Abnormal optical waveform profiles in coagulation assays from patients with antiphospholipid antibodies.

Transmittance waveforms are the optical data generated during clot formation on photo-optical coagulation analyzers and are used to define specific events of the clotting reactions. Thus, a prothrombin time (PT) or an activated partial thromboplastin time (aPTT) can be divided into a pre-coagulation phase, a coagulation phase, and a post-coagulation phase. These phases are further characterized by parameters that define the timing, the rate, the 'slope', and the magnitude of the signal change of the reactions. We investigated the transmittance waveform parameters obtained during PT and aPTT of patients with antiphospholipid antibodies (APLA) who were or were not taking warfarin, normal donors, and non-APLA patients taking warfarin. An abnormal deflection in the pre-coagulation phase of the PT (called slope 1) was observed in 61.5% of the patients with APLA, in contrast to 5.9% of non-APLA patients taking warfarin (P= 0.0015). The presence of an abnormal PT slope 1 was reagent specific and was inversely correlated with the anticardiolipin antibody immunoglobulin G (IgG) level, which suggests that the abnormal PT slope 1 may reflect interactions between patient IgG and components from the thromboplastin, possibly phospholipids. The abnormal PT slope 1 values may be of diagnostic utility in the identification of patients with antiphospholipid syndromes.

Classification of factor deficiencies from coagulation assays using neural networks.

Activated partial thromboplastin time (APTT) and prothrombin time (PT) assays are widely used to screen for coagulation disorders and to monitor administration of therapeutic drugs. The analysis of data from coagulation assays has traditionally concentrated on determination of clot times (for APTT and PT) and magnitude of signal change during coagulation (e.g. for PT-based fibrinogen quantitation). The purpose of this study was to determine if the diagnostic power of these assays could be increased by using neural networks to interpret multiple parameters from these assays. Error back-propagation neural networks were trained using multiple variables derived from APTT and PT optical data for 200 normal and abnormal patient specimens. These networks were used to: (1) classify samples as either deficient or non-deficient with respect to individual blood components; and (2) estimate the approximate concentration of specific coagulation factors. Results indicated that these networks could be successfully trained to identify specific factor deficiencies at less than 30% normal levels with good specificity and variable sensitivity, but that they estimated actual concentrations poorly in most cases. These results support possible applications for neural networks identifying specific coagulation abnormalities from non-specific APTT and PT assays using expanded data parameter sets.

Properties of optical data from activated partial thromboplastin time and prothrombin time assays.

Changes in characteristics of optical transmittance data from coagulation assays were examined as a function of concentration of coagulation proteins or anticoagulants. Transmittance data were collected for activated partial thromboplastin time (APTT) and prothrombin time (PT) assays from: 1) plasmas prepared by mixing normal plasmas with deficient plasmas to give varying levels of coagulation proteins; 2) plasmas containing added heparin; and 3) 200 specimen plasmas that were also assayed for fibrinogen, coagulation factors, and other components. Optical profiles were characterized using a set of parameters describing onset and completion of coagulation, magnitude of signal change, rate of coagulation and other properties. Results indicated that parameters other than those typically reported for APTT and PT are associated with individual deficiencies, but that diagnosis of specimen status on the basis of optical data is complex. These results suggest possibilities for expanded interpretation of PT/APTT optical data for clinical or research applications.

Relationship between total prothrombin, native prothrombin and the International Normalized Ratio (INR).

Plasma levels of total prothrombin and fully-carboxylated (native) prothrombin were compared with results of prothrombin time (PT) assays for patients undergoing oral anticoagulant therapy. Mean concentrations of total and native prothrombin in non-anticoagulated patients were 119 +/- 13 micrograms/ml and 118 +/- 22 micrograms/ml, respectively. In anticoagulated patients, INR values ranged as high as 9, and levels of total prothrombin and native prothrombin decreased with increasing INR to minimum values of 40 micrograms/ml and 5 micrograms/ml, respectively. Des-carboxy-prothrombin increased with INR, to a maximum of 60 micrograms/ml. The strongest correlation was observed between native prothrombin and the reciprocal of the INR (1/INR) (r = 0.89, slope = 122 micrograms/ml, n = 200). These results indicated that native prothrombin varied over a wider range and was more closely related to INR values than either total or des-carboxy-prothrombin. Levels of native prothrombin were decreased 2-fold from normal levels at INR = 2, indicating that the native prothrombin antigen assay may be a sensitive method for monitoring low-dose oral anticoagulant therapy. The inverse relationship between concentration of native prothrombin and INR may help in identification of appropriate therapeutic ranges for oral anticoagulant therapy.

Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin.

Regions of hirudin important for its inhibitory activity with thrombin have been examined by site-directed mutagenesis. Since thrombin has a primary specificity for basic amino acids, each of the three basic residues and the histidine in hirudin were mutated to glutamine. Mutation of Lys-47 caused a small increase (9-fold) in the dissociation constant whereas the other mutations were without effect. These results indicate that hirudin is different from most other inhibitors of serine proteases in that interactions with the primary specificity pocket of its target enzyme are not crucial to its inhibitory activity. The acidic nature of the carboxyl region of hirudin was found to be important for its interaction with thrombin. Single and multiple mutations of carboxyl-terminal glutamate residues (57, 58, 61, and 62) to glutamine caused increases in the dissociation constant. This value increased with the number of mutations and reached a maximum of 61-fold when all four glutamate residues were mutated. Kinetic studies indicated that in all cases where an increase in dissociation constant was observed, it was predominantly due to a decrease in the association rate constant.

Preparation and characterization of proteolyzed forms of human alpha-thrombin.

The kinetics of the tryptic digestion of human alpha-thrombin were studied. Based on the results of these studies a procedure for the preparation of highly purified, active human beta-thrombin was developed. This beta-thrombin contained less than 5% of other thrombin forms, was active towards tripeptidyl paranitroanilide substrates, but had lost more than 99% of its fibrinogen cleaving activity. Protein-chemical characterization of beta-thrombin showed that it had been cleaved at a single site (Arg73-Asn74) in the beta-chain, in contrast to human beta-thrombin obtained by autolysis, which is cleaved at both Arg-62 and Arg-73.

Enzymatic properties of proteolytic derivatives of human alpha-thrombin.

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of regions of alpha-thrombin involved in its interaction with hirudin.

The contributions of various regions of human alpha-thrombin to the formation of the tight complex with hirudin have been assessed by using derivatives of thrombin. alpha-Thrombin in which the active-site serine was modified with diisopropyl fluorophosphate was able to bind hirudin, but its affinity for hirudin was decreased by 10(3)-fold compared to unmodified alpha-thrombin. Modification of the active-site histidine with D-Phe-Pro-Arg-CH2Cl resulted in a form of thrombin with a 10(6)-fold reduced affinity for hirudin. gamma-Thrombin is produced by proteolytic cleavage of alpha-thrombin in two surface loops corresponding to residues 65-83 and 146-150 in alpha-chymotrypsin [Berliner, L. J. (1984) Mol. Cell. Biochem. 61, 159-172; Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. The gamma-thrombin-hirudin complex had a dissociation constant that was 10(6)-fold higher than that of alpha-thrombin. Treatment of alpha-thrombin with pancreatic elastase resulted in a form of thrombin only cleaved in the loop corresponding to residues 146-150 in alpha-chymotrypsin, and this form of thrombin had only a slightly reduced affinity for hirudin. By using limited proteolysis with trypsin, it was possible to isolate beta-thrombin which contained a single cleavage in the loop corresponding to residues 65-83 in alpha-chymotrypsin. This form of thrombin had a 100-fold decrease in affinity for hirudin. Kinetic analysis of the binding of hirudin to beta-thrombin indicated that the 100-fold decrease in affinity was predominantly due to a decrease in the rate of association of the two molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine-pancreatic alpha amylase hydrolysis of substrates containing 6-deoxy-D-glucose and 6-deoxy-6-fluoro-D-glucose and the specificity of subsite binding.

Hydrolysis of 6-deoxyamylose and mono-6-deoxy-6-fluorocyclomaltoheptaose by porcine-pancreatic alpha amylase produces low-molecular-weight modified products, which have been analyzed by chemical and chromatographic techniques. Results for both substrates show that modified D-glucose and two isomers of modified maltoses are produced in the enzyme reaction. In addition, the formation of maltoses modified in the nonreducing residue is more favored than the formation of maltoses modified in the reducing residue. These results indicate that productive binding of 6-fluoro- and 6-deoxy-D-glucose residues is permitted at subsites 1 through 4 of the amylase-active site but that binding of these modified residues may be less favorable at subsite 3, the subsite at which catalytic attack occurs.

The effect of substrate modification on porcine pancreatic alpha-amylase subsite binding: hydrolysis of substrates containing 2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose.

Modified alpha-D-(1----4)-glucans containing a small proportion of 14C-labeled 2-deoxy-D-glucose or 2-amino-2-deoxy-D-glucose were examined as substrates for porcine pancreatic alpha-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-D-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-D-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-D-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [14C]-2-amino-2-deoxy-D-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-D-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs.

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues.

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.

Porcine pancreatic alpha-amylase hydrolysis of hydroxyethylated amylose and specificity of subsite binding.

Hydrolysis of partially hydroxyethylated amylose by porcine pancreatic alpha-amylase gives rise to a number of hydroxyethylated di-, tri-, and tetrasaccharides, as well as larger products. No modified monosaccharides were detected. The structures of the products containing two to four D-glucose residues have been analyzed by chromatographic and enzymatic techniques. In no instance were these oligosaccharides modified in the reducing-end residue. The location of hydroxyethylated glucose residues within the oligosaccharides has been interpreted in terms of the ability of that (hydroxyethyl)glucose to bind productively at each of the five subsites of the enzyme active site. Results indicate that subsite 3, the subsite at which catalytic attack occurs, is especially sensitive to changes in the substrate and that unmodified glucose is required for productive binding at this subsite. Other subsites specifically allow binding of some (hydroxyethyl)glucose isomers, but not others. Hydroxyethylation is permitted at C-2, C-3, and C-6 for residues bound at subsite 1 and is permitted at C-6 and possibly at C-2 and C-3 for residues bound at subsite 5. However, substitution is permitted only at C-3 and C-6 for binding at subsite 2 and at C-2 and C-3 for binding at subsite 4.