The presence of γ′ chain impairs fibrin polymerization

Abstract

Introduction 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. Materials and Methods We expressed three fibrinogen variants in Chinese hamster ovary (CHO) cells: γ/γ homodimer, γ/γ’ heterodimer, and γ’/γ’ homodimer. We observed thrombin-catalyzed fibrinopeptide release by HPLC, fibrin polymerization by turbidity, and clot structure by scanning electron microscopy. We characterized post-translational modifications by mass spectrometry. Results Fibrinopeptide A was released at the same rate for all three fibrinogens, while fibrinopeptide B was released faster from the γ’/γ’ homodimer. The rise in turbidity was slower and final absorbance was lower during polymerization of γ’-containing fibrinogens than for γ/γ fibrinogen. Micrographs showed that γ’/γ’ fibrin clots are composed of very thin fibers, while the diameter of γ/γ’ fibers is similar to γ/γ fibers. Further, the fiber networks formed from γ’-containing samples were non-uniform. Mass spectrometry showed heterogeneous addition of N-glycans and tyrosine sulfation in the γ’ chain. Conclusions The presence of γ’ chains slows lateral aggregation and alters fibrin structure. We suggest these changes are likely due to charge-charge repulsion, such that polymerization of the γ’/γ’ homodimer is more impaired than the heterodimer since these repulsions are partially offset by incorporation of γ chains in the γ/γ’ heterodimer.

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…Gersh et al synthesized all 3 fibrinogens recombinantly: ␥A/␥A homodimer, ␥A/␥Ј heterodimer, and ␥Ј/␥Ј homodimer. 76 In agreement with our study, 33 they found that the rate of thrombin-catalyzed FpA release was similar for all 3 variants, whereas FpB release from ␥Ј/␥Ј fibrinogen was faster than from either ␥A/␥Ј fibrinogen or ␥A/␥A fibrinogen. Fibrin polymerization studies suggested that the ␥Ј chain slowed lateral aggregation and reduced fiber diameter.…”

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

“…Although previous work suggested that this difference reflects alterations in the structure of ␥Ј-Fn clots and/or enhanced stability because of increased FXIIIa-mediated crosslinking (6,10,11,16,17), we explored additional explanations. We show that (a) thrombinmediated release of FPB from ␥Ј-Fg is slower than that from ␥ A -Fg; (b) when ␥Ј-Fg is clotted with thrombin, but not with batroxobin, the rate of binding of Pg is reduced, and Pg activation is delayed; (c) when the Pg activation step is bypassed by using Pn in place of tPA and Pg, ␥Ј-Fn and ␥ A -Fn clots degrade at similar rates; and (d) with FXIIIa addition, degradation of ␥Ј-Fn and ␥ A -Fn clots is slowed to a similar extent.…”

“…The unique interaction of thrombin with ␥Ј-Fg not only attenuates the reactivity of the enzyme with its inhibitors, but also with its substrates, thereby reducing the activity and availability of thrombin. With less free thrombin, FPB release from ␥Ј-Fg is slower than that from ␥ A -Fg (6), which results in delayed clotting of ␥Ј-Fg relative to ␥ A -Fg (6,10,11).…”

“…Lysis of ␥Ј-Fn is delayed relative to that of ␥ A -Fn, a phenomenon that has been attributed to altered thickness of ␥Ј-Fn fibers and/or enhanced cross-linking of ␥Ј-Fn monomers by activated factor XIII (FXIIIa) (6,10,11,16,17). We hypothesized that the slower release of FPB from ␥Ј-Fg delays the exposure of cryptic Pg binding sites (15) and that this, in turn, delays Pg binding and activation and subsequent fibrinolysis.…”

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ′ as thrombomobulin II

“…This effect on clot structure is caused by direct interference of the negatively charged ' extension with fibrin polymerisation and protofibril formation. Gersh et al showed increased ends or "capping" of ' fibers [13]. The ' fibers pack significantly less protofibrils and are less stiff than A fibers [14].…”