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Biomedical subjects

K A Bauer

Publications and source records attributed to K A Bauer.

At least 127 records · Page 7Linked to original sources
PubMed1987

Expression and characterization of human antithrombin III synthesized in mammalian cells.

Antithrombin III (ATIII) has been expressed in transiently transfected COS-1 monkey cells and in stably transformed Chinese hamster ovary cells, and the resultant protein has been characterized for biological activity. Both cell types efficiently secrete high levels of heterogeneous molecular weight forms of ATIII antigen. The heterogeneity results from differences in post-translational modifications. However, only a small percentage (5-10%) of the total antigen expressed is biologically active. The fraction of biologically active ATIII has been purified from total ATIII by affinity fractionation on heparin-Sepharose. This fractionation indicates that the differences in the active and inactive forms of expressed ATIII result from differences in their ability to bind heparin. Purified ATIII has a specific activity very similar to that of plasma-derived ATIII and exhibits typical heparin-accelerated ATIII activity. The biologically active fraction of ATIII appears to represent the higher molecular weight forms of the ATIII expressed and is likely not a result of altered asparagine-linked glycosylation; however, the nature of the post-translational modification required for ATIII activity remains unclear. The ability to express biologically active ATIII at such high levels should allow further investigations of the structural requirements for ATIII activity.

Antithrombin III↗
PubMed1987

Dysfunctional activated protein C (PC Cádiz) in a patient with thrombotic disease.

The partial characterization of a dysfunctional protein C (PC), provisionally named "PC Cádiz", in a 45-year-old male patient suffering from recurrent venous thrombosis is described. The only defect found in laboratory assays for haemostasis and hepatic function was a half normal level of both amidolytic and anticoagulant protein C activity, measured by different functional assays that use thrombin-thrombomodulin complex and a snake venom to activate protein C. Protein C antigen was always found to be within normal levels. Two young daughters of the propositus were found to have the same defect. Double-crossed immunoelectrophoresis, performed in the presence and absence of Ca2+ in the first dimension, showed no clear differences between patient and control PC. PC adsorption to barium salts was also found to be normal. Measurement of the PC activation peptide in the barium citrate eluates after PC activation showed no significant differences between patient and 10 normal controls, the concentration of this peptide being very similar to that of PC zymogen in the same eluates before PC activation. These results indicate that this abnormal PC is able to be normally activated by thrombin-thrombomodulin complex but does not exhibit serine protease activity, probably due to a defect in the PC molecule near the active site center.

Humans↗
PubMed1987

Thrombosis in inherited deficiencies of antithrombin, protein C, and protein S.

The coagulation cascade can be pictured as a series of reactions in which a zymogen, a cofactor, and a converting enzyme interact to form a multimolecular complex on a natural surface. In each case, the four reactants must be present if the conversion of a zymogen to the corresponding serine protease is to take place at any significant rate. The principal natural anticoagulant systems that are able to exert damping effects on the various steps of the cascade are the heparin-antithrombin and protein C-thrombomodulin mechanisms that regulate the serine proteases and the cofactors or activated cofactors, respectively. Inherited thrombotic disorders associated with specific deficiencies of antithrombin, protein C, and protein S have been described. This review describes the biochemistry and physiology of these endogenous anticoagulant systems. The development of specific radioimmunoassay techniques for prothrombin activation fragment F1 + 2, fibrinopeptide A, and protein C activation peptide has allowed us to carry out studies of these endogenous regulatory mechanisms involved in thrombin generation in patients with deficiencies of antithrombin or protein C. This information is then used to construct a framework for understanding the pathophysiology of the prethrombotic and actively thrombotic states in humans with these clinical disorders.

Antithrombins↗
PubMed1987

Aging-associated changes in indices of thrombin generation and protein C activation in humans. Normative Aging Study.

In view of the known association of vascular disease with increasing age, we have conducted an analysis of hemostatic system activity with respect to perturbations induced by aging phenomena. We have utilized an immunochemical assay for prothrombin fragment F1 + 2 to quantify Factor Xa activity upon prothrombin in the plasma of 199 healthy males between the ages of 42 and 80. The levels of F1 + 2 in this population generally increased as a function of age (P less than 0.0001). The metabolic behavior of this marker was determined in 10 individuals greater than 65 yr of age with varying levels of F1 + 2, which ranged from 1.28 to 5.85 nM. The elevations in the concentration of this component were not due to diminished clearance of the fragment. Radio-immunoassays for fibrinopeptide A (FPA) and the protein C activation peptide (PCP) were subsequently employed to measure thrombin activity upon fibrinogen and thrombin-thrombomodulin activity upon protein C, respectively, in 82 members of this population ranging in age from 42 to 80. Significant positive correlations were again observed between increasing age and the level of F1 + 2 (P less than 0.0001) as well as FPA (P less than 0.01) and PCP (P less than 0.002). The results of this cross-sectional study indicate that many apparently normal males of increasing age with normal immunologic levels of antithrombin III and protein C exhibit a biochemical defect that denotes the presence of an acquired prethrombotic state.

Adult↗
PubMed1987

Suppression of hemostatic system activation by oral anticoagulants in the blood of patients with thrombotic diatheses.

RIAs for hemostatic system activation were employed to study patients who were anticoagulated with warfarin. The mean prothrombin fragment F1 + 2 concentration in stably anticoagulated individuals without an inherited thrombotic diathesis (mean prothrombin time [PT] ratio [PT of patient/PT of normal plasma pool] = 1.74) was 0.231 nM as compared with a mean plasma F1 + 2 level of 1.68 nM for a nonanticoagulated control group (P less than 0.0001). The initiation of oral anticoagulants in two subjects who did not exhibit protein C deficiency led to a paradoxical increase in F1 + 2 levels during the first day of therapy. We have also shown that a relatively low intensity regimen of warfarin (PT ratio less than 1.2) may reduce elevated concentrations of F1 + 2 into the normal range in patients with a history of recurrent thromboembolism. The mean F1 + 2 level in antithrombin-deficient individuals on warfarin was significantly elevated (mean = 0.714 nM) as compared with that in anticoagulated subjects with protein C deficiency (mean = 0.205 nM) or in those without an inherited thrombotic disorder (P less than 0.01) at equivalent levels of intensity of oral anticoagulation. We therefore conclude that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-antithrombin mechanism.

Administration, Oral↗
PubMed1987

The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation.

Numerous investigators have postulated that a hypercoagulable state exists in humans for a period of time before the development of thrombotic episodes. A clear biochemical definition of the prethrombotic state, however, has proved elusive due in part to the lack of reliable techniques for monitoring pertinent changes in blood coagulability. Based on recent advances in our knowledge of the biochemistry of the coagulation system, a series of highly sensitive and specific immunochemical tools has been developed that can quantitate the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for F1+2 and the protein C activation peptide, which measure the cleavage of the prothrombin molecule by factor Xa and the scission of protein C by the thrombin-thrombomodulin complex, respectively. Nossel and coworkers had previously constructed similar assays for fibrinopeptide A (FPA) and fragment B beta 1-42, which monitor the cleavage of fibrinogen by thrombin and the proteolysis of fibrin I by plasmin, respectively. Substantial elevations in the levels of these markers have been found in patients with disseminated intravascular coagulation and many subjects with acute deep venous thrombosis. The F1+2 and FPA assays have been used to demonstrate that significant increments in factor Xa activity but not thrombin activity regularly occur in the blood of nonanticoagulated individuals with congenital deficiencies of antithrombin or protein C. These two disorders are known to be correlated with the subsequent development of thrombosis. Patients with protein C deficiency have also been noted to have significantly reduced plasma levels of protein C activation peptide. By using the immunoassays for FPA and B beta 1-42 in studies of postoperative patients, it has been shown that an imbalance between the procoagulant action of thrombin and the anticoagulant effect of plasmin on fibrin I polymer may induce an acquired thrombotic diathesis. Finally, we have recently demonstrated that prothrombin activation as measured by the F1+2 assay is suppressed by oral anticoagulants in the blood of patients with thrombotic diatheses. These investigations suggest that these assay techniques can be used to improve our understanding of the hypercoagulable state as well as to develop more effective treatment strategies for the prevention of thromboembolic events.

Administration, Oral↗
PubMed1987

Development of a radioimmunoassay for quantitating prethrombin 2 in human plasma.

We have developed a radioimmunoassay (RIA) for prethrombin 2 (Pr2), a potential intermediate in the transformation of prothrombin to thrombin. Antisera against human Pr2 were raised in rabbits and the respective immunoglobulin G fractions were chromatographed on prothrombin-Sepharose. The specific antibody population obtained was used to construct a double-antibody RIA capable of measuring as little as 0.05 nmol/L of this component. The immunoreactivity of prothrombin was approximately 40,000 times less than that of Pr2 on a molar basis. Because of nonspecific contributions of plasma constituents to the immunoreactive signal, the measurement of Pr2 in this milieu required the use of a titration curve in which Pr2 was added back to Pr2-depleted plasma. This assay was then used to determine the levels of this species in two patient populations with increased prothrombin activation as determined by the prothrombin fragment F1 + 2 RIA, a measure of the in vivo cleavage of prothrombin by factor Xa. The mean Pr2 concentrations in eight patients with disseminated intravascular coagulation and six asymptomatic individuals with congenital antithrombin deficiency not receiving antithrombotic therapy were not significantly elevated as compared with those of normal controls (0.244 nmmol/L and 0.242 nmol/L vs. 0.184 nmol/L, respectively). Our studies show that Pr2 is cleared from the plasma of dogs with a t1/2 of approximately 25 minutes. Given that the t1/2 of F1 + 2 is estimated to be approximately 90 minutes, the low plasma levels of Pr2 observed in patients with thrombophilia cannot result from rapid clearance of this component.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency.

The presence of congenital antithrombin deficiency has been consistently shown to predispose patients to venous thrombosis. We have utilized the prothrombin fragment F1+2 radioimmunoassay to quantitate factor Xa activity in the blood of 22 asymptomatic individuals with this clinical disorder not receiving antithrombotic therapy. The mean level of F1+2 was significantly elevated in these patients as compared to normal controls (3.91 vs. 1.97 nM, P less than 0.001). The metabolic behavior of 131 I-F1+2 was found to be similar in antithrombin-deficient subjects and normal individuals. The hemostatic system hyperactivity as measured by the F1+2 assay could be specifically corrected by raising the plasma antithrombin levels of the above asymptomatic individuals into the normal range. This study provides the first demonstration that the prethrombotic state can be biochemically defined as an imbalance between the production and inhibition of factor Xa enzymatic activity within the human circulation. It is known that antithrombin and alpha 1-proteinase inhibitor (PI) are the major inhibitors of factor Xa in human plasma in the absence of heparin. To further evaluate the mechanism by which antithrombin functions as an inhibitor of factor Xa in humans, we studied five patients who exhibited severe congenital deficiencies of alpha 1-PI. Our results indicated that the plasma of these subjects showed virtually identical decreases in plasma antifactor Xa activity in the absence of heparin when compared to antithrombin-deficient individuals, but the plasma F1+2 levels in the alpha 1-PI deficient population were not significantly different than normal. This data suggests that alpha 1-PI does not function as a major inhibitor of factor Xa in vivo, and that a tonically active heparin-dependent mechanism exists in humans for accelerating the neutralization of this enzyme by antithrombin.

Adolescent↗
PubMed1985

Abnormal factor VIII coagulant antigen in patients with renal dysfunction and in those with disseminated intravascular coagulation.

Factor VIII antigen (VIII:CAg) exhibits molecular weight heterogeneity in normal plasma. We have compared the relative quantities of VIII:CAg forms present in normal individuals (n = 22) with VIII:CAg forms in renal dysfunction patients (n = 19) and in patients with disseminated intravascular coagulation (DIC; n = 7). In normal plasma, the predominant VIII: CAg form, detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was of molecular weight 2.4 X 10(5), with minor forms ranging from 8 X 10(4) to 2.6 X 10(5) D. A high proportion of VIII:CAg in renal dysfunction patients, in contrast, was of 1 X 10(5) mol wt. The patients' high 1 X 10(5) mol wt VIII: CAg level correlated with increased concentrations of serum creatinine, F1+2 (a polypeptide released upon prothrombin activation), and with von Willebrand factor. Despite the high proportion of the 1 X 10(5) mol wt VIII:CAg form, which suggests VIII:CAg proteolysis, the ratio of Factor VIII coagulant activity to total VIII:CAg concentration was normal in renal dysfunction patients. These results could be simulated in vitro by thrombin treatment of normal plasma, which yielded similar VIII:CAg gel patterns and Factor VIII coagulant activity to antigen ratios. DIC patients with high F1+2 levels but no evidence of renal dysfunction had an VIII:CAg gel pattern distinct from renal dysfunction patients. DIC patients had elevated concentrations of both the 1 X 10(5) and 8 X 10(4) mol wt VIII:CAg forms. We conclude that an increase in a particular VIII:CAg form correlates with the severity of renal dysfunction. The antigen abnormality may be the result of VIII:CAg proteolysis by a thrombinlike enzyme and/or prolonged retention of proteolyzed VIII:CAg fragments.

Aged↗
PubMed1985

Catalytic activity and platelet reactivity of heparin covalently bonded to surfaces.

Heparin was covalently bound to solid substrate surfaces by means of four different chemistries. It was coupled to polymethylacrylate (PMA) beads with glutaraldehyde, carbodiimide, or radical polymerization initiated by Ce4+, or to agarose beads with cyanogen bromide. Each of these chemistries produced measurable amounts of surface-bound heparin, which was minimally elutable in contact with plasma. Antithrombin (AT) binding by heparinized PMA materials (compared with PMA control beads) ranged from no AT binding for the material heparinized with carbodiimide (PMA-Alb-Hep(EDC] to 3.6 micrograms/ml packed beads for the material heparinized by radical polymerization (PMA-MA-Hep). Heparin-like catalytic activity of these materials (assayed by measuring the generation of thrombin-antithrombin complex in plasma) correlated well with the amount of heparin bound, but not as well with AT binding capacity. Heparinized agarose, which exhibited a large AT binding capacity (2.2 mg AT per milliliter of packed gel), had virtually no catalytic activity because of its inability to release thrombin-antithrombin complex from the surface. Platelet interaction with heparinized materials that exhibit high AT binding capacity was reduced by pretreatment with normal plasma but not by pretreatment with AT-depleted plasma. Platelet interaction with heparinized materials with low AT binding capacities was not reduced by pretreatment with normal plasma. We conclude that AT binding by heparin reduces the platelet reactivity of heparinized surfaces.

Antithrombin III↗
PubMed1984

Detection of protein C activation in humans.

We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the coagulation system at concentrations considerably less than 1.0 nM.

Amino Acid Sequence↗
PubMed1984

Thrombin generation in acute promyelocytic leukemia.

Six patients with disseminated intravascular coagulation (DIC) in association with acute promyelocytic leukemia (APL) were studied with sensitive radioimmunoassays that are able to quantitate the extent of thrombin generation within the human circulation. The levels of prothrombin activation fragment, F1 + 2, and thrombin-antithrombin complex (TAT) were obtained at clinical presentation and were then followed serially in several patients during induction chemotherapy. The antileukemic therapy often resulted in a rise in the plasma levels of these molecular species. Simultaneous measurements of fibrinopeptide A (FPA) were also obtained, and the concentrations of this polypeptide were correlated with the levels of F1 + 2 and TAT in patients who were not receiving heparin. Nine individuals with other morphological subtypes of acute nonlymphocytic leukemia (ANLL) were investigated and were usually found to have increased levels of F1 + 2, TAT, and FPA at clinical presentation. However, the magnitude of the elevations was considerably greater and the correlation between TAT and FPA levels was stronger in APL than in ANLL. These studies provide direct evidence that patients with APL, as well as ANLL, generate excessive amounts of thrombin within their vascular system. Furthermore, the data suggest that the concentrations of F1 + 2, compared with the levels of FPA, may be a more sensitive indicator of hemostatic system hyperactivity in individuals with DIC.

Adult↗
PubMed1983

Molecular heterogeneity of inherited antithrombin III deficiency.

Inherited antithrombin III deficiency is associated with an increased risk of thromboembolism. Using recombinant-DNA techniques, we isolated a molecular probe for the antithrombin III structural gene and identified a common DNA polymorphism within the gene. We found that there is genetic heterogeneity in this disorder. In one family, the antithrombin III gene was deleted in affected members, whereas in another no deletion occurred. Use of the DNA polymorphism should allow identification and further characterization of abnormal antithrombin III genes.

Adolescent↗
PubMed1983

L-asparaginase induced antithrombin III deficiency: evidence against the production of a hypercoagulable state.

L-asparaginase, a chemotherapeutic agent employed in the treatment of acute lymphocytic leukemia (ALL), is known to depress the synthesis of numerous plasma proteins. The plasma concentration of the major protease inhibitor of the coagulation mechanism, antithrombin III, is substantially decreased in patients receiving this drug. This observation has generated speculation that L-asparaginase may induce a hypercoagulable state in humans. To examine this hypothesis, we studied ten patients with ALL in remission who were being treated with the above chemotherapeutic agent. Our data revealed that infusion of this enzyme leads to a marked decrease in the plasma concentrations of prothrombin as well as antithrombin III. However, we have also observed a constant level of thrombin generation during the same period of time as monitored by plasma levels of prothrombin activation fragment (F1 + 2) and thrombin-antithrombin complex (TAT). Based upon these findings we suggest that administration of L-asparaginase does not usually lead to the induction of a hypercoagulable state.

Adolescent↗