The alphaC domains of fibrinogen affect the structure of the fibrin clot, its physical properties, and its susceptibility to fibrinolysis

The functions of the alphaC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aalpha chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of alpha251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of alpha251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the alphaC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of alpha251 fibrin clots differed markedly from control clots; alpha251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the alphaC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked alpha251 and control clots showed that gamma chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of alpha251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the alphaC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.     

Effects of cryoglobulin on fibrin clot formation and fibrinolysis

It was confirmed that activation of the kallikrein-kinin enzyme system in cryoglobulinemia might be initiated by activation of factor XII to factor XIIa by cryoglobulin. It was also demonstrated that cryoglobulin or fibrin clots lost their ability to redissolve on warming to 37 degrees C, and consequently the lysis time of fibrin clots was increased.     

End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bbeta gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient. (Blood. 2000;96:3779-3785)     

Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure

Fibrinogen Dusart is a congenital dysfibrinogenemia (A-alpha 554 Arginine-->Cysteine) associated with severe thrombotic disorder, high incidence of thrombotic embolism, and abnormal fibrin polymerization. This thrombotic disorder was attributed to an abnormal clot thrombolysis with reduced plasminogen binding to fibrin and defective plasminogen activation by tissue plasminogen activator. The purpose of this work was to assess whether clot architecture could be involved in the thromboresistance of the fibrin Dusart and the high incidence of embolism. An important change in Dusart fibrin clot structure was identified with dramatic decrease of gel porosity (Ks), fiber diameters (d), and fiber mass-length ratios (mu) derived from permeation analysis. In addition, rigidity of the Dusart clot was found to be greatly increased compared with normal fibrin. We provide evidence that both thrombolysis resistance and abnormal rigidity of the fibrin Dusart are related to this abnormal architecture, which impairs the access of fibrinolytic enzymes to the fibrin and which is responsible for a brittle clot that breaks easily, resulting in a high incidence of embolism. Indeed, when restoring a normal clot structure by adding dextran 40 (30 mg/mL) before coagulation, clot thrombolysis and clot rigidity recovered normal values. This effect was found to be dose- dependent. We conclude that clot architecture is crucial for the propensity of blood clot to be degraded and that abnormal clot structure can be highly thrombogenic in vivo. The alpha-C domains of fibrinogen are determinant in fibrin clot structure.     

Evolutionary stability of fibrinogen epitopes implicated in fibrin polymerization and in fibrinolysis

In this work, fibrinogen evolution was analysed by testing the reactivity of fibrinogen from different species with monoclonal antibodies against human fibrinogen fragment D. One epitope concerning the fibrin polymerization site 'a' and two epitopes responsible for tPA binding to fibrin were conserved in all mammalian fibrogens tested but not in crab coagulogen or pleurodella fibrinogen. In these two species, some epitopes which are not implicated in fibrinogen function were conserved. Therefore, we can conclude that polymerizing site 'a' and tPA binding sites have not been modified for at least 80 million years.     

Genetics of fibrin clot structure: a twin study

Coronary artery thrombosis following plaque rupture is an important feature of myocardial infarction, and studies have highlighted the role of coagulation in this condition. Although genetic and environmental influences on the variance in coagulation protein concentrations have been reported, there are no data on the heritability of structure/function of the final phenotype of the coagulation cascade, the fibrin clot. To assess genetic and environmental contributions to fibrin structure, permeation and turbidity studies were performed in 137 twin pairs (66 monozygotic, 71 dizygotic). The environmental influence (e2) on pore size (Ks) (e2 = 0.61 [95% confidence interval (CI), 0.45-0.80]) and fiber size (e2 = 0.54 [95% CI, 0.39-0.73]) was greater than the heritability (h2 = 0.39 [95% CI, 0.20-0.55] and 0.46 [95% CI, 0.27-0.62], respectively). After correction for fibrinogen levels, the environmental effect persisted for Ks (e2 = 0.61), but genetic influence assumed a greater importance in determining fiber size (h2 = 0.73). Multivariate analysis revealed an overlap in the influence of genetic and environmental factors on fibrinogen levels, Ks, and fiber size. Factor XIII B subunit showed environmental and genetic correlation with fibrinogen and fiber size and a genetic correlation with Ks. The results indicate that genetic and environmental influences are important in determining fibrin clot structure/function.     

The in-vitro effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor on clot formation and susceptibility to tissue plasminogen activator-induced fibrinolysis in hemodilution model

Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringer's solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl? and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and α-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.     

Effects of actin filaments on fibrin clot structure and lysis.

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament-severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.     

A novel mutation (deletion of Aalpha-Asn 80) in an abnormal fibrinogen: fibrinogen Caracas VI. Consequences of disruption of the coiled coil for the polymerization of fibrin: peculiar clot structure and diminished stiffness of the clot.

An abnormal fibrinogen was identified in a 10-year-old male with a mild bleeding tendency; several years later, the patient developed a thrombotic event. Fibrin polymerization of plasma from the propositus and his mother, as measured by turbidity, was impaired. Plasmin digestion of fibrinogen and thrombin bound to the clot were both normal. The structure of clots from both plasma and purified fibrinogen was characterized by permeability, scanning electron microscopy and rheological measurements. Permeability of patients' clots was abnormal, although some measurements were not reliable because the clots were not mechanically stable. Consistent with these results, the stiffness of patients' clots was decreased approximately two-fold. Electron microscopy revealed that the patients' clots were very heterogeneous in structure. DNA sequencing of the propositus and his mother revealed a new unique point mutation that gives rise to a fibrinogen molecule with a missing amino acid residue at Aalpha-Asn 80. This new mutation, which would disrupt the alpha-helical coiled-coil structure, emphasizes the importance of this part of the molecule for fibrin polymerization and clot structure. This abnormal fibrinogen has been named fibrinogen Caracas VI.     

Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting

The effects of varying concentrations of platelets, fibrinogen and thrombin on platelet aggregation and on fibrin clotting were investigated. The results indicated that a threshold thrombin to platelet concentration ratio may be required to cause platelet activation. Above the threshold ratio, platelets exhibited properties which enhanced thrombin action in causing aggregation and fibrin clotting. At T/P ratios below the threshold level, the presence of platelets reduced thrombin activity, in other words, platelets exerted an antithrombin action. Fibrinogen at low concentrations (0.02-1.5 mg/ml) enhanced platelet aggregation induced by thrombin; whereas, at high concentrations of fibrinogen (2.0-4.0 mg/ml), aggregation was markedly inhibited. Continuous mixing of samples of paltelets and fibrinogen at physiological concentrations with thrombin at low concentrations (less than 2.0 U/ml) resulted in platelet aggregation. On the other hand, fibrin clots formed in samples without mixing or with high thrombin concentrations (greater than or equal to 5.0 U/ml). These results suggested that the quantitative relationships between platelets, fibrinogen and thrombin, and the presence or absence of cell contact may be important factors in determining the overall hemostasis.     

Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms

Factor XIII and fibrinogen are unusual among clotting factors in that neither is a serine protease. Fibrin is the main protein constituent of the blood clot, which is stabilized by factor XIIIa through an amide or isopeptide bond that ligates adjacent fibrin monomers. Many of the structural and functional features of factor XIII and fibrin(ogen) have been elucidated by protein and gene analysis, site-directed mutagenesis, and x-ray crystallography. However, some of the molecular aspects involved in the complex processes of insoluble fibrin formation in vivo and in vitro remain unresolved. The findings of a relationship between fibrinogen, factor XIII, and cardiovascular or other thrombotic disorders have focused much attention on these 2 proteins. Of particular interest are associations between common variations in the genes of factor XIII and altered risk profiles for thrombosis. Although there is much debate regarding these observations, the implications for our understanding of clot formation and therapeutic intervention may be of major importance. In this review, we have summarized recent findings on the structure and function of factor XIII. This is followed by a review of the effects of genetic polymorphisms on protein structure/function and their relationship to disease.     

Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not β-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.     

In vitro evaluation of clot quality and stability in a model of severe thrombocytopenia: effect of fibrinogen,factor XIII and thrombin-activatable fibrinolysis inhibitor

Background

The treatment options in severe thrombocytopenia (platelet count ≤20×10⁹/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia.

Materials and methods

Thrombocytopenia (platelets [16±4]×10⁹/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl₂ and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI).

Results

The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability.

Discussion

Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.