Impaired Protofibril Formation in Fibrinogen γN308K Is Due to Altered D:D and “A:a” Interactions ,

“A:a” knob–hole interactions and D:D interfacial interactions are important for fibrin polymerization. Previous studies with recombinant γN308K fibrinogen, a substitution at the D:D interface, showed impaired polymerization. We examined the molecular basis for this loss of function by solving the crystal structure of γN308K fragment D. In contrast to previous fragment D crystals, the γN308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b =95Å, c = 448.3 Å). Alignment of the normal and γN308K structures showed the global structure of the variant was not changed and the knob “A” peptide GPRP was bound as usual to hole “a”. The substitution introduced an elongated positively charged patch in the D:D region. The structure showed novel, symmetric D:D crystal contacts between γN308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to γN308K in solution by plasmin protection assay. The results showed weaker peptide binding, suggesting that “A:a” interactions were altered. We examined fibrin network structures by scanning electron microscopy and found the variant fibers were thicker and more heterogeneous than normal fibers. Considered together, our structural and biochemical studies indicate both “A:a” and D:D interactions are weaker. We conclude that stable protofibrils cannot assemble from γN308K monomers, leading to impaired polymerization

Dissecting Clot Retraction and Platelet Aggregation: CLOT RETRACTION DOES NOT REQUIRE AN INTACT FIBRINOGEN 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

The molecular origins of the mechanical properties of fibrin

When normal blood circulation is compromised by damage to vessel walls, clots are formed at the site of injury. These clots prevent bleeding and support wound healing. To sustain such physiological functions, clots are remarkably extensible and elastic. Fibrin fibers provide the supporting framework of blood clots, and the properties of these fibers underlie the mechanical properties of clots. Recent studies, which examined individual fibrin fibers or cylindrical fibrin clots, have shown that the mechanical properties of fibrin depend on the mechanical properties of the individual fibrin monomers. Within the fibrin monomer, three structures could contribute to these properties: the coiled-coil connectors the folded globular nodules and the relatively unstructured αC regions. Experimental data suggest that each of these structures contributes. Here we review the recent work with a focus on the molecular origins of the remarkable biomechanical properties of fibrin clots

Dynamic Regulation of Fibrinogen: Integrin αIIbβ3 Binding

This study demonstrates that two orthogonal events regulate integrin αIIbβ3’s interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the αIIb–β3 interface and (2) flexibility in the carboxy terminus of fibrinogen’s γ-module. The first postulate was tested by capturing αIIbβ3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed αIIbβ3 was characterized by a kon of ~2 × 104 L mol−1 s−1 and a koff of ~8 × 10−5 s−1 at 37 °C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting αIIbβ3. Eptifibatide competitively inhibited fibrinogen’s interactions with primed αIIbβ3 (Ki ~ 0.4 nM), while a synthetic γ-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (Ki > 10 µM). The second postulate was tested by measuring αIIbβ3’s interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the γ-chain AGDV sites (rFgn γΔ408–411). Normal rFgn bound rapidly, tightly, and specifically to primed αIIbβ3; no interaction was detected with rFgn γΔ408–411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed αIIbβ3, while enthalpy-favorable, must overcome an entropy-dominated activation energy barrier. The hypothesis that fibrinogen binding is enthalpy-driven fits with structural data showing that its γ-C peptide and eptifibatide exhibit comparable electrostatic contacts with αIIbβ3’s ectodomain. The concept that fibrinogen’s αIIbβ3 targeting sequence is intrinsically disordered may explain the entropy penalty that limits its binding rate. In the hemostatic milieu, platelet–platelet interactions may be localized to vascular injury sites because integrins must be activated before they can bind their most abundant ligand

The presence of γ′ chain impairs fibrin polymerization

A fraction of fibrinogen molecules contain an alternatively spliced variant chain called γ’. Plasma levels of this variant have been associated with both myocardial infarction and venous thrombosis. Because clot structure has been associated with cardiovascular risk, we examined the effect of γ’ chain on clot structure

Hypodysfibrinogenemia causing mild bleeding and thrombotic complications in a compound heterozygote of AαIVS4+1G>T mutation and Aα4841delC truncation (AαPerth)

3 páginas, 1 figura -- PAGS nros. 770-77

Substitution of the Human αC Region with the Analogous Chicken Domain Generates a Fibrinogen with Severely Impaired Lateral Aggregation: Fibrin Monomers Assemble into Protofibrils but Protofibrils Do Not Assemble into Fibers

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob ‘A’ peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 μM normal and 0.15 M HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 μM. Considered altogether our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures but these either cannot assemble into fibers or assemble into very thin fibers

Does Topology Drive Fiber Polymerization?

We have developed new procedures to examine the early steps in fibrin polymerization. First, we isolated fibrinogen monomers from plasma fibrinogen by gel filtration. Polymerization of fibrinogen monomers differed from that of plasma fibrinogen. The formation of protofibrils was slower and the transformation of protofibrils to fibers faster for the fibrinogen monomers. Second, we used formaldehyde to terminate the polymerization reactions. The formaldehyde-fixed products obtained at each time point were examined by dynamic light scattering and transmission electron microscopy (TEM). The data showed the formaldehyde-fixed products were stable and representative of the reaction intermediates. TEM images showed monomers, short oligomers, protofibrils, and thin fibers. The amount and length of these species varied with time. Short oligomers were less than 5% of the molecules at all times. Third, we developed models that recapitulate the TEM images. Fibrin monomer models were assembled into protofibrils, and protofibrils were assembled into two-strand fibers using Chimera software. Monomers were based on fibrinogen crystal structures, and the end-to-end interactions between monomers were based on D-dimer crystal structures. Protofibrils assembled from S-shaped monomers through asymmetric D:D interactions were ordered helical structures. Fibers were modeled by duplicating a protofibril and rotating the duplicate 120° around its long axis. No specific interactions were presumed. The two protofibrils simply twisted around one another to form a fiber. This model suggests that the conformation of the protofibril per se promotes the assembly into fibers. These findings introduce a novel mechanism for fibrin assembly that may be relevant to other biopolymers