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T L Ortel

Publications and source records attributed to T L Ortel.

65 records · Page 4Linked to original sources
PubMed1990

Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region.

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

"Heparin-free" cardiopulmonary bypass: first reported use of heparinoid (Org 10172) to provide anticoagulation for cardiopulmonary bypass.

Org 10172 provided adequate anticoagulation for this patient. An excellent correlation between anti-factor Xa activity and ACT was observed at the doses used for CPB. If high-dose Org 10172 is used, these data suggest that it may be possible to circumvent the measurement of anti-factor Xa activity by using the ACT as an index of this heparinoid's anticoagulant effect. Because postoperative bleeding may be excessive, however, development of a method of reversal of Org 10172 is desirable. Although the optimal ACT, dose, plasma concentration, and means of reversal (e.g., protamine vs. heparinase) remains to be determined, heparinoids provide an alternate means of anticoagulation for CPB in patients unable to receive standard heparin.

Cardiopulmonary Bypass↗
PubMed1988

Antifibrinolytic therapy in the management of the Kasabach Merritt syndrome.

The Kasabach Merritt syndrome consists of thrombocytopenia, microangiopathic hemolytic anemia, and a localized consumption coagulopathy that develops within the abnormal vascular channels of a hemangioma. In general, these patients demonstrate only mild abnormalities of screening clotting tests, but they can potentially develop life-threatening complications. We present a patient who developed a severe anemia that was refractory to erythrocyte transfusions. Treatment with epsilon-aminocaproic acid to inhibit fibrinolysis and cryoprecipitate to replenish his deficient circulating fibrinogen interrupted the cycle of his systemic coagulopathy and enabled us to transfuse him to a normal hematocrit.

Adult↗
PubMed1984

Purification of glycopeptides of human plasma proteins by high-performance liquid chromatography.

The combination of gel permeation chromatography and high-performance liquid chromatography proves to be very effective for the purification of high-molecular-weight glycopeptides containing a single glycan, that have been difficult to separate by other procedures. In order to facilitate comparison of the chromatographic properties of glycopeptides derived from a variety of proteins and having different structures, identical procedures were used for their purification. The method was applied to a series of human plasma proteins, including immunoglobulin D, ceruloplasmin, hemopexin, beta-2-glycoprotein I, 3.1S alpha-2-leucine-rich glycoprotein, and alpha-1-B-glycoprotein. All the purified glycopeptides were placed in the protein structure of these plasma proteins. In several cases the carbohydrate structure has been determined by collaborating groups. Immunoglobulin D is the first example of a glycoprotein whose entire primary structure has been defined by utilizing a a single protein source. Furthermore, hemopexin and 3.1S alpha-2-leucine-rich glycoprotein were both found to contain GalN oligosaccharide, which had not previously been identified in these proteins. The method was also used to identify the oligosaccharide that is missing in a carbohydrate variant of ceruloplasmin.

Amino Acid Sequence↗
PubMed1984

Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.

A molecular model for the structure of human ceruloplasmin is proposed that is based on the determination of the complete amino acid sequence, studies of the products of limited proteolytic cleavage, calculations of the hydrophilic/hydrophobic character (hydropathy profile), and predictions of the local secondary structure. This multicopper oxidase (Mr approximately 132,000) consists of a single polypeptide chain (1046 amino acid residues) with four attached glucosamine oligosaccharides. Computer-assisted statistical analysis of the internal repetition in the amino acid sequence confirms that the entire polypeptide chain is divided into three contiguous homology units, each containing about 350 amino acid residues. Each homology unit is subdivided into three domains, designated A1, A2, and B, that differ in structure and probably in function. Calculations of the hydropathy profile and predictions of the secondary structure support a molecular model based on internal repetition of three homology units and help to identify characteristic features of the interdomain junctions. The alignment scores for internal duplication of pairings of the three homology units of ceruloplasmin exceed the scores yet reported for contiguous internal duplication of any other protein. This highly significant evidence for intragenic repetition suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes coding for a primordial copper oxidase.

Amino Acid Sequence↗
PubMed1984

Single-chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule.

We have determined the amino acid sequence of the amino-terminal 67,000-dalton (67-kDa) fragment of human ceruloplasmin and have established overlapping sequences between the 67-kDa and 50-kDa fragments and between the 50-kDa and 19-kDa fragments. The 67-kDa fragment contains 480 amino acid residues and three glucosamine oligosaccharides. These results together with our previous sequence data for the 50-kDa and 19-kDa fragments complete the amino acid sequence of human ceruloplasmin. The polypeptide chain has a total of 1,046 amino acid residues (Mr 120,085) and has attachment sites for four glucosamine oligosaccharides; together these account for the total molecular mass of human ceruloplasmin (132 kDa). The sequence analysis of the peptides overlapping the fragments showed that one additional amino acid, arginine, is present between the 67-kDa and 50-kDa fragments, and another, lysine, is between the 50-kDa and 19-kDa fragments. Only two apparent sites of amino acid interchange have been identified in the polypeptide chain. Both involve a single-point interchange of glycine and lysine that would result in a difference in charge. The results of the complete sequence analysis verified that human ceruloplasmin is composed of a single polypeptide chain and that the subunit-like fragments are produced by proteolytic cleavage during purification (and possibly also in vivo).

Amino Acid Sequence↗
PubMed1983

Separation of limited tryptic fragments of human ceruloplasmin by gel-permeation high-performance liquid chromatography.

Limited tryptic proteolysis of human ceruloplasmin rapidly produces several large, protease-resistant fragments, suggesting that the molecule consists of several domains. In order to locate the sites of proteolytic cleavage in the whole molecule, we used gel-permeation high-performance liquid chromatography to determine the optimum conditions for fragment separation. Using a buffer containing 8 M urea, the 67,000-daltons tryptic fragment from single-chain ceruloplasmin was isolated in a sufficiently pure state for amino acid sequence analysis to determine its location in the uncleaved molecule. These results have been used in conjunction with amino acid sequence data to develop a schematic model of the domain structure of human ceruloplasmin.

Ceruloplasmin↗
PubMed1983

Internal triplication in the structure of human ceruloplasmin.

Amino acid sequence analysis of the 67,000-dalton (67-kDal) fragment that is the amino-terminal half of human ceruloplasmin has revealed internal triplication in the primary structure of the entire molecule. This is illustrated by comparison of 620 residues representing homologous domains of the 67-kDal fragment and of the 50-kDal and 19-kDal fragments that together comprise the carboxyl-terminal half of the molecule. The polypeptide chain is divided into three covalently linked homologous segments, each of about 340 residues. All three homology units have about 30% identity in sequence, and each pair exhibits at least 40% identity. The statistical significance of the 3-fold internal duplication was established by computerized analysis of the sequence. These results and studies of the sites of limited proteolytic cleavage support a model for the ceruloplasmin molecule consisting of an alternating structure of six domains of two different kinds (or possibly nine domains of three kinds). The 3-fold internal homology suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes.

Amino Acid Sequence↗
PubMedDate not supplied

Group C streptococcal arthritis: case report and review.

Streptococci account for approximately 15%-20% of cases of nongonococcal septic arthritis. The majority of these are due to group A streptococci, but group B and group G streptococci are being isolated more frequently. We present a case of group C streptococcal arthritis and summarize nine additional cases reported in the literature. The group C streptococci include the large colony of Voges-Proskauer-negative bacteria (Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, and Streptococcus dysgalactiae) as well as as the minute colony of Voges-Proskauer-positive Streptococcus anginosus ("Streptococcus milleri") group C organisms. Any joint may become infected, but joints affected by preexisting rheumatologic abnormalities are more frequently involved. Bacteremia was documented in five of the 10 patients. One patient had an associated pneumonia, and another patient had an associated acute aortic valve endocarditis. None of the infections involved a prosthetic joint or an overlying cellulitis, associations reported for group G streptococcal arthritis. Surgical drainage of the infected joint was required in six of the 10 patients. We concluded that the presence of two groups of organisms sharing the same Lancefield group antigen necessitates the careful identification of isolates to determine potential clinical differences.

Aged↗
PubMedDate not supplied

Antibodies to prothrombin, factor V, and beta2-glycoprotein I and vascular access thrombosis.

We studied 88 hemodialysis patients for the presence of antibodies to human factor II (hFII), bovine factor V (bFV), and human beta2-glycoprotein 1 (beta2GPI). Forty-one patients had elevated anti-hFII antibodies, 17 had elevated anti-bFV antibodies, and 9 had elevated anti-beta2GPI antibodies. Fifty-two patients had elevated antibodies to one or more protein. Patients with PTFE grafts had elevated antibodies most frequently (21 [75%] vs. 20 fistulas [45%; p = 0.016 compared with PTFE] and 11 tunneled catheters [68.8%]). Twelve of 13 patients (92.3%) with PTFE grafts and thrombosis had elevated antibody levels, compared with 9 of 15 without thrombosis (60%; p = 0.049). The number of thromboses and mean thrombosis rates were significantly higher in PTFE patients with antibodies (1.24 vs. 0.14 thromboses, p < 0.01; 42.67 vs. 6.44 thromboses/100 patient years, p < 0.05). When analyzed individually, thrombotic complications occurred more frequently in patients with PTFE grafts and elevated anti-bFV antibodies (p = 0.016), but did not correlate with anti-hFII or anti-beta2GPI antibodies. Thrombotic complications did not correlate with elevated antibody levels in patients with AV fistulas or cuffed catheters. In conclusion, hemodialysis patients with PTFE grafts frequently have elevated antibodies to FII, FV, and beta2GPI, and the presence of elevated antibody levels to one or more of these proteins is associated with an increased thrombotic risk. Further studies are necessary to determine whether limiting exposure to bovine thrombin preparations will decrease the incidence of these antibodies and PTFE graft thrombosis.

Aged↗