Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.     

Rabeprazole in treatment of acid peptic diseases: results of three placebo-controlled dose-response clinical trials in duodenal ulcer, gastric ulcer, and gastroesophageal reflux disease (GERD). The Rabeprazole Study Group

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.     

A binding site for heparin in the apple 3 domain of factor XI

Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. Chem. 272, 26139-26144), we examined binding of factor XI (FXI) and FXIa to heparin. FXIa binds to heparin (Kd approximately 0.7 x 10(-9) M) >150-fold more tightly than FXI (Kd approximately 1.1 x 10(-7) M). To localize the heparin-binding site on FXI, rationally designed conformationally constrained synthetic peptides were used to compete with 125I-FXI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn235-Arg266) inhibited FXI binding to heparin (Kd approximately 3.4 x 10(-6) M), whereas peptides from the A1 domain (Phe56-Ser86), A2 domain (Ala134-Ala176), and A4 domain (Ala317-Gly350) had no such effect. The recombinant A3 domain (rA3, Ala181-Val271) inhibited FXI binding to heparin (Ki approximately 1.4 x 10(-7) M) indicating that all the information necessary for FXI binding to heparin is contained entirely within the A3 domain. The A3 domain also contains a platelet-binding site (Asn235-Arg266), consisting of three surface-exposed loop structures, Pro229-Gln233, Thr741-Leu246, and Thr249-Phe260 (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1995) J. Biol. Chem. 270, 6734-6740). Only peptide Thr249-Phe260 (which contains a heparin binding consensus sequence, RIKKSKA) inhibits FXI binding to heparin (Ki = 2.1 x 10(-7) M), whereas peptides Pro229-Gln233 and Thr241- Leu246 had no effect. Fine mapping of the heparin-binding site using prekallikrein analogue amino acid substitutions of the synthetic peptide Thr249-Phe260 and alanine scanning of the recombinant A3 indicated that the amino acids Lys252 and Lys253 are important for heparin binding. Thus, the sequence Thr249-Phe260 which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.     

The insulin-like growth factor (IGF)binding protein 1 binding epitope on IGF-I probed by heteronuclear NMR spectroscopy and mutational analysis

NMR spectroscopy studies and biosensor interaction analysis of native and site-directed mutants of insulin-like growth factor I (IGF-I) was applied to identify the involvement of individual residues in IGF-I binding to IGF-binding protein 1 (IGFBP-1). Backbone NMR chemical shifts were found to be affected by IGFBP-1 binding in the following residues: Pro2, Glu3, Cys6, Gly7, Gly19, Pro28-Gly30, Gly32, Arg36, Arg37, Gln40-Gly42, Pro63, Lys65, Pro66, and Lys68-Ala70. Three IGF-I arginine side chains were identified by NMR to participate in IGFBP-1 binding. All IGF-I arginine residues were replaced by alanines, using site-directed mutagenesis, in four single substituted variants, IGF-I(R21A), IGF-I(R50A), IGF-I(R55A), and IGF-I(R56A), and one double replacement mutant, IGF-I(R36A/R37A). Biosensor interaction analysis binding studies demonstrate the involvement of Arg36-Arg37 and Arg50 in IGFBP-1 binding, while experiments with the IGF-I receptor implicate Arg21, Arg36-Arg37, and Arg56 as part of the receptor binding epitope. These overlapping binding surfaces explain why IGF-I receptor and IGFBP-1 binding to IGF-I is competitive. The C terminus of free, but not IGFBP-1-bound, IGF-I is found to exist in two distinct, NMR-detectable conformations at 30 degreesC. One possible explanation for this structural heterogeneity could be cis-trans isomerization of the Cys6-Cys48 disulfide bond.     

Activation of prothrombin by factor Xa bound to the membrane surface of human umbilical vein endothelial cells: its catalytic efficiency is similar to that of prothrombinase complex on platelets

Upon incubation of human prothrombin with factor Xa bound to human umbilical vein endothelial cells (HUVEC) (0.5-0.6 fmol factor Xa/10(5) cells), three bonds at Arg273-Thr274, Arg286-Thr287, and Arg322-Ile323 were cleaved, yielding and releasing fragment 1-2 and a degraded form of alpha-thrombin, but not meizothrombin, into the fluid phase. The apparent Km for prothrombin and the Vmax were 0.25 +/- 0.07 microM and 210 +/- 40 fmol thrombin/min/10(5) cells, respectively. For the maximally bound factor Xa, the calculated catalytic efficiency (kcat = 6-7 s-1) was similar to those reported for the prothrombinase complex formed on the phospholipid vesicles and natural membrane surfaces. The prothrombin derivatives lacking the 10 gamma-carboxyglutamic acid (Gla) residues-containing region were not activated by the cell-bound factor Xa. The activation rate of prothrombins with Gla residues variously modified to gamma-methyleneglutamic acids was reduced in accordance with the number of modified residues. For the inhibition of prothrombin activation, intact fragment 1 was needed; the Gla-domain alone did not affect the reaction. Binding of monoclonal antibodies to the region of 1-48 or the kringle 1 region of prothrombin also interfered with the prothrombin activation. Prothrombin activation on the surface of HUVEC appeared to proceed via formation of a cellular prothrombinase complex composed of phospholipids of HUVEC membrane, endogenous factor Va, factor Xa, and prothrombin. The Gla-domain and kringle 1 regions are indispensable for the molecule to serve as an effective substrate for the cell-bound factor Xa.     

Polyserositis in a female patient with hypothyroidism

Advances in our knowledge of the biochemistry of coagulation have facilitated the development of sensitive and specific assays that are able to detect the generation of coagulation enzymes in vivo. It has been demonstrated that the factor VII-tissue factor pathway functions under normal conditions to generate factor Xa and convert prothrombin to thrombin. Furthermore, the factor VII-tissue factor pathway is also mainly responsible for the activation of factor IX with minimal contribution from the contact phase. However the relatively high levels of factor IXa generated are unable to convert factor X to factor Xa under basal conditions. Prospective studies are required to determine whether "biochemically" hypercoagulable individuals (i.e., those with elevated levels of free factor VIIa, activation peptides of factor IX, factor X, or prothrombin) are more likely to develop arterial or venous thrombosis.     

Mutation of amino acids 39-44 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.     

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.     

Genetic analysis and functional characterization of prothrombins Corpus Christi (Arg382-Cys), Dhahran (Arg271-His), and hypoprothrombinemia

The structural abnormalities and functional characteristics of dysfunctional prothrombin variants in two new kindreds have been determined. Prothrombin Corpus Christi (family 1) was purified and found to have markedly reduced fibrinogen clotting activity, yet normal amidolytic and near-normal platelet aggregating activity. A transition (C to T) at nucleotide position 8885, present in the heterozygous form in affected family members, resulted in the substitution of Cys for Arg 382. This substitution results in the loss of a positive charge within the fibrinogen-binding exosite of thrombin, thus accounting for the observed functional defect. A heterozygous C to T transition was also present at position 19994 in other family members with a hypoprothrombinemic phenotype. This mutation results in the replacement of Gln 541 (CAA) by a premature stop codon (TAA). Prothrombin Dhahran (family 2) was found to have markedly reduced fibrinogen clotting activity, but normal amidolytic activity. Affected family members were found to have a G to A transition at nucleotide position 7312 resulting in the substitution of His for Arg 271. This substitution results in the abolition of a factor Xa cleavage site, yielding meizothrombin rather than thrombin, on activation of prothrombin Dhahran by factor Xa. All but one of the above mutations occur at CpG dinucleotides, thus further supporting the observation of a high incidence of CpG transitions in hereditary dysprothrombinemia. The significant bleeding tendencies of individuals homozygous for prothrombin Dhahran (prothrombin clotting activity 5% to 7%) contrast sharply with the absence of significant chronic bleeding in the proband expressing prothrombin Corpus Christi (prothrombin clotting activity 2%). Our findings underscore the capacity of thrombin to contribute to clinical hemostasis by mechanisms other than its fibrinogen clotting activity.     

Human factor Va1 and factor Va2: properties in the procoagulant and anticoagulant pathways

Human plasma factor V is heterogeneous and yields two forms of activated factor V that bind with low (factor Va1) and high affinity (factor Va2) to phospholipids. The properties of factor Va1 and factor Va2 in the anticoagulant and procoagulant pathways were evaluated by comparing their sensitivity for inactivation by APC and their ability to act as cofactor in prothrombin activation. At low phospholipid concentrations and on membranes containing low amounts of phosphatidylserine (PS), factor Va1 was inactivated by APC at 15-fold lower rates than factor Va2, both in the absence and in the presence of protein S. At high phospholipid concentrations and on membranes with more than 15 mol % PS, factor Va1 and factor Va2 were inactivated with equal efficiency. Differences between cofactor activities of factor Va1 and factor Va2 in prothrombin activation were only observed on membranes with less than 7.5 mol % PS. Due to the different phospholipid requirements of APC-catalyzed factor Va inactivation and of expression of factor Va cofactor activity in prothrombin activation, the thrombin-forming capacity of factor V1 was 7-fold higher than that of factor V2 in a reaction system containing factor Xa, prothrombin, APC, protein S, vesicles with a phospholipid composition resembling that of activated platelets, and traces of thrombin to initiate prothrombin activation. This shows that in the process of generation, expression, and down-regulation of factor Va cofactor activity on physiological membranes, the overall procoagulant activity of factor V1 can considerably exceed that of factor V2.     

A case of cytophagic histiocytic panniculitis: successful treatment of recurrent attacks with steroid pulse therapy and oral cyclosporin A

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesis of several vascular and connective diseases. A key step in the discovery of bFGF receptor antagonists to mitigate these actions is to define the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential residue in this epitope using site-directed mutagenesis. Here we examined the role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor binding affinity. Replacement of Asn104 of bFGF by alanine reduced receptor binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor binding affinity-Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, Thr105 and Pro141 were individually replaced by alanine exhibited receptor binding similar to wild-type bFGF. By contrast, substitution of Tyr103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conclude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fold diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may stabilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.     

Thrombin activates factor XI on activated platelets in the absence of factor XII

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.     

Conformational conversion of antithrombin to a fully activated substrate of factor Xa without need for heparin

Regulation of the inhibitory activity of antithrombin, the principal inhibitor of the blood-clotting proteinases factor Xa and thrombin, is accomplished by binding to heparin. We report here an antithrombin variant in which serine at position 380, 14 residues N-terminal from the reactive bond and at a hinge point in the structure, was replaced by cysteine to test a proposed mechanism of heparin activation of antithrombin as an inhibitor of factor Xa. By derivatization of this cysteine with a bulky group, fluorescein, the antithrombin became permanently and fully activated toward reaction with factor Xa in a manner analogous to heparin activation, albeit as a substrate. These findings establish a structural basis for the mechanism of heparin activation of antithrombin against factor Xa in agreement with that proposed from an X-ray structure of antithrombin.     

Coagulation initiated on herpesviruses

Herpesviruses have been previously correlated to vascular disease and shown to cause thrombogenic and atherogenic changes to host cells. Herein we show that even in the absence of cells, purified cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can initiate thrombin production. Functional assays demonstrated that purified HSV-1 and HSV-2 provide the necessary phospholipid (proPL) for assembling the coagulation factors Xa and Va into prothrombinase, which is responsible for generating thrombin. These observations are consistent with our earlier studies involving CMV. The presence of proPL on all three herpesviruses was confirmed directly by flow cytometry and electron microscopy by using annexin V and factor Va, respectively, as proPL-specific probes. Of equal importance, we found that CMV, HSV-1, and HSV-2 were also able to facilitate factor Xa generation from the inactive precursor factor X, but only when factor VII/VIIa and Ca2+ were present. Monoclonal antibodies specific for tissue factor (TF), the coagulation initiator, inhibited this factor X activation and, furthermore, enabled identification of TF antigen on each virus type by flow cytometry and electron microscopy. Collectively, these data show that CMV, HSV-1, and HSV-2 can initiate the generation of thrombin by having essential proPL and TF activities on their surface. Unlike the normal cellular source, the viral activity is constitutive and, therefore, not restricted to sites of vascular injury. Thus cell-independent thrombin production may be the earliest event in vascular pathology mediated by herpesviruses.     

Effect on dissolution from halving methylphenidate extended-release tablets

Factor V is a single chain glycoprotein that plays an essential role in the regulation of blood coagulation. After initiation of coagulation, factor V is converted into factor Va through limited proteolysis. Factor Va acts as protein cofactor in the prothrombin-activating complex, which is comprised of the serine protease factor Xa, Ca2+ ions and a procoagulant membrane surface. Factor Va accelerates factor Xa-catalysed conversion of prothrombin into thrombin more than 10(4)-fold. The cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C (APC). The physiological importance of this regulatory pathway is demonstrated by the occurrence of hereditary thrombophilia in individuals with a genetic defect that makes factor Va less sensitive to proteolytic inactivation by APC (APC resistance).     

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.     

The assembly of the prothrombinase complex on adherent platelets

Prothrombinase complex assembly, in real time, on platelets adherent to immobilized von Willebrand Factor (vWf) was examined by total internal reflection fluorescence spectroscopy (TIRFS). Electron microscopy showed that the platelets adhered to vWf in a largely unactivated state and could be activated by thrombin. Antibody binding to glycoprotein (GP) Ib and functional GPIIb-IIIa receptor molecules on adherent platelet membranes monitored by TIRFS also indicated that platelets adhered in a largely unactivated state. Maximal expression of the receptor form of GPIIb-IIIa detected by antibody binding was seen only after thrombin stimulation of the adherent platelets. Antibody binding to GPIb was detected on adherent platelets. A reduction in antibody binding was observed after thrombin stimulation of the platelets, indicating a change in GPIb as a consequence of thrombin stimulation of the platelets. The binding of the protein components of the prothrombinase complex to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets was then studied individually. Factor Va bound to adherent and thrombin-stimulated adherent platelets with an estimated Kd of 58 nmol/L. Minimal factor Xa binding was observed on adherent platelets before thrombin stimulation. Factor Xa binding was, however, readily observed on thrombin-stimulated adherent platelets. This factor Xa binding was not saturable, and no Kd value could be estimated. Direct measurement of prothrombinase complex assembly was demonstrated by using an energy transfer phenomenon between fluorescein-labeled factor Va and rhodamine-labeled factor Xa. Prothrombinase complex assembly was observed on both adherent and thrombin-stimulated adherent platelets. The estimated Kd for the factor Va/factor Xa interaction was 4 nmol/L. TIRFS demonstrated that adherent platelets have the ability to support prothrombinase complex assembly, as shown by a direct energy transfer reaction between fluorescently labeled factors Va and Xa.     

The measurement of thrombin in clotting blood by radioimmunoassay

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.     

Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.     

Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats

Adult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dansyl Glu Gly Arg chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase. Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.