Rehana S. Lovely

Elevated plasma fibrinogen γ′ concentration is associated with myocardial infarction: Effects of variation in fibrinogen genes and environmental factors

Summary.  Background:  Fibrinogen γ′, a fibrinogen γ‐chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease.

Fibrinogen γ′ chain binds thrombin exosite II

Summary.  A high‐affinity thrombin‐binding site in an alternately processed fibrinogen variant, the γA/γ′ isoform, is characterized in this report. The binding site has been shown to be situated between γ′ 414 and 427, and Tyr418 and 422 in this part of the γ′ chain are known to be sulfated. A synthetic peptide corresponding to the γ′ chain carboxyl terminus is shown to bind thrombin with a Kd = 0.63 ± 0.16 µmol L−1. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the γ′ peptide via exosite II. Thus, thrombin binding to the γ′ chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin‐bound thrombin can retain enzymatic activity, and why fibrin‐bound thrombin is heparin‐resistant.

Assessment of Genetic Determinants of the Association of γ′ Fibrinogen in Relation to Cardiovascular Disease

Objective—&ggr;′ fibrinogen is a newly emerging biomarker that is associated with cardiovascular disease (CVD). However, the genetic determinants of &ggr;′ fibrinogen levels are unknown. We therefore conducted a genome-wide association study on 3042 participants from the Framingham Heart Study Offspring Cohort. Methods and Results—A genome-wide association study with 2.5 million single-nucleotide polymorphisms (SNPs) was carried out for &ggr;′ fibrinogen levels from the cycle 7 examination. Fifty-four SNPs in or near the fibrinogen gene locus demonstrated genome-wide significance (P<5.0×10−8) for association with &ggr;′ fibrinogen levels. The top-signal SNP was rs7681423 (P=9.97×10−110) in the fibrinogen gene locus near FGG, which encodes the &ggr; chain. Conditional on the top SNP, the only other SNP that remained genome-wide significant was rs1049636. Associations between SNPs, &ggr;′ fibrinogen levels, and prevalent CVD events were examined using multiple logistic regression. &ggr;′ fibrinogen levels were associated with prevalent CVD (P=0.02), although the top 2 SNPs associated with &ggr;′ fibrinogen levels were not associated with CVD. These findings contrast those for total fibrinogen levels, which are associated with different genetic loci, particularly FGB, which encodes the B&bgr; chain. Conclusion—&ggr;′ fibrinogen is associated with prevalent CVD and with SNPs exclusively in and near the fibrinogen gene locus.

Fibrinogen γ′ chain carboxy terminal peptide selectively inhibits the intrinsic coagulation pathway

The minor γA/γ′ isoform of fibrinogen contains a high affinity binding site for thrombin exosite II that is lacking in the major fibrinogen isoform, γA/γA fibrinogen. The biological consequences of γ′ chain binding to thrombin were therefore investigated. Coagulation assays, thrombin activity assays, and a primate thrombosis model were used to characterize the biological effects of the γ′ 410–427 peptide. The γ′ peptide had little effect on thrombin cleavage of the small peptidyl substrate tosyl‐glycyl‐prolyl‐arginine‐4‐nitranilide acetate. However, in vitro assays demonstrated that the γ′ peptide inhibited thrombin cleavage of larger proteinaceous substrates, including fibrinogen and factor VIII. The γ′ peptide inhibited the activated partial thromboplastin time in plasma and showed greater inhibition of activated partial thromboplastin time assays than prothrombin time assays, consistent with the inhibition of factor VIII cleavage. Studies in a baboon thrombosis model showed that the γ′ 410–427 peptide inhibited fibrin‐rich thrombus formation (typical of venous thrombi) and, to a lesser extent, platelet‐rich thrombus formation (typical of arterial thrombi). These results indicate that binding of thrombin exosite II by the γ′ peptide has selective effects on the intrinsic pathway.

Fibrinogen Hershey IV: A novel dysfibrinogen with a γV411I mutation in the integrin αIIbβ3 binding site

The carboxyl terminal segment of the fibrinogen gamma chain from gamma408-411 plays a crucial role in platelet aggregation via interactions with the platelet receptor alpha(IIb)beta(3). We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, gammaV411I and gammaR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet alpha(IIb)beta(3) with a lower affinity than wild-type fibrinogen. These findings show that the gammaV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.

Fibrinogen Hershey IV: a novel dysfibrinogen with a gammaV411I mutation in the integrin alpha(IIb)beta(3) binding site.

The carboxyl terminal segment of the fibrinogen gamma chain from gamma408-411 plays a crucial role in platelet aggregation via interactions with the platelet receptor alpha(IIb)beta(3). We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, gammaV411I and gammaR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet alpha(IIb)beta(3) with a lower affinity than wild-type fibrinogen. These findings show that the gammaV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.

Fibrinogen Hershey IV: A novel dysfibrinogen with a γV4 I I I mutation in the integrin αIIbβ3 binding site

The carboxyl terminal segment of the fibrinogen gamma chain from gamma408-411 plays a crucial role in platelet aggregation via interactions with the platelet receptor alpha(IIb)beta(3). We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, gammaV411I and gammaR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet alpha(IIb)beta(3) with a lower affinity than wild-type fibrinogen. These findings show that the gammaV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.