The ₉₅RGD₉₇ sequence on the A alpha chain of fibrinogen is essential for binding to its erythrocyte receptor

Background: Erythrocyte aggregation, a cardiovascular risk factor, is increased by high plasma fibrinogen levels. Here, the effect of different fibrinogen mutations on binding to its human erythrocyte receptor was assessed in order to identify the interaction sites. Methods: Three fibrinogen variants were tested, specifically mutated in their putative integrin recognition sites on the Aα chain (mutants D97E, D574E and D97E/D574E) and compared with wild-type fibrinogen. Results: Atomic force microscopy-based force spectroscopy measurements showed a significant decrease both on the fibrinogen–erythrocyte binding force and on its frequency for fibrinogen with the D97E mutation, indicating that the corresponding arginine–glycine–aspartate sequence (residues 95–97) is involved in this interaction, and supporting that the fibrinogen receptor on erythrocytes has a β3 subunit. Changes in the fibrin clot network structure obtained with the D97E mutant were observed by scanning electron microscopy. Conclusion: These findings may lead to innovative perspectives on the development of new therapeutic approaches to overcome the risks of fibrinogen-driven erythrocyte hyperaggregation

BβArg448Lys polymorphism is associated with altered fibrin clot structure and fibrinolysis in type 2 diabetes

Both type 2 diabetes (T2DM) and Bβ448Lys variant of fibrinogen are associated with dense fibrin clots, impaired fibrinolysis and increased cardiovascular risk. It was our objective to investigate whether BβArg448Lys adds to vascular risk by modulating fibrin network structure and/or fibrinolysis in diabetes. The primary aim was to study effects of BβArg448Lys on fibrin network characteristics in T2DM. Secondary aims investigated interactions between gender and BβArg448Lys substitution in relation to fibrin clot properties and vascular disease. Genotyping for BβArg448Lys and dynamic clot studies were carried out on 822 T2DM patients enrolled in the Edinburgh Type 2 Diabetes Study. Turbidimetric assays of individual plasma samples analysed fibrin clot characteristics with additional experiments conducted on clots made from purified fibrinogen, further examined by confocal and electron microscopy. Plasma clot lysis time in Bβ448Lys was longer than Bβ448Arg variant (mean ± SD; 763 ± 322 and 719 ± 351 seconds [s], respectively; p<0.05). Clots made from plasma-purified fibrinogen of individuals with Arg/Arg, Arg/Lys and Lys/Lys genotypes showed differences in fibre thickness (46.75 ± 8.07, 38.40 ± 6.04 and 25 ± 4.99 nm, respectively; p<0.001) and clot lysis time (419 ± 64, 442 ± 87 and 517 ± 65 s, respectively; p=0.02), directly implicating the polymorphism in the observed changes. Women with Bβ448Lys genotype had increased risk of cerebrovascular events and were younger compared with Bβ448Arg variant (67.2 ± 4.0 and 68.2 ± 4.4 years, respectively; p=0.035). In conclusion, fibrinogen Bβ448Lys variant is associated with thrombotic fibrin clots in diabetes independently of traditional risk factors. Prospective studies are warranted to fully understand the role of BβArg448Lys in predisposition to vascular ischaemia in T2DM with the potential to develop individualised antithrombotic management strategies

Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network

Summary Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of ‘knots’ interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin ‘knots’. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin’s viscoelastic properties by rheometry. PolyP reduced the stiffness (G’) and ability of the fibrin network to deform plastically G”, but to different extents. Consequently, the relative plastic component (loss tangent (G”/G’)) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing ‘knotted’ regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically. Supplementary Material to this article is available online at www.thrombosis-online.com

Alterations in Fibrin Structure in Patients with Liver Diseases

The hemostatic balance in patients with liver diseases is relatively well preserved due to concomitant alterations in pro- and antihemostatic pathways. Thrombin generation studies support the notion of hemostatic competence in liver diseases, but in such tests alterations in fibrinogen level and function are not taken into account. We have recently studied structural and functional properties of the fibrin clot in patients with liver diseases. Although we have confirmed previous findings that hypersialylation of the fibrinogen molecule in patients with liver diseases contributes to a defective fibrinogen-to-fibrin conversion, we have found that once the clot has been formed, it has a thrombogenic nature as assessed by permeability assays. These thrombogenic properties of the fibrin clot in cirrhosis relate to incompletely characterized intrinsic changes in the fibrinogen molecule, which may include oxidation and hypersialylation. In addition, in patients with nonalcoholic fatty liver disease thrombogenic properties of the fibrin clot are not only due to liver disease but also to obesity and the metabolic syndrome. During liver transplantation, the clot normalizes and becomes increasingly permeable, and the functional properties of the fibrin clot are markedly normalized by fibrinogen concentrate, when added to plasma samples in vitro. These new insights in the functional properties of the fibrin clot in patients with liver diseases facilitate a more rational approach to treatment and prevention of both bleeding and thrombotic complications

The (Patho)physiology of Fibrinogen γ′

Fibrinogen γ′ is a splice variant of the fibrinogen γ-chain, which leads to a negatively charged extension at the C-terminus of the γ-chain. In fibrinogen, the splice variant appears mainly as a heterodimer with the common γA chain, as γA/γ′ fibrinogen. This variant has been shown to modulate thrombin and factor XIII (FXIII) activity, influence clot architecture, and lack a platelet-binding site. Clinically γA/γ′ fibrinogen levels have been associated with arterial and venous thromboses, indicating that the functional effects of γA/γ′ fibrinogen may contribute to the pathology of thrombosis. In view of the fact that the splice variant has several functional effects and is found so far in all individuals, this review provides an up-to-date summary of the key biologic aspects of this fibrinogen variant and discusses any inconsistencies in current reports

Urban Particulate Matter Induces Changes in Gene Expression in Vascular Endothelial Cells that Are Associated with Altered Clot Structure In Vitro

Background Particulate matter contained in ambient air pollution has been associated with cardiovascular diseases in several epidemiological studies. Objective The aim of this study was to investigate the potential for urban particulate matter to induce changes in clot structure through interaction with vascular endothelial cells. Methods We examined the structure of clots formed on human umbilical vascular endothelial cells that had been treated with various types of particles versus those formed on untreated cells. Particles used were standard reference particulate matter from diesel engine emissions (SRM2975) and urban ambient collection (SRM2787). Results There was a dose-dependent increase in fibre density in clots formed on particle-treated endothelial cells. It was also found that exposure to the particles induced increased expression of tissue factor and reduced expression of thrombomodulin genes as measured by real-time polymerase chain reaction and increased expression of von Willebrand factor and plasminogen activation inhibitor-1 as measured by ELISA. Conclusion These changes are consistent with increased procoagulant activity of air pollution particulate matter–treated endothelial cells and suggest that particulate matter has the potential to promote clot formation through changes induced in endothelial genes controlling clot formation

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted due to high cost and/or risk of infection. We present a novel methodology employing non-antibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis using turbidimetric and permeation analyses, confocal and electron microscopy. Binding studies and molecular modelling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8±1.1 in the absence of Affimers, to 172.6±7.4 and >180 min (P< .0001) respectively, and from 7.6±0.2 to 28.7±5.8 (P< .05) and 149.3±9.7 (P< .0001) min in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy controls and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure, G2 profoundly altered fibrin fiber arrangement while F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing one potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk

Factor XIII deficiency as a potential cause of supratentorial haemorrhage after posterior fossa surgery

BACKGROUND: Postoperative intracranial haemorrhage can be a dramatic event, carrying significant morbidity and mortality. Bleeding at sites remote from the operation area represents a small percentage of haemorrhages whose aetiology remains unclear (Harders et al. Acta Neurochir (Wien) 74(1-2):57-60, 1985). AIM: We present the case of a 60-year-old patient who underwent posterior fossa craniotomy for the removal of a space-occupying lesion and suffered supratentorial haemorrhage soon after the operation. RESULTS: A thorough postoperative investigation revealed low levels of factor XIII (FXIII), the factor mainly responsible for fibrin clot stabilisation. CONCLUSION: We suggest that reduced FXIII activity may be an important but preventable predisposing factor to remote postoperative haemorrhage in neurosurgical patients