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The use of antithrombin III (ATIII) replacement in combination with low-dose heparin therapy in prevention of postoperative venous thrombosis in patients following total hip replacement or total knee replacement was evaluated. A randomized prospective venographically controlled trial in hip replacement compared treatment with dextran 40 with a regimen of ATIII (1,500 units preoperatively and 1,000 units daily for five days) and low-dose heparin. Patients receiving ATIII/heparin had significantly higher postoperative ATIII concentrations than dextran-treated patients and also had a low incidence of venous thromboembolic disease (7 percent). The ATIII/heparin regimen was well tolerated with no increase in bleeding or significant prolongation of the activated partial thromboplastin time. Two cohorts of patients undergoing total knee replacement were studied using different doses of ATIII in combination with heparin. An initial 10 patients were treated with the same ATIII dose used for patients undergoing total hip replacement, with a 50 percent incidence of venous thrombosis. A second group of 11 patients was treated with twice the dose of ATIII, and an incidence of venous thrombosis of 27 percent was found. The higher ATIII dose resulted in significantly higher ATIII concentrations and maintained the postoperative ATIII concentration above normal. Among patients receiving prophylaxis with either warfarin. dextran, or ATIII/heparin, no clear association was found between reduced ATIII concentrations and occurrence of venous thrombosis. It is concluded that ATIII replacement following total hip or knee replacement corrects the postoperative ATIII deficiency and that the combination of ATIII and low-dose heparin is an effective prophylactic regimen following total hip replacement.

HTLV-I (human T cell lymphotropic virus type 1) is the retrovirus causally related to adult T cell leukemia/lymphoma and is also associated with a neurological disorder, tropical spastic paraparesis, or HTLV-I-associated myelopathy. The development of these two different diseases among HTLV-I-infected individuals may depend in part on differences in their T cell immunity associated with a difference of HLA phenotype. Peptides corresponding to 17 sites in the HTLV-I envelope protein were tested for their antigenicity for lymph node cells from B10.BR, B10.D2, B10.A(5R), and B10.HTT congenic mice, representing four independent MHC haplotypes, immunized with the native envelope protein. Ten of the 17 tested sites were predicted to be amphipathic alpha-helical sites and all of them were found to be antigenic for at least one of the four MHC congenic strains of mice. Three of the 17 sites were amphipathic 3(10)-helical sites and four sites were predicted to be non-helical sites: none of the 3(10)-helical sites were antigenic and only one of four non-predicted sites was found to be immunodominant. Furthermore, three potent immunodominant peptides, V1E1 (342-363), V1E8/SP4a (191-209), and V1E10 (141-156) were also shown to be immunogenic; i.e., these peptides could be used to immunize mice to elicit proliferative responses of lymph node cells to the native HTLV-I envelope protein. Furthermore, these three peptides were able to prime animals for an enhanced antibody response to the native protein. Because this priming followed the same Ir gene control as the proliferative response, it probably reflects the ability of these peptides to prime helper T cells. The localization of immunodominant sites in HTLV-I envelope protein in mice may be useful for finding antigenic and immunogenic sites in humans, for developing a peptide vaccine for the virus, and possibly for aiding in prognosis for the development of different disease manifestations of HTLV-I infection.

The function of the A element (nucleotides 5107 to 5130) of the polyomavirus enhancer is augumented in NIH 3T3 cells by a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). One of its targets is an AP1 consensus sequence motif recognized by a nuclear factor, PEBP1. In Ha-ras-transformed NIH 3T3 cells, however, A element function was not enhanced by TPA treatment, and at the same time PEBP1 was not detected in the nuclear extract by a mobility shift assay. PEBP1 was not detected in either the extract from NIH 3T3 cells treated in vivo with a protein kinase inhibitor, staurosporine, or the extract from NIH 3T3 cells after treatment in vitro with phosphatase. These results suggest that PEBP1 is required to be properly phosphorylated for DNA binding and that it is underphosphorylated, possibly due to the downregulation of protein kinase C in Ha-ras-transformed cells. In addition, we observed that PEBP2, which bound to the A element adjacent to PEBP1, was converted to apparently related PEBP3 when conditions favored underphosphorylation.

Antithrombin III (ATIII) clearance from blood occurs by redistribution into the extravascular compartment and by binding to the endothelial surface. When, however, ATIII reacts with a proteinase such as alpha-thrombin, the complex is rapidly cleared from the circulation (half-life is approximately five minutes) by a receptor present on hepatocytes. This receptor binds a number of other serine proteinase inhibitors that are members of the class designated as the "serpins." ATIII, alpha 1-proteinase inhibitor, heparin cofactor II, and alpha 1-antichymotrypsin proteinase complexes bind to the same hepatic receptor, now designated as serpin receptor 1. Proteinase complexes with alpha 2-antiplasmin, another member of the serpin class, do not bind to serpin receptor 1. Recent studies suggest that the specificity of the receptor for serpins may reside in the so-called D helix (nomenclature based on the structure of alpha 1-proteinase inhibitor). The presence of ATIII on the surface of endothelial cells offers a unique mechanism for regulating proteinases formed during coagulation. Since this ATIII is probably associated with heparin-like substances and exists in a high-affinity state, the inhibitor rapidly binds proteinases such as alpha-thrombin. Once the complex forms, its affinity for heparinoids is decreased compared with ATIII, allowing the complex to dissociate from the endothelial surface for rapid clearance by the liver.

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.

The cloning of antithrombin III (ATIII) complementary deoxyribonucleic acids and the determination of the ATIII gene structure have permitted a systematic evaluation of the molecular basis for inherited ATIII deficiencies. Sixteen kindreds with the most common form of the deficiency, in which plasma ATIII antigen levels and activity are proportionately reduced, were studied. Two polymorphic deoxyribonucleic acid markers were used to resolve parental ATIII alleles and to trace their inheritance patterns. In 15 of 16 cases, the structure of the affected ATIII allele was indistinguishable from normal, suggesting that relatively small mutations, resulting in gene inactivation, are responsible for the lower ATIII levels in these affected families. In the remaining kindred, complete deletion of one ATIII allele was seen. Also investigated was the molecular basis for a qualitative form of ATIII deficiency in a French-Canadian family with normal levels of immunoreactive protein but only half the expected levels of serine protease inhibitor activity. Using polymorphic markers, the abnormal allele was identified, cloned, and partially sequenced from the propositus. A single G----A transition was seen in the first base of codon 382, resulting in an alanine----threonine substitution in the defective protein. This mutation, together with others in this vicinity, defines a minimal length for a fully functional thrombin-binding domain.

The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver.

A 38-year-old man of recurrent episodes of multiple cerebral infarctions with protein C abnormality was reported. He had six episodes of visual disturbance within a year. The neurological examination revealed bilateral visual loss and impaired visual field except for small central area. However, there were no abnormalities in the deep tendon reflexes and muscle strength of all extremities and cerebellar examinations. CT scan revealed multiple low density areas in the bilateral posterior lobes, and cerebral angiography demonstrated the severe stenosis of the ganglionic portion and the supraclinoid portion of the right internal carotid artery. Plasma levels of antithrombin-III activity, plasminogen activity, protein C activity, protein C antigen and protein S antigen were 105, 106, 28, 166 and 120%, respectively. Because only protein C activity decreased markedly, a diagnosis of protein C abnormality was established. We started warfarin therapy under the administration of concentrates of factor IX complex, since then there has been no cerebral accident.

A novel multifunctional controlled pore glass, MF-CPG (Fig. 1), has been synthesized and used to incorporate 3' terminal primary aliphatic amines into synthetic oligonucleotides. MF-CPG consists of a unique succinic acid linking arm which possesses both a masked primary amine for label attachment and a dimethoxytrityl protected hydroxyl for nucleotide chain elongation. Using MF-CPG, we have devised a simple and convenient technique to attach non-radioactive labels to the 3' terminus of oligonucleotides. Bifunctional probes can then be constructed by 32P labeling the 5' terminus with T4 kinase and gamma 32P-ATP. Using such bifunctional oligonucleotide probes in conjunction with polymerase chain reaction (PCR) amplification, we were able to detect single base substitutions in a target segment of the human H-ras protooncogene employing either functionality. Our technique thus expands the potential applications for oligonucleotides as hybridization probes.

Plasma antithrombin III (ATIII) levels decrease early during gram-negative septicemia, and even a moderate decrease in this major inhibitor of the coagulation system is associated with serious disseminated intravascular coagulation (DIC). Herein the efficacy of high-dose (at least 250 units/kg) ATIII supplementation in animal models of Escherichia coli endotoxemia or bacteremia is reported. An endotoxemic rat model demonstrated that: (1) DIC occurs very early, before the appearance of deleterious cardiovascular abnormalities; (2) ATIII prophylaxis attenuates DIC, metabolic dysfunction, and organ damage; (3) ATIII prophylaxis increases permanent survival; (4) ATIII treatment one hour after endotoxin challenge attenuates DIC, metabolic dysfunction, and organ damage, although not as well as when given prophylactically, and survival is not increased. An endotoxemic sheep pulmonary dysfunction model demonstrated that: (1) ATIII prophylaxis prevents the typical decrease in arterial oxygen partial pressure; (2) ATIII prophylaxis combined with alpha-1-proteinase inhibitor significantly attenuates indices of pulmonary dysfunction. An E. coli bacteremic baboon model demonstrated that ATIII prophylaxis and treatment significantly attenuate indices of DIC and organ damage and prevent death in an otherwise completely lethal dose bacterial challenge. In conclusion, prophylactic treatment with high doses of ATIII may be efficacious in disease states of impending disseminated intravascular coagulation, such as primary or secondary gram-negative septicemia.

Antithrombin III (ATIII) deficiency is inherited as an autosomal dominant trait. Three types of ATIII deficiency are recognized clinically. The prevalence of ATIII deficiency is uncertain; it has been estimated to occur in between one in 2,000 and one in 20,000 subjects. ATIII deficiency is found in between 4 and 6 percent of young patients with venous thrombosis, similar to but slightly lower than the prevalence of protein C and protein S deficiency in young subjects with thrombosis. The chances of finding a deficiency is increased if there is a history of familial or recurrent venous thrombosis. Cross-sectional reports in the literature are that between 30 and 80 percent of carriers have thrombosis. Thrombosis is uncommon in the first decade, but the risk rises sharply between the ages of 15 and 30. The major clinical manifestations of ATIII deficiency are young age at onset, idiopathic thrombosis, family history, and recurrent venous thromboembolism. Pregnancy and surgery are predisposing factors. Approaches to prophylaxis and treatment are discussed.

Hereditary antithrombin III (ATIII) deficiency predisposes patients to venous thrombosis. The prothrombin fragment F1+2 radioimmunoassay demonstrates that many asymptomatic patients with this disorder not receiving antithrombotic therapy have elevated plasma factor Xa activity. The hemostatic system hyperactivity as measured by this assay could be specifically corrected by rising plasma ATIII levels of several persons into the normal range. This indicates that the prethrombotic state can be defined as an imbalance between the production and inhibition of factor Xa enzymatic activity. The effects of warfarin on factor Xa enzymatic activity in persons with congenital ATIII deficiency have also been evaluated. At equivalent intensities of oral anticoagulation, the mean plasma F1+2 level in patients with ATIII deficiency was significantly elevated as compared with anticoagulated persons without this inherited thrombotic disorder. It is concluded that the effect of warfarin on hemostatic system activation is modulated by the endogenous heparan sulfate-ATIII mechanism. This suggests that the F1+2 radioimmunoassay can be employed to improve the understanding of the hypercoagulable state associated with antithrombin III deficiency as well as to develop more effective treatment strategies to prevent thromboembolic events in patients with this disorder.

Antithrombin III (ATIII) is the predominant naturally occurring inhibitor of serine proteases generated during blood coagulation [Rosenberg RD: Annu Rev Med 1978; 29: 367-378]. Since 1965, several assays have been developed that allow rapid and precise determination of ATIII in plasma. As a consequence, the existence of acquired ATIII deficiency in many pathologic conditions has been described. Acquired ATIII deficiency is based on decreased synthesis, increased loss or increased consumption, or induced by drugs. An inherited ATIII deficiency is associated with a lifelong tendency to venous thromboembolism. In contrast, the clinical significance of acquired ATIII deficiency has been less well defined. A precise estimate of the risk of thromboembolism in the acquired ATIII deficiency state cannot easily be provided, owing to the lack of studies in consecutive patients. In 1978, a purified human ATIII concentrate became available for clinical investigation. Despite numerous small studies, the value of ATIII replacement therapy in patients with acquired deficiency remains to be demonstrated.

Hemorrhagic and thrombotic complications are common in sick preterm infants and may reflect inadequate regulation of coagulation. All neonates have low levels of the pivotal regulator antithrombin III (ATIII) compared with adults. Plasma levels of ATIII are very low in preterm infants and are further diminished in infants with respiratory distress, necrotizing enterocolitis, sepsis, or disseminated intravascular coagulation. Babies with lower levels of ATIII in the cord blood have been shown to have a worse outcome than neonates with levels appropriate for gestational age, including higher mortality and increased incidence of intracranial hemorrhages and catheter-related thromboses. The origin of severe ATIII deficiency is unknown. Therapies with plasma replacement or anticoagulation have decreased the incidence and severity of hemorrhagic and thrombotic complications in high-risk infants in several clinical trials. These data lay the groundwork and rationale for potential use of ATIII replacement in deficient preterm infants.

Phase I clinical studies of antithrombin III (ATIII) concentrate demonstrated a mean in vivo incremental recovery of functional activity of 1.4 percent per unit/kg administered, an initial 50 percent disappearance time of 22 hours, and a biologic half-life of 3.8 days. Based on these observations, a treatment regimen designed to maintain plasma ATIII levels between 75 and 120 percent of normal has been developed. None of 10 subjects with congenital ATIII deficiency treated prophylactically had evidence of thromboembolism, including four pregnant women at the time of delivery. Five subjects treated for acute thrombosis and/or thromboembolism, four of whom were pregnant, recovered without further thrombotic extension or recurrence. Heparin resistance was reversed in two subjects, both pregnant. Nine subjects with acquired ATIII deficiency also received ATIII treatment for venous or arterial thrombosis or disseminated intravascular coagulation, all with low plasma ATIII levels. Two subjects with disseminated intravascular coagulation demonstrated improvement, one clinically, the other biochemically. All patients with congenital ATIII deficiency survived, but only five of nine with acquired deficiency survived, highlighting the importance in acquired ATIII deficiency of the underlying disease in prognosis. Survival rate was especially poor in subjects with arterial thrombosis in the setting of low plasma ATIII. Administration of ATIII concentrate was well tolerated. None of the subjects who received ATIII concentrate demonstrated evidence of an infectious transmissible agent. These studies demonstrate that it is now feasible to safely replace the deficient protein in congenital ATIII deficiency, either prophylactically or therapeutically.

Purified radiolabeled fibrinogen and antithrombin III (ATIII) were injected intravenously into rabbits before a deendothelializing injury to the aorta, and allowed to circulate for 0.1 to 6 hours before exsanguination, excision of the aorta, and quantification of each protein/unit area of subendothelium (intima-media). Uptake of fibrinogen was rapid (saturation 10 minutes after injury was approximately 13.0 pmol/cm2) compared with that of ATIII (45 to 60 minutes; 3.5 to 4.3 pmol/cm2). Both proteins associated primarily (greater than 90%) with the subendothelium rather than the platelet monolayer. The avidity of the deendothelialized vessel of these proteins was measured after a 20-minute circulation time at various intervals after injury. Whereas turnover of fibrinogen was fairly constant (approximately 100% per hour), that of ATIII was maximal (approximately 200% per hour) at 1 hour, decreasing to approximately 105% per hour at 5 hours after injury. The profile of ATIII turnover mirrored that of thrombin released in vitro from the deendothelialized aorta up to 10 days after injury, whereas the uninjured aorta and the aorta deendothelialized ex vivo adsorbed fibrinogen poorly and released negligible thrombin. Pretreatment of the aorta, deendothelialized ex vivo with thrombin in vitro increased fibrinogen uptake significantly. It is possible that, after deendothelialization in vivo, fibrinogen adsorption is determined largely by thrombin generation at the vessel wall. ATIII binding is limited by the availability of binding sites in the subendothelium, although the rate of thrombin generation influences ATIII turnover.

Cutter Biological, Miles Inc., has implemented large-scale isolation and purification of antithrombin III (ATIII) from human blood plasma. The starting material, Cohn fraction IV-1, is suspended and ATIII separated from other proteins by heparin-affinity chromatography. The concentrate is subsequently heated for 10 hours at 60 degrees C in a solution of 0.5 M sodium citrate to inactivate the causative agents of viral hepatitis B and acquired immune deficiency syndrome. After heating, a second heparin-affinity chromatography step is employed to isolate the active ATIII and to remove heat-denatured protein. The purified solution is compounded with 0.1 M alanine and 0.15 M sodium chloride, filled in vials of 500 or 1,000 IU each, and freeze dried. The final product has a specific activity of not less than 6.4 IU ATIII per mg protein, of which over 90 percent binds to heparin on crossed immunoelectrophoresis. Preclinical and clinical testing have shown this product to be safe for prophylactic and therapeutic administration to persons with congenital deficiency of ATIII.