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.

gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation.

The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

Serpin-glycosaminoglycan interactions.

Serpins (serine protease inhibitors) have traditionally been grouped together based on structural homology. They share common structural features of primary sequence, but not all serpins require binding to cofactors in order to achieve maximal protease inhibition. In order to obtain physiologically relevant rates of inhibition of target proteases, some serpins utilize the unbranched sulfated polysaccharide chains known as glycosaminoglycans (GAGs) to enhance inhibition. These GAG-binding serpins include antithrombin (AT), heparin cofactor II (HCII), and protein C inhibitor (PCI). The GAGs heparin and heparan sulfate have been shown to bind AT, HCII, and PCI, while HCII is also able to utilize dermatan sulfate as a cofactor. Other serpins such as PAI-1, kallistatin, and α(1)-antitrypsin also interact with GAGs with different endpoints, some accelerating protease inhibition while others inhibit it. There are many serpins that bind or carry ligands that are unrelated to GAGs, which are described elsewhere in this work. For most GAG-binding serpins, binding of the GAG occurs in a conserved region of the serpin near or involving helix D, with the exception of PCI, which utilizes helix H. The binding of GAG to serpin can lead to a conformational change within the serpin, which can lead to increased or tighter binding to the protease, and can accelerate the rates of inhibition up to 10,000-fold compared to the unbound native serpin. In this chapter, we will discuss three major GAG-binding serpins with known physiological roles in modulating coagulation: AT (SERPINC1), HCII (SERPIND1), and PCI (SERPINA5). We will review methodologies implemented to study the structure of these serpins and those used to study their interactions with GAG's. We discuss novel techniques to examine the serpin-GAG interaction and finally we review the biological roles of these serpins by describing the mouse models used to study them.

Severe bleeding in a woman heterozygous for the fibrinogen gammaR275C mutation.

The dysfibrinogen gammaR275C can be a clinically silent mutation, with only two out of 17 cases in the literature reporting a hemorrhagic presentation and four cases reporting a thrombotic presentation. We describe here a particularly severe presentation in 54-year-old female patient who required a hysterectomy at 47 years of age due to heavy menstrual bleeding. Coagulation studies revealed a prolonged prothrombin time and thrombin time, a normal fibrinogen antigen level, and a low fibrinogen activity level. Molecular analysis of the patient's DNA revealed a gamma chain gene mutation resulting in an amino acid substitution at residue 275 (gammaR275C). Protein sequencing of the fibrinogen gamma chain confirmed this mutation, which was named Fibrinogen Portland I. This case demonstrates that the gammaR275C mutation can lead to a severe hemorrhagic phenotype.