Model for surface-dependent factor XII activation: the roles of factor XII heavy chain domains.

Factor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged surfaces such as polymeric orthophosphate. FXII is composed of noncatalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation but restricts activation when FXII is not surface bound. From the N terminus, the heavy chain contains fibronectin type 2 (FN2), epidermal growth factor-1 (EGF1), fibronectin type 1 (FN1), EGF2, and kringle (KNG) domains and a proline-rich region. It shares this organization with its homolog, pro-hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains in FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog ε-aminocaproic acid. A model is proposed in which an ε-aminocaproic acid-sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region.

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with KD ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR-R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR-R and inter-repeat junctions identified critical conserved residues. Full-length PR-(R1 → R7) bound frag D with KD ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR-(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR-R and R-R junctions with modest inter-repeat sequence variability. CD of PR-R7 and PR-(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks.

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis.

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Protease activity in single-chain prekallikrein.

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

A non-circulating pool of factor XI associated with glycosaminoglycans in mice.

BACKGROUND: The homologous plasma proteins prekallikrein and factor XI (FXI) circulate as complexes with high molecular weight kininogen. Although evidence supports an interaction between the prekallikrein-kininogen complexes and vascular endothelium, there is conflicting information regarding FXI binding to endothelium. OBJECTIVE: To study the interaction between FXI and blood vessels in mice. METHODS: C57Bl/6 wild-type or F11-/- mice in which variants of FXI were expressed by hydrodynamic tail vein injection, received intravenous infusions of saline, heparin, polyphosphates, protamine, or enzymes that digest glycosaminoglycans (GAGs). Blood was collected after infusion and plasma was analyzed by western blot for FXI. RESULTS AND CONCLUSIONS: Plasma FXI increased 5- to 10-fold in wild-type mice after infusion of heparin, polyphosphates, protamine, or GAG-digesting enzymes, but not saline. Similar treatments resulted in a much smaller change in plasma FXI levels in rats, and infusions of large boluses of heparin did not change FXI levels appreciably in baboons or humans. The releasable FXI fraction was reconstituted in F11-/- mice by expressing murine FXI, but not human FXI. We identified a cluster of basic residues on the apple 4 domain of mouse FXI that is not present in other species. Replacing the basic residues with alanine prevented the interaction of mouse FXI with blood vessels, whereas introducing the basic residues into human FXI allowed it to bind to blood vessels. Most FXI in mice is noncovalently associated with GAGs on blood vessel endothelium and does not circulate in plasma.

A mechanism for hereditary angioedema with normal C1 inhibitor: an inhibitory regulatory role for the factor XII heavy chain.

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.

Proteases: Pivot Points in Functional Proteomics.

Proteases drive the life cycle of all proteins, ensuring the transportation and activation of newly minted, would-be proteins into their functional form while recycling spent or unneeded proteins. Far from their image as engines of protein digestion, proteases play fundamental roles in basic physiology and regulation at multiple levels of systems biology. Proteases are intimately associated with disease and modulation of proteolytic activity is the presumed target for successful therapeutics. "Proteases: Pivot Points in Functional Proteomics" examines the crucial roles of proteolysis across a wide range of physiological processes and diseases. The existing and potential impacts of proteolysis-related activity on drug and biomarker development are presented in detail. All told the decisive roles of proteases in four major categories comprising 23 separate subcategories are addressed. Within this construct, 15 sets of subject-specific, tabulated data are presented that include identification of proteases, protease inhibitors, substrates, and their actions. Said data are derived from and confirmed by over 300 references. Cross comparison of datasets indicates that proteases, their inhibitors/promoters and substrates intersect over a range of physiological processes and diseases, both chronic and pathogenic. Indeed, "Proteases: Pivot Points …" closes by dramatizing this very point through association of (pro)Thrombin and Fibrin(ogen) with: hemostasis, innate immunity, cardiovascular and metabolic disease, cancer, neurodegeneration, and bacterial self-defense.

An update on factor XI structure and function.

Factor XI (FXI) is the zymogen of a plasma protease, factor XIa (FXIa), that contributes to thrombin generation during blood coagulation by proteolytic activation of several coagulation factors, most notably factor IX (FIX). FXI is a homolog of prekallikrein (PK), a component of the plasma kallikrein-kinin system. While sharing structural and functional features with PK, FXI has undergone adaptive changes that allow it to contribute to blood coagulation. Here we review current understanding of the biology and enzymology of FXI, with an emphasis on structural features of the protein as they relate to protease function.

A novel antithrombin domain dictates the journey's end of a proteinase.

Antithrombin (AT) is an anticoagulant serpin that irreversibly inactivates the clotting proteinases factor Xa and thrombin by forming covalent complexes with them. Mutations in its critical domains, such as those that impair the conformational rearrangement required for proteinase inactivation, increase the risk of venous thrombosis. Águila et al. characterize for the first time the destabilizing effects of mutations in the region of AT that makes contact with the proteinase in the final acyl-enzyme complex. Their work adds new insight into the unique structural intricacies of the inhibitory mechanism.

Proteolytic properties of single-chain factor XII: a mechanism for triggering contact activation.

When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.

The hyperglycemic byproduct methylglyoxal impairs anticoagulant activity through covalent adduction of antithrombin III.

INTRODUCTION: The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition. METHODS: MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition. RESULTS AND CONCLUSIONS: MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.

Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism.

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys(414) deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-D-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK ·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys(414) deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys(414) to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK · Pg complex compared with SK · Pm is characterized by a ∼ 25-fold weaker encounter complex and ∼ 40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys(414) engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK · Pg complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared with SK · Pm.

The dimeric structure of factor XI and zymogen activation.

Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit.

A sequential mechanism for exosite-mediated factor IX activation by factor XIa.

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg(145) to form fIXα, and then after Arg(180) to form the protease fIXaß. FIXα is released from fXIa, and must rebind for cleavage after Arg(180) to occur. Catalytic efficiency of cleavage after Arg(180) is 7-fold greater than for cleavage after Arg(145), limiting fIXα accumulation. FXIa contains four apple domains (A1-A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases K(m) for fIX cleavage after Arg(145) and Arg(180) 25- and ≥ 90-fold, respectively, and markedly decreases k(cat) for cleavage after Arg(180). Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca(2+). Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg(180) than for cleavage after Arg(145), resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca(2+)-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain.

Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex.

Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4'F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54-65)(SO3⁻), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4'F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54-65)(SO3⁻) binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T·heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin·heparin complex binds HCII and allowing for interaction of thrombin·(heparin)2 complexes with HCII. Hir-(54-65)(SO3⁻) displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin·heparin complex is dramatically enhanced compared with heparin binding alone and that exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.

Activation of factor XI by products of prothrombin activation.

The prothrombinase complex converts prothrombin to α-thrombin through the intermediate meizothrombin (Mz-IIa). Both α-thrombin and Mz-IIa catalyze factor (F) XI activation to FXIa, which sustains α-thrombin production through activation of FIX. The interaction with FXI is thought to involve thrombin anion binding exosite (ABE) I. α-Thrombin can undergo additional proteolysis to ß-thrombin and γ-thrombin, neither of which have an intact ABE I. In a purified protein system, FXI is activated by ß-thrombin or γ-thrombin, and by α-thrombin in the presence of the ABE I-blocking peptide hirugen, indicating that a fully formed ABE I is not absolutely required for FXI activation. In a FXI-dependent plasma thrombin generation assay, ß-thrombin, γ-thrombin, and α-thrombins with mutations in ABE I are approximately 2-fold more potent initiators of thrombin generation than α-thrombin or Mz-IIa, possibly because fibrinogen, which binds to ABE I, competes poorly with FXI for forms of thrombin lacking ABE I. In addition, FXIa can activate factor FXII, which could contribute to thrombin generation through FXIIa-mediated FXI activation. The data indicate that forms of thrombin other than α-thrombin contribute directly to feedback activation of FXI in plasma and suggest that FXIa may provide a link between tissue factor-initiated coagulation and the proteases of the contact system.

Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition.

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.

Irreversible inactivation of alpha-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K(D)) of 6+/-1 and 7+/-1 microM, respectively, approximately 6-fold tighter than plasma HCII, with K(D) 40+/-4 microM. Binding of recombinant and deglycosylated HCII to DS, both with K(D) 4+/-1 microM, was approximately 4-fold tighter than for plasma HCII, with K(D) 15+/-4 microM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated alpha-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn(169)N-glycan with the HCII heparin-binding site.

Sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor II.

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH(2)-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K(D)) of 10 +/- 4 microm and 400 +/- 300 microm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k(obs)). SOS bound HCII with K(D) 1.45 +/- 0.30 mm, and this binding was tightened in the T.SOS.HCII complex, characterized by K(complex) of approximately 0.20 microm. Inactivation data were incompatible with a model solely depending on HCII.SOS but fit an equilibrium linkage model employing T.SOS binding in the pathway to higher order complex formation. Hirudin-(54-65)(SO(3)(-)) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54-65)(SO(3)(-)) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K(D) = 1600 +/- 300 microm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.