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Biomedical subjects

M A Blajchman

Publications and source records attributed to M A Blajchman.

At least 19 recordsLinked to original sources
PubMed1992

Molecular basis of inherited human antithrombin deficiency.

Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.

Alleles↗
PubMed1992

Partial deletion of an antithrombin III allele in a kindred with a type 1 deficiency.

This report details the precise mapping of a partially deleted human antithrombin III (AT-III) allele, found in a kindred with an inherited type 1 AT-III deficiency. Using truncated AT-III probes generated by polymerase chain reaction (PCR) amplification from a full-length AT-III cDNA, as well as other genomic probes specific for the 5' upstream region of the AT-III gene, we were able to characterize a partial deletion on an AT-III allele encompassing exons 1 and 2 of the AT-III gene, and a region 5' to the coding sequences. The absence of the 5' upstream region in the affected AT-III allele was confirmed directly by the PCR amplification of a 1.5-kb polymorphic fragment of genomic DNA samples from family members. The precise determination of the 5' breakpoint of the affected allele was made possible by two different approaches: (1) subcloning plus biotin capture PCR, or (2) inverse PCR. This allowed us to confirm the mapping of the deletion obtained by Southern analysis; to show that the 3' region of the mutant AT-III allele, including exons 3 to 7, was intact; and to sequence approximately 0.7 kb upstream to the breakpoint in the mutant allele. Furthermore, PCR amplification of the region of the breakpoint provided unique products detectable only in affected members of this kindred. The breakpoint in the partially deleted allele is 480 bp upstream from the 5' boundary of exon 3. No significant homology was found between the 0.7-kb sequence upstream to the breakpoint of the mutant allele and known human sequences.

Alleles↗
PubMed1992

Molecular cloning and cell-free expression of mouse antithrombin III.

The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

Amino Acid Sequence↗
PubMed1992

Measurement of markers of activated coagulation in antithrombin III deficient subjects.

Functional antithrombin III levels were measured by factor Xa inhibition in 63 members of a large family with type 2 antithrombin III deficiency and individuals were classified as antithrombin III deficient or non-deficient according to the results. F1 + 2 and TAT complexes were measured using an ELISA and FPA levels were measured by radioimmunoassay. Thirty subjects (48%) were classified as antithrombin III deficient and 33 (52%) as antithrombin III non-deficient. The mean level of F1 + 2 was significantly higher in the deficient adults (0.87 +/- 0.26) compared to both the non-deficient adults (0.70 +/- 0.21) (p = 0.03) and the deficient adults receiving warfarin (0.16 +/- 0.01) (p less than 0.001). The differences in the mean values of TAT complexes and FPA between deficient and non-deficient individuals were not statistically significant. These findings suggest that untreated antithrombin III deficient subjects generate more thrombin than their non-deficient family members and that warfarin inhibits this thrombin formation. In this cross-sectional study, it is not possible to correlate the levels of the surrogate makers with future clinical outcome.

Adolescent↗
PubMed1992

Inhibition of the amplification reactions of blood coagulation by site-specific inhibitors of alpha-thrombin.

Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.

Amino Acid Sequence↗
PubMed1992

Thrombosis in antithrombin-III-deficient persons. Report of a large kindred and literature review.

PURPOSE: To estimate the prevalence of objectively proven thrombotic complications in antithrombin-III-deficient persons. STUDY DESIGN: Cross-sectional study and a critical review of the literature. DATA SOURCES AND EXTRACTION: The prevalence of thrombosis in antithrombin III-deficient and -nondeficient family members of a large kindred was estimated by history, review of diagnostic tests, and examination for venous reflux by Doppler ultrasonography, as an indicator of previous venous thrombosis. A MEDLINE search and literature review of the published English- and French-language literature from 1966 to 1990 that described antithrombin-III-deficient families was done, and the following information was obtained: the prevalence of thrombosis in deficient and nondeficient family members, the presence or absence of risk factors for thrombosis (surgery, pregnancy, the postpartum state, use of oral contraceptives, immobilization, metastatic cancer, major trauma) at the time of the thrombotic event, and age of onset of the first episode of thrombosis. The validity of the studies was assessed according to predetermined criteria. RESULTS: Sixty-seven research subjects were evaluated. Six of 31 (19.4%) antithrombin-III-deficient subjects compared with none of 36 (0%) nondeficient subjects had had one or more thrombotic events. The initial episode in five of six subjects had occurred in association with risk factors for thrombosis. The literature search indicated that the pooled prevalence of symptomatic venous thrombosis among the deficient subjects was 51%, but objective testing was done in only 17% of these subjects at the time of presentation. CONCLUSION: Based on the data from this antithrombin-III-deficient kindred, lifelong anticoagulant prophylaxis does not appear to be warranted in asymptomatic carriers, and prophylaxis could be limited to periods of high risk for thrombosis.

Adolescent↗
PubMed1992

Molecular cloning and expression of rabbit antithrombin III.

A cDNA containing the complete open-reading frame encoding rabbit antithrombin III (AT-III) was isolated from a rabbit liver cDNA expression library, using a specific antibody as a probe. Sequence analysis showed 84% identity between the deduced amino acid sequences of the rabbit and human proteins. A previously described cell-free expression system was used to verify the identity of the clone. The full-length cDNA was inserted into an expression vector, and messenger RNA (mRNA) transcripts generated. In vitro translation of these transcripts, in the presence of [35S]methionine, in an mRNA-dependent rabbit reticulocyte lysate system resulted in the synthesis of a 51-Kd polypeptide, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This nonglycosylated protein was capable of forming SDS-stable complexes with human alpha-thrombin. Complex formation was significantly enhanced following the deletion of nucleotides encoding the signal peptide, and the resultant generation of a 47-Kd nonglycosylated mature protein product. When the template DNA giving rise to this product was internally truncated, two rabbit AT-III deletion mutants were generated that lacked the ability to interact with thrombin, but retained the ability to bind heparin. Cell-free expression plasmids encoding the human and rabbit AT-III mature molecules were manipulated to produce two interspecies fusion proteins. For the first, human codons were used to replace rabbit codons from residue 369-433, while in the second human codons replaced rabbit codons from residue 217-433. Both fusion proteins exhibited less efficient thrombin-complexing ability than the original cell-free-derived mature rabbit AT-III. Thus, portions of AT-III molecules from the two species, despite their high degree of homology, are not interchangeable. Knowledge of the structure of rabbit AT-III, combined with the availability of the rabbit cDNA, will permit defined experimentation aimed at understanding antithrombin III structure relative to its function in vivo.

Amino Acid Sequence↗
PubMed1992

Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity.

Antithrombin-III-Stockholm is a new structural variant of antithrombin-III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT-III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha-thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

Adult↗
PubMed1992

The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, alpha-thrombin.

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.

Antithrombin III↗
PubMed1992

An animal model of allogeneic donor platelet refractoriness: the effect of the time of leukodepletion.

Approximately 50% of multi-transfused individuals become refractory to random donor platelets. Recent clinical data suggest that those patients receiving leukocyte-depleted blood products are less likely to become refractory to random donor platelets than recipients of non-leukocyte-depleted products. Leukocyte depletion can be performed immediately after collection of a unit of whole blood before its storage (prestorage leukodepletion) or just before the transfusion of the blood product to a recipient, after its storage (poststorage leukodepletion). However, the most appropriate time for the leukodepletion of blood products has not been established. The present study was undertaken to establish an animal model of allogeneic platelet refractoriness, and to compare the effect of prestorage and poststorage leukodepletion on the frequency of refractoriness to allogeneic donor platelets. In this model, two strains of rabbits were used: California Black rabbits were used as blood donors, while New Zealand White rabbits were used as recipients. Eight weekly infusions of nonleukodepleted allogeneic fresh blood resulted in an allogeneic platelet refractory rate of 91.2% (31/34). The prestorage leukodepletion of the donor blood was associated with a significantly higher allogeneic platelet survival and lower refractory rate (33.3%) to allogeneic platelets than poststorage leukodepletion (66.7%). Furthermore, the data suggest that cell-free plasma products are capable of inducing refractoriness to allogeneic donor platelets; the stored plasma having a greater likelihood of inducing such refractoriness than fresh plasma. Thus, these data provide evidence that the prestorage leukodepletion of allogeneic donor blood is associated with a lower frequency of refractoriness and better allogeneic platelet survival than poststorage leukodepletion.

Animals↗
PubMed1992

The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor.

The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51-88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the beta-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.

Antibodies, Monoclonal↗
PubMed1992

Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082.

The minimum concentrations of heparin, dermatan sulfate, hirudin, and D-Phe-Pro-ArgCH2Cl required to delay the onset of prothrombin activation in contact-activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin III is an octadecasaccharide with high affinity for antithrombin III. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor II can catalyze thrombin inhibition by heparin cofactor II. A highly sulfated bis(lactobionic acid amide), LW10082 (Mr 2288), which catalyzes thrombin inhibition by heparin cofactor II and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.

Antithrombin III↗
PubMed1992

Effect of allogeneic blood transfusion on solid tumor growth and pulmonary metastases in mice.

The effect of allogeneic blood transfusions on solid tumor growth and pulmonary metastases was examined in two different strains of mice. Recipient mice (C57B1 or DBA/2) were given transfusions from allogeneic donors (Balb/c or B6AF1, respectively). The effect of allogeneic blood transfusion on solid tumor growth (B16 in C57B1 mice and P815 in B6AF1 mice) as well as the number of pulmonary metastases (B16 in C57B1 mice) was examined utilizing inoculations of varying numbers of tumor cells. In both solid tumor models, allogeneic transfusion resulted in significant enhancement of tumor growth when smaller (1.25 x 10(5), 2.5 x 10(5)) numbers of tumor cells were inoculated into the host animal. In contrast, no effect of allogeneic transfusion on tumor growth was observed when higher (4.5 x 10(5)) numbers of tumor cells were inoculated. Similarly, increased numbers of pulmonary metastases following allogeneic blood transfusion were observed when lower numbers (1 x 10(5)) of B16 tumor cells were administered; whereas no effect was observed with higher (4.5 x 10(5)) tumor cell numbers. The data in the present study suggest that the number of tumor cells inoculated into the recipient animal has a strong bearing in the allogeneic blood-transfusion-induced tumor growth effect.

Animals↗
PubMed1992

The low molecular weight heparin Enoxaparin inhibits the consumption of factor VII and prothrombin activation in vivo associated with elective knee replacement surgery.

Patients over 40 years of age who undergo elective orthopaedic surgery have a relatively high risk for developing post-surgical deep vein thrombosis (DVT). Prophylactic use of heparin or low molecular weight heparins can reduce the incidence of post-operative DVT by up to 80%. It is not known whether prophylaxis is achieved by inhibition of prothrombin activation or catalysis of thrombin inhibition in vivo. We determined the changes in concentrations of factor VII zymogen and thrombin-antithrombin III (the latter as an index of prothrombin activation) in the plasmas of 129 patients randomized to receive two daily subcutaneous injections of placebo or 30 mg of Enoxaparin after elective knee surgery. Enoxaparin reduced the frequency of post-surgical DVT by 70%. The concentration of factor VII zymogen had decreased by approximately 50% within 24 h after the knee surgery, followed by a gradual increase to near presurgical values. Additionally, post-Enoxaparin plasmas had statistically significant higher concentrations of factor VII zymogen than post-placebo plasmas. Post-Enoxaparin plasmas had significantly lower concentrations of endogenous thrombin-antithrombin III than comparable post-placebo plasmas. Finally, post-Enoxaparin plasmas inactivated exogenous factor Xa and thrombin more effectively than comparable post-placebo plasmas. As Enoxaparin moderated the generation of endogenous thrombin-antithrombin III after elective knee surgery, inhibition of prothrombin activation in vivo by Enoxaparin may be important for its prophylactic antithrombotic effect.

Adult↗
PubMed1992

Prophylactically equivalent doses of Enoxaparin and unfractionated heparin inhibit in vivo coagulation to the same extent.

This study compared how Enoxaparin and unfractionated (UF) heparin influenced in vivo coagulation in patients randomized to receive, by twice daily subcutaneous injections, either 30 mg of Enoxaparin or 7500 I.U. of UF heparin after elective hip surgery. These two regimens were equally effective in reducing the incidence of post-operative deep vein thrombosis DVT. We compared the concentrations of endogenous thrombin-antithrombin III in pre- and post-surgical plasmas to determine how each prophylactic regimen influenced prothrombinase activity in vivo, and found the same concentrations of endogenous thrombin-antithrombin III in post-heparin and post-Enoxaparin plasmas. However, significantly higher concentrations of endogenous thrombin-antithrombin III were found in pre- and post-surgical plasmas of patients who developed post-operative DVT than the levels found in comparable plasmas of patients who remained DVT-negative, regardless of the drug received for prophylaxis. Human factor Xa was added to an equal volume of each patient's plasmas and the amount of added enzyme inactivated by antithrombin III measured using an enzyme-linked immunosorbent assay for factor Xa-antithrombin III. Post-heparin and post-Enoxaparin plasmas inactivated approximately 4 times more factor Xa than the pre-surgical plasmas, regardless of the clinical outcome. Thus, before and after surgery, a higher than normal in vivo prothrombinase activity may be a significant risk factor for developing post-operative DVT.

Antithrombin III↗
PubMed1992

A novel congenital haemostatic defect: combined factor VII and factor XI deficiency.

Isolated deficiencies of factors VII and XI are both rare. Not surprisingly, therefore, combined factor VII and XI deficiency has not been reported previously. We report here a kindred with a combined heterozygous deficiency for both factors VII and XI. The proposita is a 28-year-old woman who had both a prolonged prothrombin time (PT) and a prolonged activated partial prothrombin time (APTT) associated with a mild bleeding tendency. Coagulation studies were performed on the six available members of this kindred. The PT and APTT were normal or mildly abnormal in five of these individuals. Factor VII coagulant activity (VII:C) varied from 0.33 to 0.77 units/ml in affected subjects. In contrast, the concentration of factor VII-related antigen for the six individuals ranged from 0.68 to 2.10 units/ml. Comparable factor VII:C levels were obtained when each subject's plasma was tested with either a rabbit or a human thromboplastin reagent. Factor XI coagulant activity was less than 0.5 units/ml in three of the six subjects and normal (approximately 1.0 units/ml) in the other three. The concentrations of thrombin-antithrombin-III and prothrombin fragment 1.2 were within normal limits for all individuals. In addition to being associated with heterozygous factor XI deficiency, the abnormal factor VII molecule in the plasma of affected individuals in this kindred appears to represent a newly described mutation. This is suggested by the pattern of reactivity with thromboplastin from different species, the normal tissue factor binding and the bleeding tendency in heterozygous individuals in this kindred.

Adult↗
PubMed1992

Pseudopolycythemia, pseudothrombocytopenia, and pseudoleukopenia due to overfilling of blood collection vacuum tubes.

A patient blood sample with an unexpectedly high hemoglobin level, high hematocrit, low white blood cell count, and low platelet count was recognized as being spurious based on previously available data. Repeated testing of the original sample showed a gradual return of all parameters to expected levels. We provide evidence that the overfilling of blood collection vacuum tubes can lead to inadequate sample mixing and that, in combination with the settling of the cellular contents in the collection tubes, can result in spuriously abnormal hematological parameters as estimated by an automated method.

Aged↗