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S T Lord

Publications and source records attributed to S T Lord.

At least 55 records · Page 3Linked to original sources
PubMed1996

Dissecting clot retraction and platelet aggregation. Clot retraction does not require an intact fibrinogen gamma chain C terminus.

Fibrinogen mediates the processes of platelet aggregation and clot retraction. Previous studies have demonstrated that fibrinogen binding to the platelet receptor alphaIIbbeta3 requires the C-terminal residues of the fibrinogen gamma chain. We made a recombinant human fibrinogen that lacks the gamma chain C-terminal four residues (AGDV). As expected this fibrinogen did not support platelet aggregation. Unexpectedly, this variant did support clot retraction that was indistinguishable from retraction with normal recombinant or plasma fibrinogen. These results suggest that the site on fibrinogen that is required for platelet aggregation differs from the site on fibrin that is required for clot retraction.

Adenosine Diphosphate↗
PubMed1996

Strategy for recombinant multichain protein synthesis: fibrinogen B beta-chain variants as thrombin substrates.

Thrombotic disease has been found in patients with congenital dysfibrinogens that have abnormalities in the amino terminal domain of the fibrinogen B beta-chain. Surprisingly, these fibrinogens are poor substrates for thrombin. In order to examine the molecular basis for this impaired thrombin-fibrinogen interaction, we synthesized three fibrinogens with single amino acid substitutions in this domain: B beta A68T, B beta P70S, and B beta L72S. B beta-chain expression vectors were altered by PCR-directed mutagenesis of the B beta cDNA. The altered vectors were transfected into a Chinese hamster ovary (CHO) cell line that was constructed as a first step in recombinant fibrinogen synthesis, this CHO line synthesizes fibrinogen A alpha- and gamma-chains. More than 86% of the stably selected clones expressed significant levels of fibrinogen, confirming that a two-step strategy permitted efficient synthesis of variant fibrinogens. In large-scale cultures variant fibrinogen accumulation in serum-free medium fluctuated between 1 and 15 micrograms/mL. Normal and variant recombinant fibrinogens were compared to plasma fibrinogens by following the time course of thrombin-catalyzed release of fibrinopeptides. Only the variant B beta A68T, a change identified in a congenital dysfibrinogen, showed significantly impaired kinetics. The rate of fibrinopeptide A release was decreased 27-fold, and the rate of fibrinopeptide B release was decreased 45-fold relative to normal fibrinogen. Fibrinopeptide release was not significantly altered by the substitutions B beta P70S or B beta L72S. These results suggest that B beta residue Ala68 has a novel and critical role in the interaction between thrombin and fibrinogen.

Amino Acid Sequence↗
PubMed1996

Construction, properties and specific fluorescent labeling of a bovine prothrombin mutant engineered with a free C-terminal cysteine.

To define the role of phosphatidylserine-induced conformational changes in prothrombin activation during blood coagulation, a recombinant bovine prothrombin was constructed, characterized and shown to have a globally native-like conformation. We introduced a cysteine to replace the penultimate residue (Gly581) of a previously constructed active site mutant, and expressed the double mutant in Chinese hamster ovary cells at the level of 0.6 microgram/ml of cell culture medium. Specific labeling with fluorescein maleimide was accomplished by limited reduction with dithiothreitol to free the engineered cysteine while maintaining the native-like functional properties of the molecule. The average stoichiometry of labeling was 0.84 probe/protein. The location of the probe at the C-terminus was confirmed by proteolysis by native thrombin, by Taipan venom, and by carboxypeptidase Y. Both the double mutant and labeled prothrombin could be activated by snake venoms and the prothrombinase but, as expected, the double mutant meizothrombin did not autolyze as does native meizothrombin. Thus, for the first time, a native-like but specifically labeled prothrombin has been constructed. This molecule will be an essential tool for elucidating the structural role of membranes during prothrombin activation. In addition, the methods described might be usefully applied to labeling of an odd, engineered cysteine in other disulfide bond-containing proteins.

Amino Acid Sequence↗
PubMed1995

Binding of fibrinogen A alpha 1-50-beta-galactosidase fusion protein to thrombin stabilizes the slow form.

The interaction of fibrinogen A alpha1-50-beta-galactosidase fusion protein with the slow and fast forms of thrombin was studied and compared to thrombin-fibrinogen interaction under identical solution conditions. At equilibrium, the affinity of the fusion protein for the slow form of thrombin is 3 times higher than its affinity for the fast form. The fusion protein and fibrinogen have the same affinity for the fast form. On the other hand, the affinity of the fusion protein for the slow form of thrombin is 40 times tighter than that of fibrinogen. In the transition state, binding of the fusion protein has the same properties as fibrinogen, with the fast form showing higher specificity. The N-terminal fragment of the fibrinogen A alpha chain thus contains residues that are responsible for the preferential binding of the fusion protein to the slow form at equilibrium and to the fast form in the transition state. If this fragment binds to thrombin in a similar way for fibrinogen and the fusion protein, then the N-terminal domains of the B beta and gamma chains of fibrinogen, that are not present in the fusion protein, must play a key role in the binding of fibrinogen to thrombin at equilibrium. These chains may destabilize binding to the slow form by nearly 2.4 kcal/mol, thereby favoring binding of fibrinogen to the fast form. We propose that the three chains of fibrinogen play different roles in the thrombin-fibrinogen interaction, with the A alpha chain containing residues for preferential binding to the fast form in the transition state and the B beta and gamma chains containing residues that destabilize binding to the slow form at equilibrium.

Enzyme Stability↗
PubMed1994

A correlation between thrombotic disease and a specific fibrinogen abnormality (A alpha 554 Arg-->Cys) in two unrelated kindred, Dusart and Chapel Hill III.

For the first time, a correlation between a specific fibrinogen abnormality and the clinical symptoms of thrombosis has been found in unrelated families. These abnormal fibrinogens have been designated Dusart and Chapel Hill III. The abnormal fibrinogen Chapel Hill III was identified previously in a patient with thrombotic disease. We purified fibrinogen from small aliquots of patient and normal plasmas by a simple, rapid procedure. Coomassie stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that fibrinogen Chapel Hill III contained several high molecular weight forms in addition to the two forms seen with normal fibrinogen. Immunoblot analysis of Chapel Hill III fibrinogen demonstrated that essentially all the high molecular weight forms react with antiserum to albumin. Immunoblot analysis of plasmin digests of Chapel Hill III fibrinogen demonstrated that albumin is linked to the C-terminus of the A alpha chain. Using DNA analysis, we found that the patient is heterozygous for a single base change, resulting in the substitution A alpha Arg 554-->Cys. This is the same change identified in fibrinogen Dusart. The Dusart family members who are heterozygous for this substitution also suffer from recurrent thrombotic disorders.

Afibrinogenemia↗
PubMed1993

Characterization of purified recombinant fibrinogen: partial phosphorylation of fibrinopeptide A.

Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.

Animals↗
PubMed1993

Molecular basis for fibrinogen Dusart (A alpha 554 Arg-->Cys) and its association with abnormal fibrin polymerization and thrombophilia.

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C-->T) in the A alpha-chain gene, resulting in the amino acid substitution A alpha 554 Arg-->Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (A alpha 554 Arg-->Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals.

Adult↗
PubMed1993

Purification and characterization of recombinant human fibrinogen.

Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function. In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells. The cDNA for each fibrinogen chain was individually cloned into the same expression vector. Each vector was cotransfected with the selection vector pRSVneo into CHO cells. In addition, the plasmids encoding A alpha and gamma were cotransfected with pRSVneo. Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed. Analysis of these lines demonstrated that CHO cells express and secrete free gamma chain, and an A alpha-gamma complex. To obtain recombinant fibrinogen, the A alpha-gamma G418-resistant clones were transfected with the B beta expression plasmid and a second selection vector, pMSVhis. Colonies resistant to neomycin and histidinol were selected and clonal lines obtained. These clones secreted biologically active recombinant human fibrinogen, which was purified from serum-free culture media by protamine-Sepharose chromatography. Analysis of the purified protein on SDS-polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen. Removal of Asn-linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B beta and gamma chains, as is seen for plasma fibrinogen.

Animals↗
PubMed1992

The residues AGDV of recombinant gamma chains of human fibrinogen must be carboxy-terminal to support human platelet aggregation.

The carboxy-terminus of the gamma chain of fibrinogen contains a sequence which is believed to be one of the domains that interacts with glycoprotein (GP) IIb/IIIa to support platelet aggregation. A normal variant of fibrinogen exists in which the four carboxy-terminal amino acids are replaced by 20 amino acids. This variant, known as gamma', has been reported to bind less effectively to platelets. The purpose of the present study was to engineer novel proteins to determine what differences in amino acid sequence between the gamma and gamma' chains influence the interaction of the carboxyterminus with GPIIb/IIIa. In this regard, the gamma chain cDNA in a bacterial plasmid expression vector was modified by oligonucleotide-directed mutagenesis to produce recombinant gamma chains with amino acid changes in the carboxy-terminus which reflect the differences between gamma and gamma'. The recombinant gamma chain with an unmodified carboxy-terminus supported adenosine diphosphate (ADP)-induced platelet aggregation to the same extent as intact fibrinogen. In contrast, the ability of gamma' 427 (the recombinant gamma' variant) and gamma 427 (where the 16 amino acid gamma' extension [412-427] was added to the carboxy-terminus of gamma) to support platelet aggregation was markedly reduced. In addition, the extent of ADP-induced platelet aggregation was decreased in the presence of gamma' 411 (where amino acids 408-411 in gamma were replaced with amino acids 408-411 in gamma'), while gamma 407 (where the four carboxy-terminal amino acids were deleted) was not capable of supporting aggregation. These findings demonstrate that the four residues AGDV are not only required but must be carboxy-terminal to support platelet aggregation.

Amino Acid Sequence↗
PubMed1992

Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA).

In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461-610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino-terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.

Adult↗
PubMed1992

Abnormal fibrinogens IJmuiden (B beta Arg14----Cys) and Nijmegen (B beta Arg44----Cys) form disulfide-linked fibrinogen-albumin complexes.

The molecular defects in two congenital abnormal fibrinogens, IJmuiden and Nijmegen, were determined by sequence analysis of genomic DNA amplified by the polymerase chain reaction. Both fibrinogens were heterozygous, IJmuiden having a B beta Arg14----Cys substitution and Nijmegen having a B beta Arg44----Cys substitution. Clotting induced by thrombin or Reptilase was impaired in both fibrinogens, indicating defective fibrin polymerization. Immunoblot analysis of both purified fibrinogens demonstrated that some of the abnormal molecules were linked by disulfide bonds to albumin. In addition, abnormal high molecular weight fibrinogen complexes with Mrs between 600,000 and 700,000 were present. Fibrinogen-albumin and high molecular weight complexes were also detected in the patients' plasmas. Quantitative analysis demonstrated that of the total plasma fibrinogen in the IJmuiden patient, 20% was linked to albumin and 10% was present as high molecular weight complexes. In plasma Nijmegen, 13% was linked to albumin and 15% was present as high molecular weight complexes. These results demonstrate that the additional abnormal cysteine in fibrinogens IJmuiden and Nijmegen resulted in the formation of disulfide-linked complexes with other proteins, predominantly albumin. We also found that a significant fraction of the abnormal fibrinogen molecules contained free sulfhydryl groups. These findings complicate interpretation of functional studies of these altered fibrinogens.

Amino Acid Sequence↗
PubMed1992

Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr.

In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.

Adult↗
PubMed1991

A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization.

A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase. The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca2+ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca2+ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca2+ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca2+ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca2+ ions, whereas normal fibrinogen bound 2.0 Ca2+ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the gamma-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen gamma-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.

Adult↗
PubMed1991

A synthetic analog of fibrinogen alpha 27-50 is an inhibitor of thrombin.

Binding of the synthetic peptide AAKDSDWPFAS-DEDWNYKAPSGAR, a fibrinogen alpha 27-50 analog, to thrombin was studied by inhibition assays and affinity chromatography. Peptide alpha 27-50 corresponds to a segment of human fibrinogen downstream from the thrombin cleavage site, with cysteine residues at positions 28, 36, 45 and 49 replaced by alanine. The peptide inhibited clotting of fibrinogen with an inhibition constant of 190-400 microM. Cleavage of fibrinopeptides A and B was inhibited by the peptide and the peptide was competitive with fibrinogen for thrombin. Inhibition of the small substrate tosyl-Gly-Pro-Arg-p-nitroaniline was not observed indicating that the peptide did not block the active site of the enzyme. Peptide alpha 27-50 that was covalently linked to Sepharose bound active site-inhibited thrombin at low ionic strength and was eluted at higher salt concentration. The peptide was not cleaved on overnight exposure to thrombin as determined by reverse phase HPLC. In summary, the peptide bound to, but was not a substrate for thrombin. These results suggest that this region of fibrinogen contributes to binding of thrombin.

Amino Acid Sequence↗
PubMed1991

Mutagenesis of human fibrinogen gamma chain 259-411 synthesized in E. coli: further characterization of the role of the disulfide bond CYS326-CYS339 in calcium binding.

We have produced human fibrinogen gamma 259-411 in Escherichia coli in order to study the relationship between the calcium binding activity of the polypeptide and the integrity of the disulfide bond cysteine326-cysteine339. The polypeptide was produced from a plasmid expression vector at approximately 5 micrograms per milliliter of bacterial culture. The identity of the polypeptide was confirmed by N-terminal amino acid sequence analysis. The expression vector was modified by oligonucleotide directed mutagenesis to remove the nucleotides encoding cysteines gamma 326 and gamma 339. The calcium binding activity of wild-type and altered polypeptides were compared using a solid phase assay. Our results indicate that removal of the two cysteine residues had no appreciable effect on calcium binding activity. We conclude that the integrity of the disulfide bond cysteine326-cysteine339 is not critical for calcium binding to gamma 259-411.

Base Sequence↗
PubMed1991

The varying frequencies of five DNA polymorphisms of X-linked coagulant factor IX in eight ethnic groups.

Five RFLPS of X-linked coagulation factor IX were evaluated in more than 500 normal persons (723-804 X chromosomes) of both sexes who belonged to eight ethnic groups: Anglo-Americans, Basques, Swedes, African-Americans, East Africans, East Indians, Chinese, and Malays. The polymorphisms, 5' to 3', were BamHI, XmnI, TaqI, MnlI, and HhaI. A PCR procedure was developed for three previously described RFLPs-XmnI, TaqI, and MnlI; a PCRP procedure was developed for BamHI, and a PCRP which had been described by others was used for HhaI. Europeans were the most polymorphic, African-Americans and East Africans were intermediate, and Orientals were the least polymorphic. Extragenic 3' HhaI was highly polymorphic in most groups, and extragenic 5' BamHI was polymorphic only in persons with African ancestry. Two major haplotypes predominated among 247 men, and the expected and observed heterozygosities were concordant among women. Allelic association was very strong between the three intragenic PCRPs; it was present but weak between 5' extragenic BamHI and XmnI. No association was found between 3' extragenic HhaI and MnlI.

Asian People↗