Biomedical subjects

David A Lane

Publications and source records attributed to David A Lane.

22 records · Page 2Linked to original sources

PubMed2002

Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity.

Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)-like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (approximately 10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than "spacer" function of EGF2 was responsible. We confirmed that the variant 134 had a functional gamma-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity.

1-Carboxyglutamic Acid↗

PubMed2002

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function.

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P <.001), Pro477Ser (76.8% +/- 1%, P <.001), Pro483Leu (82.1% +/- 8%, P <.007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K(m)) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P <.0001) K(m), 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.

Adult↗

PubMed2002

Genetic and phenotypic variability between families with hereditary protein S deficiency.

While many mutations thought to result in protein S (PS) deficiency are known, there have been few attempts to relate genotype expression with plasma phenotype. We have investigated the nature and consequence of PS gene (PROS1) mutations in 17 PS-deficient families who presented with mixed type I and type III phenotypes. Seven different mutations were found in nine families: delG-34 (STOP codon at -24), Val-24Glu, Arg49Cys, Asn217Ser, Gly295Val, +5 G to A intron j and His623Pro. PS wild type (PSWT) and the five missense mutants were transiently expressed in COS-1 cells. All mutants expressed lower (p<0.05) PS antigen compared to PSWT (100%). The mutants Val-24Glu, Gly295Val and His623Pro expressed very low/undetectable PS levels. The mutant Asn217Ser produced around 30% of PSWT, while the mutant Arg49Cys had the highest PS levels (around 50%). Metabolic labelling and pulse-chase experiments showed that all of the mutants had impaired secretion, but this was of variable severity. Also, enhanced intracellular degradation of unsecreted material was found for all mutants. There was a strong correspondence between plasma free PS levels in carriers of the mutations, secreted PS from transfected COS-1 cells and labelled PS from 24 h conditioned medium in pulse-chase experiment. We conclude that the magnitude of secretion defect depends on the nature of the PROS1 mutation and influences the level of free PS in plasma. It is likely that the severity of the secretion defect will determine the risk for venous thrombosis.

Amino Acid Substitution↗

PubMedDate not supplied

Haemostatic gene polymorphisms in venous and arterial thrombosis.

Haemostatic gene polymorphisms are potential risk factors for thrombosis. Considerable attention has been focussed on identifying risk alleles. Progress has undoubtedly been made in venous thrombosis. Factor V Leiden and the prothrombin G20210A substitution are now established risk factors, and a number of other polymorphisms are candidates. The initial promise that genetic risk factors might contribute appreciably to an explanation of the development of arterial thrombotic disorders has largely been unfulfilled and the expectations raised by early reports of positive associations have been tempered by inconsistent results with almost all genes studied. The problems seen in arterial disease are replicated in investigations of other complex diseases. In the optimistic rush to show positive associations of genetic factors with diseases, sight has been lost of the need for stringent study design. Furthermore, the scale of studies needed to produce reproducible conclusions has been underestimated. The lessons learnt from accumulated experience should now enable progress to be made.

Arterial Occlusive Diseases↗