Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis
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Curated by eLife
Evaluation Summary:
This paper is of interest to a broad audience in the blood coagulation and fibrinolysis field. Previously undescribed roles in a range of blood clot properties are attributed to a region of the clotting protein fibrinogen, using state-of-the-art methodology. The data support the main conclusions of the paper, open new avenues of investigation for understanding clot properties, and have clinical implications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis.
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Author Response:
Reviewer #1:
In this paper McPherson et al investigated fibrin clot properties and fibrinolysis with recombinant fibrinogen variants lacking parts of the fibrinogen alphaC-region. The aim was to understand the contribution of two subregions, the alphaC-connecter and the alphaC-domain, which are known to be involved in the lateral aggregation of fibrin fibers and their cross-linking. The study measured the contribution of subregions to fibrin fiber growth, mechanical strength, how fibrinolysis proceeds in their absence, their impact on clot retraction, and how the variants affect whole blood clot features.
Strengths and weaknesses:
The major strengths of this report lie in the broad range of appropriately selected assays used to characterize the fibrinogen variants described, the clarity of the data presented, and …
Author Response:
Reviewer #1:
In this paper McPherson et al investigated fibrin clot properties and fibrinolysis with recombinant fibrinogen variants lacking parts of the fibrinogen alphaC-region. The aim was to understand the contribution of two subregions, the alphaC-connecter and the alphaC-domain, which are known to be involved in the lateral aggregation of fibrin fibers and their cross-linking. The study measured the contribution of subregions to fibrin fiber growth, mechanical strength, how fibrinolysis proceeds in their absence, their impact on clot retraction, and how the variants affect whole blood clot features.
Strengths and weaknesses:
The major strengths of this report lie in the broad range of appropriately selected assays used to characterize the fibrinogen variants described, the clarity of the data presented, and a clear discussion of the mechanistic implications of the findings compared to previous work. We can now understand more clearly how the alphaC-subregions contribute to clot structure, initial fibrin fiber growth, clot strength and stiffness, fibrinolysis, clot contraction linked to erythrocyte retention and platelet binding, and a clinically relevant assessment of the clotting and lysis of whole blood.
We thank reviewer 2 for the positive, detailed and constructive comments in their public review.
The methodology does not have weaknesses in the context presented. One issue that arises from such a study, that is centered on the characterization recombinant molecules assessed in vitro, is to what extent each of the characteristics described would have physiological impact in vivo. The study has the advantage of clearly separating out different roles for the fibrinogen alphaC-region, but the more complex interplay of the variants with a complete vasculature and blood composition, in an organism that produced the variants, would enhance the study claims. This issue is hinted at in the paper, albeit in vitro/ex vivo. The fibrin density and fiber thickness of alpha390 clots was different in a purified setting compared to whole reconstituted blood clots post-thromboelastography made using blood from Fga-/- mice. It therefore seems reasonable that characteristics of the alphaC-region functions described may show more or less importance when assessed in vivo.
While our study is mainly focussed on in vitro and ex vivo studies of fibrinogen with truncations in the αC-region, and while we have full confidence that similar mechanisms involving the fibrinogen αC-region are at play during haemostasis and thrombosis in whole organisms, we appreciate that future in vivo studies of fibrinogens with similar αC truncations may add further information regarding the physiological impact of this intriguing region of the fibrinogen molecule. However, such in vivo models are likely complicated by the observed reduced expression levels of fibrinogen with αC truncations, with subsequent impact resulting from both altered function and protein levels.
Reviewer #2:
McPherson H et al designed two fibrinogen variants with truncated alfa- C terminal region: the alfa-390 and alfa-220. The alfa-390 lacks the alfa-C domain, while the alfa-220 lacks both the alfa-C domain and alfa-C connector region. By using different type of optical and electronic microscopy they characterize the fibrin structure at different resolution, and the early fibrin oligomers formation of these homozygous fibrinogen variants, and compared them to the WT fibrinogen. The functional implications of removing these protein stretches in the fibrin mechanical properties and stability were studied by turbidity, FXIIIa fibrin crosslinking, fibrin microrheology, clot contraction (retraction), and rotational thromboelastometry. It was found for the first time the differential roles of these two regions: the role of the alfa-C connector in the longitudinal protofibril/fibre growth, mechanical and fibrinolytic stability, while the alfa-C domain (variant 390) was implicated in the lateral protofibrils association, since its removal gave rise to denser fibrin networks with thinner fibres, already described in the literature. Their finding has clinical implications since support the design of antithrombotic drugs that can limit the thrombus size or growth. Their conclusions are supported by the results. The confocal microscopy fibrin structure was confirmed by the scanning electron microscopy images, and highlight the importance of coupling the fibrinogen under study to the fluorophore in order to do not bias the fibrin structure.
We thank reviewer 3 for the positive comments and for appreciating the potential clinical implications of our study in their public review.
In order to study the differential implications of these alfa-C subregions on the susceptibility of fibrinogen/fibrin plasmin degradation that will support thromboelastometric and turbidity results, it would be interesting in the future to perform fibrinogen/fibrin plasmin degradation kinetic of these variants monitored by SDS/PAGE.
Our study has clearly shown the importance of the fibrinogen αC-region in the mechanical and proteolytic resistance of the blood clot. These were dramatically affected in the total truncation variant, the fibrin polymer of which showed very poor mechanical properties and was lysed extremely rapidly by the fibrinolytic system. We agree that future studies of relevant fibrin degradation products over time may further underpin these findings and the relevance of this part of the fibrinogen molecule in determining clot stability.
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Evaluation Summary:
This paper is of interest to a broad audience in the blood coagulation and fibrinolysis field. Previously undescribed roles in a range of blood clot properties are attributed to a region of the clotting protein fibrinogen, using state-of-the-art methodology. The data support the main conclusions of the paper, open new avenues of investigation for understanding clot properties, and have clinical implications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this paper McPherson et al investigated fibrin clot properties and fibrinolysis with recombinant fibrinogen variants lacking parts of the fibrinogen alphaC-region. The aim was to understand the contribution of two subregions, the alphaC-connecter and the alphaC-domain, which are known to be involved in the lateral aggregation of fibrin fibers and their cross-linking. The study measured the contribution of subregions to fibrin fiber growth, mechanical strength, how fibrinolysis proceeds in their absence, their impact on clot retraction, and how the variants affect whole blood clot features.
Strengths and weaknesses:
The major strengths of this report lie in the broad range of appropriately selected assays used to characterize the fibrinogen variants described, the clarity of the data presented, and a clear …
Reviewer #1 (Public Review):
In this paper McPherson et al investigated fibrin clot properties and fibrinolysis with recombinant fibrinogen variants lacking parts of the fibrinogen alphaC-region. The aim was to understand the contribution of two subregions, the alphaC-connecter and the alphaC-domain, which are known to be involved in the lateral aggregation of fibrin fibers and their cross-linking. The study measured the contribution of subregions to fibrin fiber growth, mechanical strength, how fibrinolysis proceeds in their absence, their impact on clot retraction, and how the variants affect whole blood clot features.
Strengths and weaknesses:
The major strengths of this report lie in the broad range of appropriately selected assays used to characterize the fibrinogen variants described, the clarity of the data presented, and a clear discussion of the mechanistic implications of the findings compared to previous work. We can now understand more clearly how the alphaC-subregions contribute to clot structure, initial fibrin fiber growth, clot strength and stiffness, fibrinolysis, clot contraction linked to erythrocyte retention and platelet binding, and a clinically relevant assessment of the clotting and lysis of whole blood.
The methodology does not have weaknesses in the context presented. One issue that arises from such a study, that is centered on the characterization recombinant molecules assessed in vitro, is to what extent each of the characteristics described would have physiological impact in vivo. The study has the advantage of clearly separating out different roles for the fibrinogen alphaC-region, but the more complex interplay of the variants with a complete vasculature and blood composition, in an organism that produced the variants, would enhance the study claims. This issue is hinted at in the paper, albeit in vitro/ex vivo. The fibrin density and fiber thickness of alpha390 clots was different in a purified setting compared to whole reconstituted blood clots post-thromboelastography made using blood from Fga-/- mice. It therefore seems reasonable that characteristics of the alphaC-region functions described may show more or less importance when assessed in vivo.
Aims, results and conclusions of the paper:
The authors achieved their aims, separating functional roles for the two alphaC-subregions, and assessing their relative impact in a broad range of relevant experimental settings. The results clearly support the author's conclusions and are a solid basis on which to design future work with additional variants, and to further assess the role of the alphaC-region in vivo.
In more detail, the authors produced and purified two novel recombinant fibrinogen variants, alpha390 and alpha220, lacking the alphaC-domain and the alphaC-region (alphaC-connector and alphaC-domain), respectfully. Using turbidity and microscopy, alpha390 gave thinner, denser fibrin clots while alpha220 clots were porous and stunted, compared to control. Alpha220 fibrin polymers formed slowly and were short, implicating directly the alphaC-region in fibrin fiber growth. Surprisingly, both alpha chain variants underwent cross-linking, but alpha220 was extremely sensitive to fibrinolysis and alpha220 clot stiffness could not be measured without FXIIIa. Using blood from Fga-/- mice, which cannot support clotting, clot retraction and size was severely affected with alpha220 supplementation whereas alpha390 behaved like wild-type fibrinogen. This highlights the importance of the alphaC-connector in this mechanism which involves FXIIIa-catalyzed cross-linking. The observation was reinforced by measuring platelet binding, which was also affected but not lost in the alpha220 variant, and similar to wild-type fibrinogen with alpha390. Finally, EXTEM and APTEM thromboelastography assays were made on reconstituted Fga-/- blood as a global assessment of clot properties and the contribution of fibrinolysis. This was important because it demonstrated that both truncations failed to reach wild-type clot firmness, clotting was slowed, and that both variants were susceptible to a decline in clot strength due to concurrent clotting and fibrinolysis. Also, imaging on these ex vivo whole blood clots confirmed the short, sparse nature of alpha220 fibers seen in vitro, and highlighted a certain "normalization" of the thinner, denser alpha390 fibrin when cells were present.
Impact and utility to the field of study:
This work has clinical relevance for patients with fibrinogen alpha chain truncation mutations and gives insights to clinicians measuring clot properties in patients. Perhaps more importantly at this stage, the study implicates the alphaC-subregions in fundamental aspects of fibrin formation, fibrinolysis and overall clot mechanics. The methods represent a state-of-the-art panel of tests and therefore a useful guide to the research field on how to approach aspects of fibrinogen function in clotting, particularly using recombinant molecules. The impact of this work therefore lies in both the discovery of roles for the fibrinogen alphaC-subregions, and a cautious extrapolation to the understanding of whole blood clot mechanics measured in patients.
Additional context:
Readers can put into context these findings by familiarizing themselves with the clinical presentations and impact on clot properties of known dysfibrinogenemia (or hypodysfibrinogenemia) mutations in the fibrinogen alphaC-region. Also, there are published experimental studies of fibrinogen alphaC variants. The significance of the variants described can be put into a broader context by comparing the thromboelastography and subsequent imaging shown here with whole blood clot analysis used for clinical haemostasis decision-making.
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Reviewer #2 (Public Review):
McPherson H et al designed two fibrinogen variants with truncated alfa- C terminal region: the alfa-390 and alfa-220. The alfa-390 lacks the alfa-C domain, while the alfa-220 lacks both the alfa-C domain and alfa-C connector region. By using different type of optical and electronic microscopy they characterize the fibrin structure at different resolution, and the early fibrin oligomers formation of these homozygous fibrinogen variants, and compared them to the WT fibrinogen. The functional implications of removing these protein stretches in the fibrin mechanical properties and stability were studied by turbidity, FXIIIa fibrin crosslinking, fibrin microrheology, clot contraction (retraction), and rotational thromboelastometry. It was found for the first time the differential roles of these two regions: the …
Reviewer #2 (Public Review):
McPherson H et al designed two fibrinogen variants with truncated alfa- C terminal region: the alfa-390 and alfa-220. The alfa-390 lacks the alfa-C domain, while the alfa-220 lacks both the alfa-C domain and alfa-C connector region. By using different type of optical and electronic microscopy they characterize the fibrin structure at different resolution, and the early fibrin oligomers formation of these homozygous fibrinogen variants, and compared them to the WT fibrinogen. The functional implications of removing these protein stretches in the fibrin mechanical properties and stability were studied by turbidity, FXIIIa fibrin crosslinking, fibrin microrheology, clot contraction (retraction), and rotational thromboelastometry. It was found for the first time the differential roles of these two regions: the role of the alfa-C connector in the longitudinal protofibril/fibre growth, mechanical and fibrinolytic stability, while the alfa-C domain (variant 390) was implicated in the lateral protofibrils association, since its removal gave rise to denser fibrin networks with thinner fibres, already described in the literature. Their finding has clinical implications since support the design of antithrombotic drugs that can limit the thrombus size or growth. Their conclusions are supported by the results. The confocal microscopy fibrin structure was confirmed by the scanning electron microscopy images, and highlight the importance of coupling the fibrinogen under study to the fluorophore in order to do not bias the fibrin structure.
In order to study the differential implications of these alfa-C subregions on the susceptibility of fibrinogen/fibrin plasmin degradation that will support thromboelastometric and turbidity results, it would be interesting in the future to perform fibrinogen/fibrin plasmin degradation kinetic of these variants monitored by SDS/PAGE.
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