed from a Palmaz balloon expandable stent and an expandable polytetrafluoroethylene (ePTFE) graft implanted in an aorto-ilio-femoral, femoral-femoral configuration. Arteriography, duplex ultrasonography and spiral CT scans were performed in each patient before and after endografting to evaluate for technical success, the presence of endoleaks, and renal artery perfusion. RESULTS: There was no statistically significant difference in patient demography, AAA size, or aortic neck length or diameter between patients who had their endografts placed below or across the renal arteries. However, significantly more proximal aortic endoleaks occurred in those patients with infrarenal endografts (P < or = .05). Median serum creatinine level before and after endografting was not significantly different between the 2 patient subgroups, with the exception of 2 patients who had inadvertent coverage of a single renal orifice by the endograft. Median blood pressure and the requirement for antihypertensive therapy remained the same after transrenal aortic stent grafting. Significant renal artery compromise did not occur after appropriately positioned transrenal stents as shown by means of angiography, CT scanning, and duplex ultrasound scan. Mean follow-up time was 10.3 months (range, 3 to 18 months). Patients who had significant renal artery stenosis (> or =50%) before aortic endografting did not show progression of renal artery stenosis after trans-renal endografting. Two patients with transrenal aortic stent grafts had inadvertent coverage of 1 renal artery by the endograft because of device malpositioning, which resulted in nondialysis dependent renal insufficiency. In addition, evidence of segmental renal artery infarction (<20% of the kidney), which did not result in an apparent change in renal function, was shown by means of follow-up CT scans in 2 patients with transrenal endografts. CONCLUSION: Transrenal aortic endograft fixation using a balloon expandable device in patients with AAAs can result in a significant reduction in the risk of proximal endoleaks. Absolute attention to precise device positioning, coupled with the use of detailed imaging techniques, should reduce the risk of inadvertent renal artery occlusion from malpositioning. Long-term follow-up is essential to determine if there will be late sequelae of transrenal fixation of endografts, which could adversely effect renal perfusion.     

Pathogenicity of human anti-platelet factor 4 (PF4)/heparin in vivo: generation of mouse anti-PF4/heparin and induction of thrombocytopenia by heparin

Acinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l-1), synthesized two beta-lactamases with pIs 6.3 and > 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, second- and, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem beta-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the beta-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (Kcat/Km) of 1500 mM-1 s-1. Moreover, this purified beta-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 beta-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.     

Pilot study of the immunologic effects of recombinant human growth hormone and recombinant insulin-like growth factor in HIV-infected patients

OBJECTIVE: To study the immunologic effects of recombinant human growth hormone (rhGH), recombinant human insulin-like growth factor type 1 (rhIGF-1), or the combination, in patients with moderately advanced HIV infection. DESIGN: Randomized but not blinded trial. SETTING: Government medical research center. PATIENTS: Twenty-four HIV-infected patients with CD4 cell counts of 100-400 x 10(6)/l who were receiving nucleoside antiretroviral therapy. INTERVENTIONS: Either rhGH, rhIGF-1, or the combination was administered subcutaneously for 12 weeks. MAIN OUTCOME MEASURES: Immunologic parameters, including T-cell subsets and assays of in vitro interleukin (IL)-2 production in response to antigens and mitogens, and safety profile. RESULTS: Plasma IGF-1 levels were low or low-normal prior to treatment and increased with all three therapies. There were no significant changes in CD4 cell counts, RA/RO CD4 cell subsets, natural killer cell function, immunoglobulin levels, or in vitro IL-2 production in response to mitogen or alloantigens. However, there was an upward trend (and for p18IIIB a statistically significant increase) in the in vitro IL-2 production in response to each of five HIV envelope peptides. Potential toxic effects included fatigue, arthralgia, edema, myalgia, and headache. Patients also were noted to have weight gain averaging 4 kg early in the course of treatment. CONCLUSIONS: These results suggest that treatment with rhGH/rhIGF-1 was reasonably well tolerated and that modest improvement in HIV-specific immune function was attained. Further studies will help clarify the therapeutic potential of rhGH/rhIGF-1 as an immunostimulator in the setting of HIV infection.     

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.     

Comparative effects of recombinant staphylokinase and streptokinase on platelet aggregation

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.     

Pharmacokinetics of heparin and its pharmacodynamic effect on plasma lipoprotein lipase activity and coagulation in healthy horses

We evaluated the pharmacokinetics of IV administered sodium heparin and the pharmacodynamic effect of heparin on lipoprotein lipase (LPL) activity. Horses were allotted to 3 groups. Plasma samples were obtained from each horse before and at various times for 6 hours after heparin administration for determination of heparin concentration, LPL activity, and activated partial thromboplastin time (APTT). The disposition of heparin was dose dependent. The area under the plasma heparin concentration vs time curve (AUC) increased more than proportionally with dose, indicating that heparin elimination was nonlinear. Total clearance of heparin was similar after the 40 and 80 IU/kg of body weight dosages, averaging 0.45 and 0.36 IU/kg/min, respectively. However, after administration of the 120 IU/kg dose, clearance was significantly less than that after the 40 IU/kg dose. The half-life of heparin averaged 53, 70, and 136 minutes after 40, 80, and 120 IU/kg, respectively, with significant differences observed between the low and high doses. In contrast to heparin, the area under the plasma concentration vs time curve for LPL activity increased less than proportionally with dose. Maximal LPL activity observed was independent of dose, averaging 4.8 mumol of free fatty acids/ml/h. The APTT was significantly prolonged for 120 minutes after administration of the 40 IU/kg dose. Correlation coefficients for LPL activity vs either plasma heparin concentration or APTT were less than 0.7, indicating that neither laboratory measure can be used to accurately predict plasma LPL activity.     

In vivo effects of recombinant human granulocyte colony-stimulating factor on neutrophil oxidative functions in normal human volunteers

The effect of daily in vivo granulocyte colony-stimulating factor (G-CSF) treatment on neutrophil function was studied over a 14-day period using a luminescence system for differential measurement of oxidase and myeloperoxidase (MPO) dioxygenation activities in whole blood. Opsonin receptor-mediated phagocyte functions were also measured with this system. G-CSF produced a dose-dependent neutrophil leukocytosis and a proportional increase in oxidase activity per volume of blood. The oxidase activity per neutrophil remained relatively constant throughout the test period. However, both chemical- and opsonin-stimulated MPO oxygenation activities per neutrophil were greatly increased by treatment with maxima correlating temporally to initial G-CSF exposure during the early mitotic phase of neutrophil development. The possibility that peroxynitrite contributes to this maximum luminol-dependent activity was tested, but neither superoxide dismutase, a competitive inhibitor of peroxynitrite production, nor N-methyl-L-arginine, an inhibitor of nitric oxide synthase, exerted a significant inhibitory effect.     

Effect of nadroparin, a low-molecular-weight heparin, on clinical and angiographic restenosis after coronary balloon angioplasty: the FACT study. Fraxiparine Angioplastie Coronaire Transluminale

BACKGROUND: Experimental studies suggest that the antiproliferative effect of heparin after arterial injury is maximized by pretreatment. No previous studies of restenosis have used a pretreatment strategy. We designed this study to determine whether treatment with nadroparin, a low-molecular-weight heparin, started 3 days before the procedure and continued for 3 months, affected angiographic restenosis or clinical outcome after coronary angioplasty. METHODS AND RESULTS: In a prospective multicenter, double-blind, randomized trial, elective coronary angioplasty was performed on 354 patients who were treated with daily subcutaneous nadroparin (0.6 mL of 10,250 anti-Xa IU/mL) or placebo injections started 3 days before angioplasty and continued for 3 months. Angiography was performed just before and immediately after angioplasty and at follow-up. The primary study end point was angiographic restenosis, assessed by quantitative coronary angiography 3 months after balloon angioplasty. Clinical follow-up was continued up to 6 months. Clinical and procedural variables and the occurrence of periprocedural complications did not differ between groups. At angiographic follow-up, the mean minimal lumen diameter and the mean residual stenosis in the nadroparin group (1.37+/-0.66 mm, 51.9+/-21.0%) did not differ from the corresponding values in the control group (1.48+/-0.59 mm, 48.8+/-18.9%). Combined major cardiac-related clinical events (death, myocardial infarction, target lesion revascularization) did not differ between groups (30.3% versus 29.6%). CONCLUSIONS: Pretreatment with the low-molecular-weight heparin nadroparin continued for 3 months after balloon angioplasty had no beneficial effect on angiographic restenosis or on adverse clinical outcomes.     

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.