Karim C. Lounes

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region*

Summary. The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12‐carboxy‐terminal residues of the γ‐chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the γ‐chain segments γ318–320 and γ408–411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to γΔ408‐411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: γΔ319‐320, γ318Asp→Ala and γ320Asp→Ala. Furthermore, antibodies raised against the sequences γ308–322 and γ316–333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment γ316–322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin‐specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

Polymerization Site a Function Dependence on Structural Integrity of Its Nearby Calcium Binding Site

Abstract: To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: γD364A in the a site and γD318A, γD320A, and γD318 +γD320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS‐PAGE analysis of the products showed that γD364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected γD318A, γD320A, or γD318 +γD320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.

Investigation of residues in the fibrin(ogen) gamma chain involved in tissue plasminogen activator binding and plasminogen activation.

In order to characterize tissue plasminogen activator (t-PA) binding to γ-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for γD316, γD318, γD320, and γK321. We measured thrombin-catalyzed polymerization and found normal polymerization with γK321A, no polymerization with γD316A, and, as reported by Lounes et al. in 2002, impaired polymerization with γD318A and γD320A. We measured t-PA binding in a solid-phase assay, and t-PA activity by the generation of plasmin. Comparing normal fibrin with fibrinogen, we found a seven-fold increase in binding and a two-fold increase in activity. Binding to all variant fibrinogens was the same as normal. In contrast, t-PA binding to all variant fibrins was weaker than binding to normal fibrin, 2.5-fold for γK321A, seven-fold for γD320A and 10-fold for γD316A and γD318A. Plasmin generation in the presence of variant fibrinogens was similar, although not identical, to normal, and plasmin generation in the presence of variant fibrins was impaired for the Asp to Ala variants. As the three variants with the weakest t-PA binding and least activity also showed impaired polymerization, our results support previous findings demonstrating the DD:E complex, found in the normal fibrin polymer, is necessary for the fibrin enhanced binding of t-PA and activation of plasminogen.

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

Abstract: BβArg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo‐Cys residues were found to be disulfide‐bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP‐peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Mutations on fibrinogen (γ316-322) are associated with reduction in platelet adhesion under flow conditions

Abstract: In this paper we report on studies of platelet adhesion to several fibrinogen γ chain variants under physiological flow conditions. Reduced platelet adhesion was found to patient dysfibrinogen Vlissingen and its recombinant form (deletion of γ319–320). Furthermore, substitutions of the amino acids 318, 320, or both in the recombinant fibrinogen γ chain showed a strong decrease in platelet adhesion under flow conditions in our perfusion system. Antibodies raised against peptides covering these sequences inhibited platelet adhesion completely, which suggested that the γ316–322 sequence could be involved in platelet adhesion in flowing blood.