S. Paul Bajaj

A Simplified Procedure for Purification of Human Prothrombin, Factor IX and Factor X

A simplified procedure is described for the purification of prothrombin, Factor X and Factor IX in overall yields of 35-40% from pooled human plasma. The initial steps, which are common to prior purification techniques, include adsorption onto and elution from barium citrate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. The procedure differs from previous techniques in that the nest step, heparin-agarose chromatography, is carried out in a (sodium) citrate buffer, pH 7.5. These chromatographic conditions permit the separation of prothrombin, Factor X and Factor IX from each other, yielding fractions with apparent homogeneity in several electrophoretic systems. The additional chromatographic steps of earlier purification procedures are therefore unnecessary. The heaprin-agrarose column chromatographic conditions consistently resulted in the separation of human prothrombin in into two fractions in a ratio of approximately 4:1. Both fractions possess similar specific activity in a one stage prothrombin assay, and also activate at the same rate in a Factor Xa, Ca2+ and phospholipid system. Both fractions of prothrombin also comigrate in sodium dodecyl sulfate gel electrophoresis with an apparent Mr integral of 70,000.

Structure–Function Relationships in Factor IX and Factor IXa

Factor IX (FIX) consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and the C-terminal serine protease domain. During physiologic coagulation, one of the activators of FIX is the FVIIa/tissue factor (TF) complex. In this reaction, the Gla and EGF1 domains of FIX are thought to interact with TF. The FIXa that is generated then combines with FVIIIa on the platelet surface to activate FX in the coagulation cascade. In this assembly, the protease domain and possibly the EGF2 domain of FIXa are thought to provide the primary specificity in binding to FVIIIa. Disruption of the interaction of FIX/FX with TF and of the FIXa:FVIIIa interface may provide a pharmacologic target as an alternative strategy for the development of antithrombotic agents.

Thermodynamic Linkage between the S1 Site, the Na+Site, and the Ca2+ Site in the Protease Domain of Human Coagulation Factor Xa STUDIES ON CATALYTIC EFFICIENCY AND INHIBITOR BINDING

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate bound FXa withK d ∼39 mm in the absence and ∼9.5 mm in the presence of Ca2+. Sodium-bound FXa (sodium-Xa) has ∼18-fold increased catalytic efficiency (∼4.5-fold decrease in K m and ∼4-fold increase ink cat) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca2+ further increases this k cat∼1.4-fold. Ca2+ binds to the protease domain of substrate bound FXa with K d ∼705 μm in the absence and ∼175 μm in the presence of Na+. Ca2+ binding to the protease domain of FXa (Xa-calcium) has no effect on the K m but increases thek cat ∼4-fold in hydrolyzing S-2222, and Na+ further increases this k cat∼1.4-fold. In agreement with the K m data, sodium-Xa has ∼5-fold increased affinity in its interaction withp-aminobenzamidine (S1 site probe) and ∼4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca2+ (±Na+) has no effect on these interactions. Antithrombin binds to Xa-calcium with a ∼4-fold faster rate, to sodium-Xa with a ∼24-fold faster rate and to sodium-Xa-calcium with a ∼28-fold faster rate. Thus, Ca2+and Na+ together increase the catalytic efficiency of FXa ∼28-fold. Na+ enhances Ca2+ binding, and Ca2+ enhances Na+ binding. Further, Na+ enhances S1 site occupancy, and S1 site occupancy enhances Na+ binding. Therefore, Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas Ca2+ site is only linked to the Na+ site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition

Kunitz domain 1 (KD1) of tissue factor pathway inhibtor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nm, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine β-trypsin were obtained that diffracted to 1.8 Å. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Oγ of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis PROPERTIES OF KD1-L17R VARIANT

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

Design, synthesis, and structure-activity relationship of a new class of amidinophenylurea-based factor VIIa inhibitors.

Selective inhibition of coagulation factor VIIa has recently gained attraction as interesting approach towards antithrombotic treatment. Using parallel synthesis supported by structure-based design and X-ray crystallography, we were able to identify a novel series of amidinophenylurea derivatives with remarkable affinity for factor VIIa. The most potent compound displays a K(i) value of 23 nM for factor VIIa.

Protease Nexin-2/Amyloid β-Protein Precursor Regulates Factor VIIa and the Factor VIIa–Tissue Factor Complex

Protease nexin-2/amyloid beta-protein precursor (PN-2/AbetaPP) and its Kunitz protease inhibitory (KPI) domain were characterized as inhibitors of factor VIIa (FVIIa) and factor VIIa-tissue factor complex (FVIIa-TF). PN-2/AbetaPP and KPI domain inhibited FVIIa with an apparent K(i) of 1.1+/-0.2x 10(-7) M and 1.5+/-0.1x10(-7) M, respectively. When soluble tissue factor (TF(1-219)) was present, there was increased FVIIa inhibition by PN-2/AbetaPP or KPI domain (K(i)=7.8+/-0.3x10(-8) M and 6.8+/-0.6x10(-8) M, respectively). When relipidated tissue factor (TF(1-243)) was present, the K(i) of FVIIa inhibition by PN-2/AbetaPP increased 4.7-fold further. PN-2/AbetaPP complexed with FVIIa, as shown on gel filtration and solid phase binding assay. The apparent second-order rate constant of inhibition of FVIIa by PN-2/AbetaPP in the absence of TF(1-219) was less than that of the FVIIa-TF(1-219) complex. Antithrombin in the absence of TF(1-219) also had a lower apparent second-order rate constant of inhibition than in its presence. In a mixture that included FVIIa, relipidated TF(1-243) and factor X, PN-2/AbetaPP or KPI domain had an IC(50) at 65 and 250 nM, respectively; antithrombin and heparin (1 U/mL) had an IC(50) of 12.8 nM. These data indicate that tissue factor promoted the inhibition of FVIIa by PN-2/AbetaPP or KPI domain, but antithrombin was a better inhibitor of soluble FVIIa-TF in extrinsic tenase.

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.

A Sequential Mechanism for Exosite-mediated Factor IX Activation by Factor XIa

Background: Factor XIa proteolytically activates factor IX. Results: XIa cleaves IX after Arg145, forming IXα, and then after Arg180, forming IXaβ. Both reactions require substrate binding to the XIa A3 domain. Conclusion: XIa activates IX by an exosite-mediated release-rebind mechanism. Efficiency of the second cleavage is enhanced by changes resulting from the first cleavage. Significance: The data support a new model for IX activation by 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 Arg145 to form fIXα, and then after Arg180 to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg180 to occur. Catalytic efficiency of cleavage after Arg180 is 7-fold greater than for cleavage after Arg145, 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 Km for fIX cleavage after Arg145 and Arg180 25- and ≥90-fold, respectively, and markedly decreases kcat for cleavage after Arg180. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca2+. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg180 than for cleavage after Arg145, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca2+-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.

The mechanism underlying activation of factor IX by factor XIa

Factor XI (fXI) is the zymogen of a plasma protease, factor XIa (fXIa), that contributes to thrombin generation during blood coagulation by proteolytic conversion of factor IX (fIX) to factor IXaβ (fIXaβ). There is considerable interest in fXIa as a therapeutic target because it contributes to thrombosis, while serving a relatively minor role in hemostasis. FXI/XIa has a distinctly different structure than other plasma coagulation proteases. Specifically, the protein lacks a phospholipid-binding Gla-domain, and is a homodimer. Each subunit of a fXIa dimer contains four apple domains (A1 to A4) and one trypsin-like catalytic domain. The A3 domain contains a binding site (exosite) that largely determines affinity and specificity for the substrate fIX. After binding to fXIa, fIX undergoes a single cleavage to form the intermediate fIXα. FIXα then rebinds to the A3 domain to undergo a second cleavage, generating fIXaβ. The catalytic efficiency for the second cleavage is ~7-fold greater than that of the first cleavage, limiting fIXα accumulation. Residues at the N-terminus and C-terminus of the fXIa A3 domain likely form the fIX binding site. The dimeric conformation of fXIa is not required for normal fIX activation in solution. However, monomeric forms of fXI do not reconstitute fXI-deficient mice in arterial thrombosis models, indicating the dimer is required for normal function in vivo. FXI must be a dimer to be activated normal by the protease fXIIa. It is also possible that the dimeric structure is an adaptation that allows fXI/XIa to bind to a surface through one subunit, while binding to its substrate fIX through the other.